Science, Faculty of
Microbiology and Immunology, Department of
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UBCV
Ifill, Gyles Anderson
Doctor of Philosophy - PhD
University of British Columbia
In many Gram-negative pathogens, gene transcription is often facilitated by post-transcriptional regulatory mechanisms. In Bordetella pertussis, several regulatory RNAs have been identified, but their targets and impacts on virulence factor production have not been assessed. RNase III and RNase E, endonucleases with dominant roles in regulatory RNA processing, were mutated in B. pertussis to determine gene loci regulated at the post-transcriptional level. RNA-Seq analysis of these strains identified that ~25% of the B. pertussis transcriptome was affected in each endonuclease mutant. Substantial impacts were observed on genes associated with amino acid uptake, bacterial secretion, and many virulence factors. Comparing these findings to the regulon of the RNA chaperone Hfq, 120 genes, and 19 operons were identified as potentially influenced at the post-transcriptional level. Amongst these, the lipid A glucosamine modification (lgm) locus was one of the most upregulated gene loci in the RNase E mutant strain. The lgm locus is a 5 gene operon consisting of four genes in one orientation (lgmA-D) and a fifth open reading frame (lgmE) overlapping in the opposite orientation. Given lipid A modifications in Gram-negative bacteria are typically controlled by complex regulatory networks, it was proposed that several overlapping systems are involved in lgm locus regulation, including the role of a cis-encoded antisense RNA. As shown by luciferase reporter assays, the lgm locus responds to Bvg phase, nutrient availability, low pH, and increased C02%. These assays also identified a reciprocal relationship in activation of the diametrically opposed lgmA and lgmE promoters, suggestive of asRNA regulation. It was then proposed that lgmE acts as dual-function RNA as the lgmE ORF has all the characteristics of a translated protein. It is shown that lgmE encodes a functional, small membrane-associated protein, and deletion and overexpression of lgmE negatively impact lgm locus activity. Furthermore, structural predictions and modelling of protein-protein interactions suggest LgmE may form a membrane anchor for localization with LgmB. Overall, the lgm locus forms a non-contiguous operon whose activity is modulated at the transcriptional, post-transcriptional, and post-translational levels, with these integrated mechanisms intersecting as a means to fine-tune lipid A modification in B. pertussis.
Lipid A modifications in Bordetella pertussis: Regulation and Function of the lgm Locus by Gyles Anderson Ifill B.Sc., The University of East Anglia, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2021 © Gyles Anderson Ifill, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Lipid A modifications in Bordetella pertussis: regulation and function of the lgm locus submitted by Gyles A. Ifill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Examining Committee: Professor Rachel C. Fernandez, Microbiology and Immunology, UBC Supervisor Professor J. Thomas Beatty, Microbiology and Immunology, UBC Supervisory Committee Member Professor Rosie J. Redfield, Zoology, UBC University Examiner Associate Professor Cara H. Haney, Microbiology and Immunology, UBC University Examiner Additional Supervisory Committee Members: Professor Charles J. Thompson, Microbiology and Immunology, UBC Supervisory Committee Member Professor Natalie C. J. Strynadka, Biochemistry and Molecular Biology, UBC Supervisory Committee Member iii Abstract In many Gram-negative pathogens, gene transcription is often facilitated by post-transcriptional regulatory mechanisms. In Bordetella pertussis, several regulatory RNAs have been identified, but their targets and impacts on virulence factor production have not been assessed. RNase III and RNase E, endonucleases with dominant roles in regulatory RNA processing, were mutated in B. pertussis to determine gene loci regulated at the post-transcriptional level. RNA-Seq analysis of these strains identified that ~25% of the B. pertussis transcriptome was affected in each endonuclease mutant. Substantial impacts were observed on genes associated with amino acid uptake, bacterial secretion, and many virulence factors. Comparing these findings to the regulon of the RNA chaperone Hfq, 120 genes, and 19 operons were identified as potentially influenced at the post-transcriptional level. Amongst these, the lipid A glucosamine modification (lgm) locus was one of the most upregulated gene loci in the RNase E mutant strain. The lgm locus is a 5 gene operon consisting of four genes in one orientation (lgmA-D) and a fifth open reading frame (lgmE) overlapping in the opposite orientation. Given lipid A modifications in Gram-negative bacteria are typically controlled by complex regulatory networks, it was proposed that several overlapping systems are involved in lgm locus regulation, including the role of a cis-encoded antisense RNA. As shown by luciferase reporter assays, the lgm locus responds to Bvg phase, nutrient availability, low pH, and increased C02%. These assays also identified a reciprocal relationship in activation of the diametrically opposed lgmA and lgmE promoters, suggestive of asRNA regulation. It was then proposed that lgmE acts as dual-function RNA as the lgmE ORF has all the characteristics of a translated protein. It is shown that lgmE encodes a functional, small membrane-associated protein, and deletion and overexpression of lgmE negatively impact lgm locus activity. Furthermore, structural predictions and modelling of iv protein-protein interactions suggest LgmE may form a membrane anchor for localization with LgmB. Overall, the lgm locus forms a non-contiguous operon whose activity is modulated at the transcriptional, post-transcriptional, and post-translational levels, with these integrated mechanisms intersecting as a means to fine-tune lipid A modification in B. pertussis. v Lay Summary The disease whooping cough is caused by the bacterium Bordetella pertussis. Recent outbreaks of whooping cough have led to increased interest in how the bacterium causes disease. One key component of infection is being able to evade the host immune system, which many bacterial pathogens achieve by changing the properties of their membranes. The work in this thesis aimed to characterize which environmental stimuli dictate changes in the membrane of B. pertussis. It is shown that an important membrane modification system is stimulated under nutrient limitation and conditions found in the human lung. This system also incorporates inputs from regulatory systems acting at the DNA, RNA, and protein levels. The work presented here highlights the complexity of membrane remodeling and provides further understanding of how B. pertussis may be responding within the host. vi Preface The research presented in this thesis was designed, performed, and analyzed by Gyles Ifill with the support and guidance of Dr. Rachel C. Fernandez. Several experiments were conducted collaboratively as specified below for each chapter. A version of Chapter 2 has been published in mSphere: Ifill, G., Blimkie, T., Lee, A. H., Mackie, G. A., Chen, Q., Stibitz, S., Hancock, R., & Fernandez, R. C. (2021). RNase III and RNase E Influence Posttranscriptional Regulatory Networks Involved in Virulence Factor Production, Metabolism, and Regulatory RNA Processing in Bordetella pertussis. mSphere, 6(4), e0065021. https://doi.org/10.1128/mSphere.00650-21. Travis M. Blimkie and Dr. Amy H. Lee were responsible for RNA-Seq and differential expression analysis in the lab of Dr. Robert E. W. Hancock. Dr. George A. Mackie contributed through discussions of phenotypes and gene expression changes observed in the generated endonuclease mutants. Dr. Qing Chen and Dr. Scott Stibitz provided plasmids from which mutagenesis constructs were assembled, and the Escherichia coli strain RHO3 for plasmid transfer by conjugation. The manuscript was written by Gyles A. Ifill and Dr. Rachel C. Fernandez with the exception of RNA-Seq analysis methods which were written by Travis M. Blimkie. All authors provided editorial comments on the published manuscript. In Chapter 3, the reporter plasmids pSS3967 and pQC1794 were a kind gift from Drs. Qing Chen and Scott. Stibitz at the U.S. Food and Drug Administration. Read counts in Figure 3.2A were plotted by Travis M. Blimkie, a bioinformatician in the lab of Dr. Robert E. Hancock at UBC. The following experiments were done in collaboration with members of Dr. Rachel C. Fernandez’ lab at UBC: vii Mass spectrometry analysis of lipid A fractions was performed by post-doctoral fellow Dr. Yasmine Fathy Mohamed. The plasmid constructs, pB4, pB4-LgmB, and pB4-LgmA + LgmB, were constructed by post-doctoral fellow Dr. Daniel Jun. The growth, maintenance of HEK293 blue cell lines and TLR4 activation assay of wild type Bordetella pertussis and BP338ΔlgmA-D compared to RNase III and RNase E mutant strains was performed by Kiruthika Manivannan, MSc. The luciferase reporter assays in Fig. 3.4A and Fig. 3.5 were performed by Ravneet Grewal under the supervision of Gyles Ifill. In Chapter 4, the plasmid pQC2241 was a kind gift from Q. Chen and S. Stibitz at the U.S. Food and Drug Administration. The plasmid pGEM-tetR10.oriApra (pT10) was obtained from Dr. Jan Burian in the laboratory of Dr. Charles Thompson at UBC. Mass spectrometry analysis of lipid A fractions was performed by post-doctoral fellow Dr. Yasmine Fathy Mohamed and the plasmid constructs pB4-LgmC, and pB4-L63MLgmC were constructed by post-doctoral fellow Dr. Daniel Jun, both in the lab of Dr. Rachel C. Fernandez at UBC. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiv List of Figures. ..............................................................................................................................xv List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................... xxi Dedication .................................................................................................................................. xxii Chapter 1: Introduction ........................................................................................................... 1 1.1 Whooping cough and Bordetella pertussis............................................................... 1 1.1.1 Pathophysiology and clinical presentation .............................................................. 2 1.2 Molecular pathogenesis ............................................................................................ 3 1.2.1 BvgAS two-component system............................................................................... 3 1.2.2 Gene expression profiles of BvgAS ........................................................................ 4 1.2.3 Virulence factors associated to BvgA~P regulation ............................................... 5 1.2.3.1 Toxins ............................................................................................................. 5 1.2.3.2 The type III secretion system .......................................................................... 7 1.2.3.3 Autotransporters .............................................................................................. 8 1.2.4 Integrated signals from other two-component systems .......................................... 8 1.3 Post-transcriptional gene regulation in bacterial pathogens .............................. 10 1.3.1 Features encoded in the 5’ untranslated region ..................................................... 10 ix 1.3.2 Cis- and trans-encoded regulatory RNAs ............................................................. 11 1.3.3 Post-transcriptional regulation by ribonucleases .................................................. 13 1.3.3.1 RNase III ....................................................................................................... 14 1.3.3.2 RNase E ........................................................................................................ 16 1.3.3.3 Other ribonucleases ....................................................................................... 19 1.3.4 Post-transcriptional regulation in B. pertussis ...................................................... 20 1.4 Structure and function of the Gram-negative outer membrane ........................ 22 1.4.1 LPS structure ......................................................................................................... 22 1.4.1.1 O-antigen....................................................................................................... 22 1.4.1.2 Core sugars.................................................................................................... 23 1.4.1.3 Lipid A .......................................................................................................... 23 1.4.1.4 Biosynthesis and export ................................................................................ 26 1.4.2 The biological function of LPS ............................................................................. 28 1.4.2.1 Membrane integrity ....................................................................................... 28 1.4.2.2 Host-pathogen interactions and Toll-Like Receptor 4 .................................. 28 1.4.2.3 Function and regulation of lipid A modifications ......................................... 31 1.4.3 B. pertussis LPS structure and modification ......................................................... 34 1.4.4 Lipid A glucosamine modification locus .............................................................. 37 1.4.5 Function and regulation of lgm locus.................................................................... 40 1.5 Hypotheses and thesis goals ................................................................................... 41 Chapter 2: RNase III and RNase E influence post-transcriptional regulatory networks involved in virulence factor production, metabolism, and regulatory RNA processing in Bordetella pertussis. ................................................................................................................. 44 x 2.1 Synopsis .................................................................................................................... 44 2.2 Introduction ............................................................................................................. 45 2.3 Materials and Methods ........................................................................................... 49 2.3.1 Bacterial strains, plasmids, and growth conditions. .............................................. 49 2.3.2 Construction of endoribonuclease mutants in B. pertussis ................................... 51 2.3.3 RNA-Seq and bioinformatic analyses ................................................................... 52 2.3.4 qPCR ..................................................................................................................... 54 2.3.5 Immunoblots ......................................................................................................... 54 2.3.6 Data availability .................................................................................................... 55 2.4 Results ...................................................................................................................... 56 2.4.1 Generation and growth characteristics of the RNase III mutant, BPrncD45A ..... 56 2.4.2 Generation and growth characteristics of the RNase E mutant, BPrneΔCT ......... 60 2.4.3 Impact of mutations in RNase III and RNase E on the B. pertussis transcriptome .. ............................................................................................................................... 64 2.4.4 Validation of RNA-Seq data set ........................................................................... 70 2.4.5 Mutations in RNase III and RNase E drove virulence factor stability in opposite directions. .......................................................................................................................... 73 2.4.6 RNase III and RNase E processed transcripts associated with metabolism in the Bvg+ mode ........................................................................................................................ 79 2.4.7 Comparative analysis of B. pertussis transcriptomic data provided insights into regulatory RNA networks ................................................................................................. 81 2.5 Discussion................................................................................................................. 83 xi Chapter 3: Regulation of Lipid A modifications in Bordetella pertussis involves several overlapping networks modulating expression at transcriptional, post-transcriptional, and translational levels ........................................................................................................... 88 3.1 Synopsis .................................................................................................................... 88 3.2 Introduction ............................................................................................................. 89 3.3 Materials and methods ........................................................................................... 94 3.3.1 Bacterial strains, plasmids, and growth conditions. .............................................. 94 3.3.2 Construction of deletion mutants .......................................................................... 97 3.3.3 Preparation of bacterial cells for TLR4-activation assays .................................... 97 3.3.4 Growth and maintenance of HEK-Blue cell lines ................................................. 98 3.3.5 TLR4-NFκB activation assays .............................................................................. 98 3.3.6 Polymyxin B assay ................................................................................................ 99 3.3.7 Extraction of lipid A and mass spectrometry analysis .......................................... 99 3.3.8 Luciferase reporter assays. .................................................................................. 100 3.3.9 Beta-galactosidase assays ................................................................................... 102 3.3.10 Reverse transcription PCR .................................................................................. 102 3.3.11 Mapping read counts and lgm locus promoter predictions ................................. 104 3.4 Results .................................................................................................................... 105 3.4.1 Endonucleases RNase III and RNase E significantly impact B. pertussis TLR4 activation ......................................................................................................................... 105 3.4.2 Transcriptional profiling of the lgm locus in endonuclease mutant backgrounds .... ............................................................................................................................. 109 xii 3.4.3 LgmA promoter is responsive to Bvg phase, iron, and divalent cation availability . ............................................................................................................................. 113 3.4.4 Ectopic plgmE reporter shows differential activation signals when compared to in situ plgmE reporter .......................................................................................................... 119 3.4.5 Low pH and CO2 % induce reciprocal activation of plgmA and plgmE. ............ 121 3.4.6 Deletion of lgmE impacts TLR4 activation without loss of glucosamine modification .................................................................................................................... 124 3.4.7 Co-translation of LgmAB is required to complement lgmB mutation ................ 128 3.5 Discussion............................................................................................................... 133 3.5.1 Post-transcriptional regulation ............................................................................ 134 3.5.2 Transcriptional regulation ................................................................................... 136 3.5.3 Genetic architecture of the lgm locus .................................................................. 140 Chapter 4: Characterization of LgmE, a putative antisense protein involved in the regulation of lgm locus .......................................................................................................... 142 4.1 Synopsis .................................................................................................................. 142 4.2 Introduction ........................................................................................................... 143 4.3 Materials and Methods ......................................................................................... 146 4.3.1 Bacterial strains, plasmids, and growth conditions. ............................................ 146 4.3.2 Construction of pIG10 gene expression system. ................................................. 149 4.3.3 LgmE protein expression .................................................................................... 152 4.3.4 Generation of constructs to detect native expression of lgmE ............................ 153 4.3.5 Membrane preparations for immunoblots ........................................................... 154 4.3.6 Immunoblots ....................................................................................................... 154 xiii 4.3.7 TLR4-NFκB activation assays ............................................................................ 155 4.3.8 Bright-field and fluorescence microscopy .......................................................... 155 4.3.9 Protein structure and interaction modelling ........................................................ 156 4.4 Results .................................................................................................................... 157 4.4.1 Recombinant expression of the antisense open reading frame, lgmE ................. 157 4.4.2 Alternative start codon for LgmC ....................................................................... 163 4.4.3 Native expression of LgmE in B. pertussis ......................................................... 167 4.4.4 Deletion and overexpression of lgmE suppress B. pertussis activation of hTLR4 ... ............................................................................................................................. 172 4.4.5 Structural modelling of LgmE ............................................................................ 176 4.5 Discussion............................................................................................................... 183 Chapter 5: Conclusion .......................................................................................................... 189 5.1 Overview ................................................................................................................ 189 5.2 Limitations and future work ................................................................................ 197 Bibliography ...............................................................................................................................200 Appendices ..................................................................................................................................228 xiv List of Tables Table 1.1 Ribonucleases present in both E. coli and B. pertussis. ................................................ 21 Table 2.1 Strain and primer list. .................................................................................................... 50 Table 2.2 Top ranking differentially expressed genes in transcriptomic analysis of endonuclease mutants in B. pertussis. ................................................................................................................. 69 Table 3.1 Strain and primer list ..................................................................................................... 95 Table 4.1 Strain and primer list ................................................................................................... 147 Appendix Table A.1 Well characterized virulence factors found to be differentially expressed in the endonuclease mutants ........................................................................................................... 230 Appendix Table A.2 Genes differentially expressed in both BPrncD45A and BPrneΔCT. ....... 232 Appendix Table A.3 sRNAs identified in RNA-Seq analysis of endonuclease mutants ............ 232 Appendix Table A.4 Gene loci found near IS elements influenced by antisense RNA found to be differentially expressed in endonuclease mutants....................................................................... 233 Appendix Table A.5 Operons differentially expressed in endonuclease mutants. ..................... 234 Appendix Table B.1 Sequences identified from lgmA and lgmE upstream regions most likely to contain sigma factor binding sites as predicted by BacPP. ......................................................... 235 xv List of Figures. Figure 1.1 General overview of regulatory RNA processing by endonucleases in Gram-negative bacteria. ......................................................................................................................................... 18 Figure 1.2 Schematic of Gram-negative membrane and General LPS structure. ......................... 25 Figure 1.3 Schematic of dimerization of TLR4-MD2 upon lipid A binding ................................ 30 Figure 1.4 Structures of lipid A from B. pertussis Tohama-1 strain BP338 ................................. 36 Figure 1.5 Genetic architecture of the lgm locus and predicted structures and function of gene products. ........................................................................................................................................ 39 Figure 2.1 Design and growth characteristics of B. pertussis endonuclease mutants. .................. 59 Figure 2.2 Complementation of endonuclease mutants. ............................................................... 63 Figure 2.3 Differential gene expression in the RNase III (BPrncD45A) and RNase E (BPrneΔCT) mutants compared to wild type as determined by RNA-Seq................................... 68 Figure 2.4 Validation of the RNA-Seq data sets using brkA and tcfA. ......................................... 72 Figure 2.5 RNase III and RNase E have opposing activities on virulence factor transcripts. ...... 78 Figure 2.6 Impact of endonuclease mutations on Bvg regulated metabolism genes. ................... 80 Figure 3.1 Impact of endonuclease mutants on lipid A modification in B. pertussis. ................ 108 Figure 3.2 Mapped reads counts of lgm locus transcripts and presence of sense and antisense transcripts. ................................................................................................................................... 112 Figure 3.3 regions upstream of lgmA and lgmE with predicted promoter elements. .................. 114 Figure 3.4 lgm locus reporter assays ........................................................................................... 118 Figure 3.5 Comparison of in situ and ectopic luciferase reporters of the lgmE upstream region...................................................................................................................................................... 120 Figure 3.6 Impact of pH and % CO2 on B. pertussis growth and lgm locus activation. ............ 123 xvi Figure 3.7 Assessment of new lgm locus mutants on glucosamine modification of B. pertussis lipid A ......................................................................................................................................... 127 Figure 3.8 Complementation of BP338lgmBΔ6-520 requires co-translation of LgmAB ............... 132 Figure 4.1 Development of pIG10, tetracycline responsive gene expression system. ............... 151 Figure 4.2 Immunoblot of LgmE expression in E. coli membrane fractions. ............................ 159 Figure 4.3 Immunoblot of LgmE expression in B. pertussis. ..................................................... 162 Figure 4.4 Identification of alternative start for LgmC. .............................................................. 166 Figure 4.5 Immunoblot of N-terminal 6×His tag inserted into chromosomal copy of lgmE...... 168 Figure 4.6 Assessment of native LgmE expression using translational fusions. ........................ 171 Figure 4.7 Deletion and overexpression of lgmE impact B. pertussis TLR4 activation without altering the transcript stability of other lgm locus genes. ........................................................... 175 Figure 4.8 Structural modelling of LgmE. .................................................................................. 178 Figure 4.9 pyDock modelled interactions between predicted protein models of LgmB and LgmE...................................................................................................................................................... 182 Figure 5.1 Proposed model of lgm locus regulation involving dual functions of lgmE. ............ 195 Appendix figure A.1 Preliminary characterization of temperature sensitive RNase E mutant, BPrneG66S .................................................................................................................................... 229 xvii List of Abbreviations ABC Adenosine Triphosphate (ATP)-Binding Cassette Abs Absorbance ADP Adenosine Diphosphate Ampr Ampicillin Resistant ANOVA Analysis Of Variance Ara4N Aminoarabinose (4-Amino-4-Deoxy-L-Arabinose) asRNA Antisense Ribonucleic Acid aTc Anhydrotetracycline ATP Adenosine Triphosphate BG Bordet-Gengou (Agar) bp Base Pair BSA Bovine Serum Albumin Bvg Bordetella Virulence Gene BvgA~P Phosphorylated (Activated) Bvga C10-OH Hydroxydecanoic Acid C14-OH Hydroxymyristoyl Chain C16-OH Hydroxypalmitoyl Chain C55P Undecaprenyl-Phosphate cAMP Cyclic Adenosine Monophosphate CAMPS Cationic Antimicrobial Peptides CD14 Cluster of Differentiation 14 c-di-GMP Cyclic Di-Guanosine-Monophosphate CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CyaA Adenylate Cyclase DAP Di-Amino Pimelic Acid DE Differentially Expressed DEAD Asp-Glu-Ala-Asp (Motif) DMEM Dulbecco's Modified Eagle Medium xviii DNA Deoxyribonucleic Acid DNT Dermonecrotic Toxin DPMS Dolichol-Phosphate Mannose Synthase EAL Glu-Ala-Leu (Motif) EDTA Ethylenediaminetetraacetic Acid FDA Food And Drug Administration FDR False Discovery Rate GAGE Generally Applicable Gene Set Enrichment GDP Guanosine Diphosphate GFP Green Fluorescent Protein GlcN Glucosamine GlcNAc N-Acetyl Glucosamine GMP Guanosine Monophosphate Gmr Gentamycin Resistant G-protein Guanine Nucleotide-Binding Protein GT Gylcosyltransferase GTP Guanosine Triphosphate HEK Human Embryonic Kidney His Histidine HRP Horseradish Peroxidase hrs Hours(s) hTLR4 Human Toll-Like Receptor 4 IM Inner Membrane IRF3 Interferon Regulatory Transcription Factor 3 IS Insertion (Element) JM Juxta-membrane Kanr Kanamycin Resistant KDO Ketodeoxyoctonic Acid KEGG Kyoto Encyclopedia of Genes and Genomes LB Lysogeny Broth LBP Lipopolysaccharide Binding Protein xix LOS Lipooligosaccharides LPS Lipopolysaccharide MALDI-ToF Matrix-Assisted Laser Desorption/Ionization-Time of Flight MD-2 Myeloid Differentiation Factor 2 MD-2* Dimerizing MD-2 MIC Minimum Inhibitory Concentration min Minute(S) mRNA Messenger Ribonucleic Acid MyD88 Myeloid Differentiation Primary Response Gene 88 nalr Nalidixic Acid Resistant NCBI National Center for Biotechnology Information NFκB Nuclear Factor Kappa-B NIH National Institutes of Health nt Nucleotide OD600nm Optical Density at 600 nanometers OM Outer Membrane ONPG Ortho-Nitrophenyl-ß-Galactoside ORF Open Reading Frame PAGE Polyacrylamide Gel Electrophoresis PAS Per-Arnt-Sim PBS Phosphate-Buffered Saline pBSII pBluescriptIIKS- PCR Polymerase Chain Reaction PG Peptidoglycan Phyre2 Protein Homology/Analogy Recognition Engine V2.0 PmB Polymyxin B PP Periplasm PT Pertussis Toxin PVDF Polyvinylidene Fluoride RBS Ribosome Binding Site RFP Red Fluorescent Protein xx RNA Ribonucleic Acid rpm Revolutions Per Minute RR Response Regulator rRNA Ribosomal Ribonucleic Acid RT-PCR Reverse Transcriptase-Polymerase Chain Reaction RT-qPCR Reverse Transcriptase- Quantitative Polymerase Chain Reaction RTX Repeat In Toxin Repeats SDS Sodium Dodecyl Sulphate secs Second (S) sRNA Small Ribonucleic Acid SS Stainer-Scholte SSM Stainer-Scholte Media STAR Spliced Transcripts Alignment To A Reference TIR Toll/Interleukin-1 Receptor TIRAP Tir Domain Containing Adaptor Protein TLR4 Toll-Like Receptor 4 TLR4* Dimerizing Toll-Like Receptor 4 TM Transmembrane TRAM TRIF-Related Adaptor Molecule TRIF TIR Domain-Containing Adaptor Protein-Inducing Interferon Β tRNA Transfer Ribonucleic Acid UBC University of British Columbia UDP Uridine Diphosphate UMP Uridine Monophosphate UTR Untranslated Region v/v Volume/ Volume vag Virulence Associated Genes VFT Venus Fly Trap (Domain) vrgs Virulence Repressed Genes xxi Acknowledgements I would first like to thank Dr. Rachel C. Fernandez for this incredible opportunity to study abroad and the drive and push to get the work recognized at conferences across the world. I was truly captured by the spiel from the first discussion of the project over skype. It really has been an experience. I am grateful for the insight and discussion over the years with my committee members: Dr. Natalie Strynadka, Dr. Tom Beatty, and Dr. Charles Thompson. Dr. George Mackie has been an invaluable collaborator in discussions about RNase E function, the phenotypes we saw in Bordetella, and also being a friendly face to see on the fifth floor of the LSI. I would like to thank members of the Fernandez lab, previous and present, for their help on the various projects over the years. Including the undergraduates that I have trained, as this provided the drive to better understand my projects to best guide others. I would also like to thank Jenny Vermeulen for training and assistance on their microscope and general borrowing of consumables and conversations throughout my PhD. Mostly, I would like to thank the bandmates for the midweek musical escapes, and some unforgettable Christmas parties. The sports teams for dodgeball, softball, and football (Goalitis most of all) and the general graduate student mass of the M&I and Biochemistry departments for all the laughs and friendships that have kept me sane. xxii Dedication For my parents and brother who have supported me throughout this adventure. Being by my side even though more than 5000 km away. 1 Chapter 1: Introduction 1.1 Whooping cough and Bordetella pertussis Bordetella pertussis is a small Gram-negative aerobe, and the causative agent of whooping cough, an acute respiratory infection (1). This small coccobacillus is around 0.8 µm by 0.4 µm in size and is an obligate human pathogen as no environmental niche has been observed. Even though infants are most likely to show clinical manifestations of whooping cough, it is believed adults and adolescents are the primary reservoir of transmission, due to short-lived and waning immunization after childhood vaccination (2, 3). B. pertussis was first isolated at the beginning of the 20th century by Jules Bordet and Octave Gengou (4). Many candidate vaccines had been developed by the 1930s and vaccines consisting of whole killed cells of B. pertussis were universally used by the 1940s (5). This led to a dramatic decline in pertussis cases, and it was almost eradicated in the developed world by the 1970s; however, despite high vaccine uptake, there has recently been a substantial increase in cases of whooping cough reported globally (6). Since the year 2000, epidemic cycles of similar periodicity to the pre-vaccine era have been observed globally with case burden peaking in 2012. Furthermore, recent estimates suggest there are approximately 24.1 million pertussis cases and 160,700 deaths in children younger than 5 years of age worldwide (7). In the late 1990s to early 2000s, the acellular pertussis vaccine was introduced in many developed countries, composed of several purified B. pertussis antigens (6, 8). This vaccine, even though sufficient at protecting vaccinated individuals against disease symptoms, has been shown to not fully protect against colonization and transmission (9). This has been postulated as one of the factors contributing to the resurgence of whooping cough with some recent outbreak strains lacking vaccine antigens (10). Alongside this evolution of specific antigens, the 2 persistence of B. pertussis is attributed to the comprehensive arsenal of virulence factors enabling modulation and evasion of host immune responses (1). 1.1.1 Pathophysiology and clinical presentation B. pertussis is transmitted by aerosols typically expelled as a cough from an infected individual. To establish infection, the bacterium must be able to adhere to ciliated epithelial cells present in the human upper respiratory tract. Here, adaptations to the host environment allow for the multiplication of bacterial cells. This induces mucus secretion from host cells, facilitating progression further into the respiratory tract. Within the lower respiratory tract, production of toxins and other virulence factors leads to the destruction and effacement of the ciliated epithelium (6, 11). Infection can last up to 12 weeks with three main phases. The catarrhal stage, lasting for 1-2 weeks presents as a mild respiratory infection with the development of a mild cough and low-grade fever. The second, paroxysmal stage, can last up to 8 weeks, characterized by episodes of intense coughs followed by the inspiratory “whoop” from which the name of the disease derives. This stage is also associated with other complications such as pneumonia. The final stage, convalescence, is 4 weeks on average, denoted by a progressive reduction of all symptoms. However, this can last up to several months depending on the severity of disease and co-morbidities. The most severe disease is often seen in neonates, where mortality is estimated at around 3%, although this is much lower in adolescents and adults (1). 3 1.2 Molecular pathogenesis The success of B. pertussis as a pathogen is in part explained by the substantial arsenal of virulence factors produced by the organism. These determinants of pathogenesis encode various toxins, adhesins, and secretion systems, allowing colonization, immune evasion, and immunomodulation (1, 12). In B. pertussis, virulence factor expression is centrally controlled by the master two-component system BvgAS (6, 13). This system coordinates expression changes of over 500 genes in response to physiological temperatures (14). As much as initiating the expression of virulence factors is imperative for host colonization, the repression of specific genes is equally as important within the host environment (6). 1.2.1 BvgAS two-component system Two-component systems are a conserved signal transduction mechanism found in prokaryotes and some eukaryotes (15, 16). These are formed of at least a membrane-bound sensor kinase and a cytoplasmic response regulator (RR). In response to environmental stimuli, the activated kinase transfers a phosphate group to the RR. In its phosphorylated form, the RR can bind defined sequence motifs near promoters of specific genes in order to coordinate gene expression changes (15-18). The Bordetella virulence locus, BvgAS, is a well-characterized two-component system critical to the regulation of virulence factor production in B. pertussis (13, 14, 19). BvgS is a 135 kDa protein sensor kinase imbedded in the inner membrane. It acts as a dimer, where each monomer is formed of two periplasmic Venus fly trap (VFT) domains, a transmembrane segment, a PAS (per-arnt-sim) domain, and a histidine-kinase which initiates the phosphorelay (20). The VFT 4 domain forms a hinged, two-lobed structure which is proposed to fold around a specific ligand (20, 21). An activating ligand has not been identified for BvgS, yet it is active at physiological temperatures such as 35-37°C. Active BvgS conducts the virulence-on state of B. pertussis, referred to as the Bvg+ mode (19). Here, the periplasmic VFT domain is loosely folded and this conformation transduces a structural signal across the inner membrane to activate the cytoplasmic phosphotransferase domains (22). Transfer of this phosphate to BvgA (BvgA~P), initiates this RR to bind promoter regions of regulated genes. BvgA is a 23 kDa protein with a C-terminal helix-turn-helix motif required for DNA-binding (13). BvgA acts as a dimer in which the phosphorylated form binds a consensus sequence denoted as an inverted heptad present in the promoter region of many genes (23). BvgA~P dimer binding modulates the transcription of the target gene, largely through interaction with the C-terminal domain of the alpha subunit of RNA polymerase (13). Importantly, BvgS kinase activity can be switched off by growing the cells in the presence of millimolar concentration of magnesium sulphate or nicotinic acid, or by lowering the growth temperature to below 25°C (19). Known as the Bvg- mode, this state is represented by stabilization of the VFT-domain and the resulting conformational change diminishing kinase activity (22). 1.2.2 Gene expression profiles of BvgAS Broadly speaking, BvgAS coordinates two main gene expression profiles known as virulence-associated genes (vags) and virulence repressed genes (vrgs) (14). These two expression profiles are further categorized into 4 main gene expression classes which are linked to the timing after BvgS activation and the cellular concentrations of BvgA~P (6, 13). The Bvg- mode can be characterized by maximal expression of class 4 genes, which are predominantly vrgs, and the 5 minimal expression of vag loci (6). As cells are shifted into the Bvg+ mode through BvgS activation and phosphorylation of BvgA, the maximal expression of vag loci occurs while vrgs are repressed. Vags are subcategorized into class 1 and class 2 genes. Class 2 genes are otherwise known as early activation vags. Promoter regions of these genes are associated to high affinity BvgA~P binding motifs, which are preferentially bound and activated by low concentrations of BvgA~P (13). Late activation vags, class 1 genes, require high concentrations of BvgA~P due to lower affinity BvgA binding motifs (6). Lastly, class 3 genes, or Bvg intermediate (Bvgi) mode genes, are maximally expressed at the same time as class 2 genes due to high-affinity BvgA binding sites upstream of their promoters. However, lower affinity BvgA~P binding sites are located downstream of the promoter region; thus, as BvgA~P concentration increases and class 1 genes are activated, class 3 genes are repressed through BvgA~P blocking RNA polymerase (13). 1.2.3 Virulence factors associated to BvgA~P regulation The B. pertussis genome encodes many bona fide virulence factors of which many have been well characterized in Bordetella spp. or other Gram-negative pathogens. These genes include adhesins, toxins, and protein secretion systems functioning to manipulate host-immune responses and promote colonization and persistence. 1.2.3.1 Toxins B. pertussis produces several Bvg-regulated toxins during host infection, such as dermonecrotic toxin (DNT), adenylate cyclase toxin (CyaA), and pertussis toxin (PT). DNT is a 160 kDa protein that inactivates small Rho-family GTPases through transglutaminase activity (24). Even 6 though expressed, DNT is not required for colonization but has recently been identified as a factor leading to neurological complications during infection (25). PT in an exotoxin required for colonization at different points in the colonization of the respiratory tract, inflicting a broad range of effects on the host cell and immune responses (26, 27). It is a secreted AB5 hexameric exotoxin in which the B subunits bind host cells, leading to entry and release of the catalytic A subunit. The A subunit, encoded by ptxA, is an adenosine diphosphate (ADP)-ribosylating enzyme, which inactivates various G-protein coupled signaling pathways. Early in B. pertussis infections, PT facilitates colonization by inhibiting neutrophil influx into the host airways (27-29) and altering mononuclear phagocyte circulation. Where, later in infection PT reduces pro-inflammatory cytokine production in host lung cells and suppresses antibody responses, providing resistance to antibody-mediated clearance (30, 31). CyaA is a 177 kDa protein with an N-terminal adenylate cyclase domain and a C-terminal RTX (repeats-in-toxin) domain required for haemolysis and entry into host cells. Calmodulin present in host cells activates the cyclase domain resulting in the production and accumulation of cyclic adenosine monophosphate. The abundance of this second messenger inhibits neutrophil functions including phagocytosis and chemotaxis. Furthermore, CyaA modulates innate immune signaling through the induction of anti-inflammatory cytokine interleukin (IL)-10. This activity promotes B. pertussis colonization later in infection by diminishing neutrophil capabilities once immune infiltration into the respirator tract has occurred (32). Both CyaA and PT influence host metabolism by greatly increasing insulin secretion by stimulation of pancreatic Beta-cells (33). This results in low blood sugar and ultimately impaired immune cell function through this restricted access to glucose (34). Even though both toxins are not reported to disseminate from the site of infection, survival within macrophage is believed to 7 facilitate the systemic spread of B. pertussis to organs including the pancreas (33). Importantly, as Bordetella spp. are unable to use glucose as a carbon source, B. pertussis is largely unaffected by this shift in host metabolism (35). 1.2.3.2 The type III secretion system The movement of proteins across compartments of the cell is a necessity for bacteria to function. Alongside the maintenance of cell physiology, bacterial pathogens use specialized protein secretion systems to promote pathogenesis and modulate the host environment (36). The type III secretion system (T3SS) is a large complex of proteins commonly referred to as an injectosome, as its structure and function resemble a needle and syringe (37). The system is found in a wide range of Gram-negative pathogens in which effector proteins are delivered from the bacterial cell cytoplasm to host cell cytoplasm to interfere with a variety of host cell pathways (36, 38). In B. pertussis the T3SS locus involves more than 30 genes with homology to T3SS genes from related bacteria (39). In conjunction with BvgA~P dependent activation, the T3SS is regulated by several additional factors at the transcriptional and post-transcriptional levels (40, 41). Little is known about the role of the B. pertussis T3SS and secreted effector proteins due to inactivation of the system in lab passaged strains (42). However, it has been confirmed that, in vivo, the T3SS is required to promote B. pertussis colonization by suppressing pro-inflammatory cytokine production in host cells, early in mouse infection (43). Also, contact with host macrophages can activate transcription and assembly of the secretion apparatus in vitro (44). 8 1.2.3.3 Autotransporters Type 5 secretion systems, also known as autotransporters, are among the largest Gram-negative protein families and have been identified to be present in most known Gram-negative pathogens (45). These proteins encode their own secretion apparatus and have a conserved general organization consisting of an N-terminal “passenger” domain and a C-terminal “translocator” domain. The translocator domain forms a 12-stranded beta-barrel which inserts into the outer membrane aided by chaperones and the beta-barrel insertion machinery (46). This then allows the passenger domain, the functional part of the protein, to be secreted to the cell surface (36, 45). Autotransporters are found throughout Bordetella spp. and have a diverse range of functions and structures. Pertactin was amongst the first autotransporters characterized in B. pertussis and is reported to act as an adhesin (47, 48). Other Bvg-regulated autotransporters, such as BrkA TcfA, and Vag8, are involved in host cell adhesion and modulating innate immune system responses. Vag8 binds C1-inhibitor, a complement regulatory protein, inhibiting complement deposition on the bacterial cell surface promoting resistance to complement-mediated killing (49, 50). BrkA contributes to host cell adherence and prevents C4b deposition (51, 52) on the cell surface protecting from complement-mediated killing (52, 53). TcfA is also an important factor, promoting survival and colonization of B. pertussis in the upper respiratory tract (54). It is also proposed to be involved in complement evasion by binding complement factor H, yet this function is still to be confirmed (55). 1.2.4 Integrated signals from other two-component systems Alongside the BvgAS system, other two-component systems have been characterized in providing further control over the expression of both vags and vrgs in B. pertussis (56). The 9 PlrRS two-component system was recently shown to be required for maximal expression of vags while in the lower respiratory tract. Induced by increased concentrations of CO2, PlrRS drives persistence and survival within the host. PlrRS acts upstream of the BvgAS, however, the factor integrating the two systems is not known. While the PlrRS system is not required for infection, clearance of the bacteria occurs faster in PlrS mutants (57). Additionally, the underlying mechanism of vrg expression in Bordetella involves the input from another two-component system regulator (56). In the Bvg- mode, two proteins were shown to be involved in the expression of vrg loci: BvgR and RisA (58). BvgR is adjacent to and transcribed with BvgAS. BvgR encodes a regulatory protein with an EAL domain involved converting the signaling molecule cyclic di-guanosine-monophosphate (c-di-GMP) into GMP during the Bvg+ mode. RisA is an OmpR family RR able to bind c-di-GMP. This RR also requires phosphorylation to be activated (58). In B. pertussis phosphorylated RisA (RisA~P) is not dependent on its cognate sensor kinase RisS, but the kinase activity of RisK is required for activation. In the Bvg- mode, in the absence of BvgR, cellular levels of c-di-GMP increase, binding and activating RisA~P which can then upregulate vrg loci. RisA in this form is required for the expression of a majority of characterized vrgs in B. pertussis (19, 58). The Bvg- mode in other Bordetella spp. is an important physiological state required for ex vivo survival, however, its role in B. pertussis pathogenesis is not known (6). Even though the requirement of RisA within the host has not been explored, it has been established that the improper repression of vrg loci during infection has deleterious effects on colonization (6, 59). 10 1.3 Post-transcriptional gene regulation in bacterial pathogens As seen in many bacterial pathogens, transcriptional regulation of gene expression in B. pertussis is complex and multifaceted. Furthermore, over the last 30 years, there has been stark interest in the importance of post-transcriptional gene regulation in Gram-negative pathogens. Post-transcriptional regulation encompasses a vast range of mechanisms to determine the fate of the target messenger RNA (mRNA) transcript, providing an extra tier of regulation allowing for additional levels of transcriptional and translational control (60). This process emmeshes the roles of RNA secondary structure, RNA binding proteins, ribonucleases, and typically non-coding regulatory RNAs. 1.3.1 Features encoded in the 5’ untranslated region RNA transcripts are able to fold and form extensive secondary structure (61). Features encoded into the 5’ untranslated region (UTR) of a transcript, such as RNA thermometers or riboswitches, have evolved to utilize this property in establishing post-transcriptional control of translation (62). RNA folding is highly susceptible to a narrow range of temperatures, and as the name suggests, RNA thermometers are responsive to this quality. Elements in the 5’ UTR fold into a “closed” conformation at a low temperature, preventing access to the ribosome binding site (RBS) or the ATG start codon. This secondary structure is then melted at a higher temperature, entering a “open” conformation allowing for ribosome binding and translation to occur (63). This is seen in pathogens of several mammalian hosts (64). For example, PfrA is a transcriptional regulator which coordinates virulence factor expression in Listeria monocytogenes. A hairpin present in the 5’ UTR of pfrA prevents translation of the gene if the cells are below 37oC (65). Indeed, the inverse is also observed in cspA, a cold shock gene in E. coli, which is more 11 efficiently translated below 20°C, regulated by an RNA thermometer that is in an open conformation at lower temperatures (66). Riboswitches are another type of regulatory element present in the 5’ UTR of some mRNA transcripts. These are characterized by a ligand-binding aptamer, and an expression platform with variable secondary structure elements (62, 67). Binding of the ligand typically leads to the target gene being switched off, as this leads to changes in RNA secondary structure of the expression platform, obscuring the RBSs or introducing a premature terminator (63). Riboswitches are primarily involved in binding metabolites resulting in rapid modulation of gene expression in the relevant metabolic pathway (68). Though not well described for virulence factors, c-di-GMP binding riboswitches have been identified suggesting a potential role in host-pathogen interactions (64). 1.3.2 Cis- and trans-encoded regulatory RNAs Regulatory RNAs have been recognized as key factors in coordinating rapid changes in gene expression. These are represented by typically non-coding transcripts derived from intergenic regions between open reading frames (ORFs) (69). Regulatory RNAs are enriched under stress conditions and play a critical role in fine-tuning gene expression in various systems across the bacterial cell (70, 71). Due to the many, and frequently hostile, environments encountered by pathogens within the host, regulatory RNAs have been identified as an important factor in regulating the expression of virulence factors (64). Broadly speaking, regulatory RNAs are categorized into two groups. 12 The first is described as RNAs that are transcribed from the opposite strand within the same genetic locus as the target mRNA. These are known as cis-encoded antisense RNAs (asRNAs) and their size can range from less than 100 bp to several thousand bases (72). As asRNAs are complementary to the sense transcript, these regulatory RNAs influence the stability of the target transcript through complementary base pairing and RNA duplex formation. The expression of the antisense transcript typically negatively correlates with the stability of the sense RNA. This often involves cleavage by RNase III, a ribonuclease with specific activity for double-stranded RNA, and subsequent degradation of both transcripts. However, asRNA-mRNA interactions can also increase transcript half-life by obscuring cleavage sites of other nucleases (73, 74). In E. coli the gadY asRNA promotes RNase III mediated cleavage of the gadXW sense transcript; this activity is associated with increased stability of the resultant gadX and gadW mRNA cleavage products (73, 75). The excludon is a new paradigm of asRNA function. Within this, a long asRNA several thousand bp in length is involved in the simultaneous transcription and translation of an operon, while also repressing the activity of a functionally related gene or operon on the opposite strand (73). This type of regulation has been described in Listeria and Clostridium spp. and is likely a feature of regulation in many bacteria (76). The other class of regulatory RNAs are encoded at a genetic locus different from its target. Known as trans-encoded small RNAs (sRNAs), these transcripts range from 50-500 bp in size and interact with sequences within the 5’ UTR (63). These are derived from promoters found in intergenic regions or cleaved from the extended 3’ UTR of other mRNAs (70, 77). As these transcripts are not perfectly complementary to the target mRNA, sRNAs require interactions to be catalyzed by an RNA chaperone (78). Hfq is an abundant RNA chaperone present in many 13 bacterial species. It forms a donut-like homohexamer, with each monomer able to bind AU-rich sequences. The “proximal face” of Hfq binds U-rich sequences and sRNAs, while the “distal face” binds A-rich sequences and mRNAs (79, 80). This structure provides a means of promoting interactions between sRNA and mRNA. Similar to asRNAs, sRNA interactions invariably alter the stability and properties of the target mRNA, commonly involving cleavage by the ribonuclease RNase E. Hfq-RNA complexes are able to interact with the C-terminal domain of RNase E, which promotes cleavage of the mRNA transcript (81). This event often leads to degradation, however, this interaction can also induce RNA secondary structure changes, modulating access to RBSs (82). 1.3.3 Post-transcriptional regulation by ribonucleases RNAs in bacteria are considered unstable, when compared to eukaryotes, with average transcript half-lives being only a few minutes (60). This instability is an inherent tool, facilitating rapid switches of gene expression profiles in responses to environmental stimuli through mRNA decay. Much of this RNA decay is dependent on the action of several bacterial ribonucleases (60). Even though largely non-specific in vitro, these ribonucleases are highly responsive and coordinated by RNA secondary structure and sequence specificity in vivo (83). Ribonucleases are broadly classed as either endo- or -exonucleases. Endonucleases cleave internal phosphodiester linkages of RNA transcripts. This is often required to initiate subsequent processing by exonucleases, which cleave from the 5’ or 3’ termini of the transcript in a processive manner to generate mononucleotides (82). However, these cleavage events do not always result in the degradation of the target transcript. In the case of ribosomal RNAs (rRNAs) and transfer RNA (tRNAs) processing by nuclease is required for maturation of the RNA species 14 for it to become functional. Importantly, the endonucleases RNase III and RNase E play a dominant role in molding the post-transcriptional landscape in many bacteria. This endonuclease activity is required for general RNA turnover and metabolism, maturation of sRNAs from UTRs of mRNAs, and the processing of regulatory RNAs bound to their targets (Figure 1.1) (84). In this context, RNase III and RNase E activity is an important feature of cell homeostasis as well as pathogenesis through post-transcriptional control of virulence factor transcript stability in bacterial pathogens (85, 86). 1.3.3.1 RNase III RNase III is a global post-transcriptional regulator which has specific activity for double stranded RNA (dsRNA). Predominantly studied in E. coli, RNase III is broadly found amongst bacterial species, yet the nuclease domain is also conserved in higher organisms in which DICER is a homologue (87). Encoded by the rnc gene, RNase III acts as a dimer, where each monomer is a 25 kDa protein with an N-terminal Mg2+ dependent nuclease domain and a C-terminal dsRNA binding domain (87, 88). Within the bacterial cell, the primary role of RNase III is in the maturation of rRNAs and tRNAs and it is also involved in processing gene transcripts associated with metabolic and cell homeostasis roles. Furthermore, RNase III is involved in the regulation of several toxin-antitoxin loci implicated in persister cell formation, and in clustered regularly interspersed short palindromic repeat (CRISPR)-Cas systems involved in bacteriophage immunity (87). Importantly, due to specificity for double stranded RNA, RNase III activity is required for post-transcriptional regulation of many coding genes. RNA duplexes formed from cis-encoded asRNAs to its cognate mRNA typically lead to rapid cleavage by RNase III (89). Also, even though not as common, RNA-RNA interactions chaperoned by Hfq are sometimes 15 preferentially cleaved by RNase III to alter the stability of the target mRNA (87, 90). In a pathogenic context, RNase III has been shown to be a major regulator of RNAIII, an antisense RNA in Staphylococcus aureus (64, 86). The 3’end of this transcript is involved in binding the mRNAs of several key virulence factors. These duplexes are then targeted for cleavage by RNase III and subsequent degradation (91, 92). RNase III is also able to cleave a hairpin structure within its 5’UTR to control its own expression. This autoregulation is used to diminish RNase III mRNA until rRNA abundance within a cell reaches a certain threshold (93). This processing of 5’ UTRs is another common regulatory mechanism of RNase III. Hairpin structures can obscure or expose RBSs where RNase III processivity is required to modulate translation. As ribosome binding can increase transcript stability by protecting transcripts from RNase E decay, translational activation by RNase III only occurs in genes with strong translation initiation sites (87). 16 1.3.3.2 RNase E RNase E is an essential endoribonuclease that sits at the center of RNA metabolism within Gram-negative bacteria. It is also involved in the maturation of rRNAs and tRNAs with this activity occurring downstream of RNase III and other nuclease pathways (94). RNase E forms a large membrane-bound tetramer, which is built from a dimer of dimers. Each monomer can be divided into an N-terminal nuclease domain and a largely unstructured C-terminal domain. The nuclease domain is dependent on Mg2+ or Mn2+ cations and preferentially cleaves monophosphorylated, single-stranded RNA. The C-terminal domain, even though unstructured, contains several regions involved in interactions with the inner membrane, other proteins, and nucleic acids (94). RNase E acts as the functional scaffold of a large complex called the degradosome. Through binding sites located in the C-terminal domain, the degradosome complex includes interactions with RhlB (A DEAD-box RNA helicase), enolase (a glycolytic enzyme), and PNPase (an exonuclease) (95). This complex is able to efficiently process and degrade cellular RNA through the presence of RNA unwinding, endo- and exo-nuclease activity. Importantly, the C-terminal domain of RNase E also interacts with the RNA chaperone Hfq, indicating a substantial role of this nuclease in sRNA processing (84). There are two models proposed for sRNA regulation involving RNase E. The first suggests that Hfq bound to the stabilized sRNA-mRNA duplex competes for access to RNA binding sites in the C-terminal domain of RNase E. The second model suggests that preference for the monophosphorylated sRNA, or changes to mRNA secondary structures make the sRNA-mRNAs substrate more susceptible to RNase E degradation (94). As RNase E is critical to general RNA turnover within the bacterial cell, the activity of this nuclease has been identified in the regulation and stability of several key virulence factors in 17 Gram-negative pathogens (85, 86). In Yersinia pseudotuberculosis, RNase E is required for the full expression of the T3SS and C-terminally truncated RNase E clones showed decreased infectivity of macrophages (96). Alongside classical virulence factors, RNase E in E. coli and Salmonella is involved in the processing of several RNAs involved in the decoration of the Gram-negative outer membrane (97). This role in RNA turnover provides a core mechanism by which RNA profiles can be rapidly adapted during several stress states. 18 Figure 1.1 General overview of regulatory RNA processing by endonucleases in Gram-negative bacteria. Antisense RNAs (asRNAs) are perfectly complementary to the target mRNA as they are encoded from the opposite strand in the same genetic locus. RNA-RNA duplexes formed from complementary asRNA-mRNA binding are readily cleaved by RNase III, an endonuclease with specific activity for double-stranded RNA. Small RNAs (sRNAs) are encoded at a different genetic locus from their target mRNA and therefore require an RNA chaperone, such as Hfq, to catalyze RNA-RNA interactions. The sRNA-mRNA-chaperone complex is then able to interact with the C-terminal domain of endonuclease RNase E. The N-terminal nuclease domain of RNase E is then involved in cleaving the target mRNA. With both RNase III and RNase E, cleavage of the target transcript does not always mean degradation. In the case of rRNAs and tRNAs, cleavage is associated with the maturation of the RNA, allowing it to become functional. Cleavage events are also associated with stabilizing the target transcript and changing RNA secondary structure, a common feature of modulating translation. Both RNase III and RNase E have also been implicated in the production of sRNAs through processing of the 3’ UTR of some mRNAs. 19 1.3.3.3 Other ribonucleases PNPase is an adenosine triphosphate (ATP)-dependent, 3’ to 5’ exonuclease, and a major component in RNA degradation pathways in bacteria. Often found in complex with RNase E, endonuclease cleavage is promptly followed by exonuclease degradation by PNPase (82). Likely due to this close association to RNase E, this exonuclease is also considered an important post-transcriptional regulator of many bacterial systems including virulence factor production. However, PNPase has also been shown to directly interact with Hfq and associated sRNA-mRNA duplexes (98). RNase R is the other major exonuclease involved in the 3’ to 5’ degradation of cellular RNAs (99). It is a multidomain protein that is particularly adept at degrading structured RNA, such as rRNAs and tRNAs (100). The protein is seemingly unstable during exponential growth yet is stabilized by post-translational modification during stationary phase or when the bacterial cell is under stress (101). Even though associated with various stress responses, RNase R has a convoluted role across bacterial pathogens. Deletion of RNase R is associated with both virulence defects as well as having no impact on virulence depending on the organism (86). RNase G is an endonuclease involved in the processing of the 5’ end of 16s RNA. The nuclease itself has substantial homology to the N-terminal domain of RNase E yet lacks any C-terminal scaffolding for degradosome assembly (86). Similarly, RNase G is involved in the stability of many mRNA transcripts (102). There is an overlapping cleavage specificity between the two enzymes, yet the motifs are not identical. In addition, overexpression of RNase G can rescue RNase E temperature-sensitive mutants when grown at inactivating temperatures (37°C - 42°C) 20 (103). RNase G is not essential but is associated with stress responses and antibiotic resistance in some bacterial pathogens (104). 1.3.4 Post-transcriptional regulation in B. pertussis There is limited understanding of B. pertussis post-transcriptional regulatory networks. Several putative sRNAs have been identified, some of which are regulated by BvgAS, yet no targets for the sRNAs have been shown (105). Investigations of the primary transcriptome identified that a majority of antisense transcripts present are derived from insertion elements which make up ~ 10% of the B. pertussis genome (106). From this work, a BvgA~P dependent sRNA involved in the regulation of glutamate uptake was also identified (107). Hfq has a substantial impact on gene expression in B. pertussis (108). Strains lacking Hfq were shown to have defects in virulence and growth compared to wild type. These Δhfq mutants also demonstrated down regulation in surface proteins and certain virulence factors such vag8, brkA, and the T3SS (44). B. pertussis contains many of the same ribonuclease present in E. coli (Table 1.1), however little is known about the roles these play in regulating the post-transcriptional landscape of the organism. 21 Table 1.1 Ribonucleases present in both E. coli and B. pertussis. The table is adapted from Table 1 in Mackie (2013) (94) which identified ribonucleases and their substrates in E. coli. Orthologs of ribonucleases in B. pertussis Tohama I were identified through protein homology searches of each E. coli enzyme using the Basic Local Alignment Search Tool (NIH). Enzyme in E. coli Substrates Presence in B. pertussis Endoribonucleases Gene Essential mRNA rRNA tRNA sRNA locus tag RNase E rne Yes ++ ++ ++ + BP0475 RNase G rng No + ++ ? ? BP2317 RNase HI rnhA No + - - ? BP1433 RNase HII rnhB No ? - - ? BP3211 RNase P rnpA, rnpB Yes + - ++ ? BP0493 RNase BN rbn No ? - + ? NA RNase I rna No - - - - NA RNase III rnc No + ++ + ? BP2431 Exoribonucleases RNase D rnd No ? - + ? NA RNase PH rph No ? - ++ ? BP1588 RNase R rnr Synthetic lethality + ? ? + BP3526 RNase T rnt No ? ++ ++ ? BP2022, low homology Oligo-RNase orn Yes ++ - - - BP2687 PNPase pnp Synthetic lethality ++ ? + + BP0795 RNase II rnb Synthetic lethality ++ - + ? NA Key; ++, the enzyme activity is required for the processing of the majority of that class of RNA; +, enzyme activity has been shown to be required for processing of some RNA species in that class; - no current evidence of enzyme involved in processing RNA class; ?, no definitive evidence at this point for cleavage of the given RNA class; NA, (not applicable) no homologue found in B. pertussis genome. 22 1.4 Structure and function of the Gram-negative outer membrane Gram-negative pathogens use a wide range of mechanisms to adapt to the host environment. Alongside virulence factors, such as toxins and adhesins, the bacterial membranes play a critical role in surviving the host environment and modulating immune responses (109). Gram-negative bacteria possess two distinct membranes, the inner membrane and outer membrane imbedded with integral membrane and lipoproteins. The inner membrane consists of a symmetrical lipid bilayer made of phospholipids. A cell wall composed of peptidoglycan is present in the periplasm to which the outer membrane is attached through peptidoglycan-associated lipoproteins (110). The outer membrane is an asymmetrical lipid bilayer. The periplasmic side is formed of phospholipids while the outer leaflet is made of lipopolysaccharide (LPS; Fig. 1.2A) (111). LPS, also known as endotoxin, is made up of 3 domains: the O antigen, the core sugars, and lipid A (Fig. 1.2B). LPS is an important cell structure in mediating host-pathogen interactions and maintaining membrane integrity during infection (112). 1.4.1 LPS structure 1.4.1.1 O-antigen The O-antigen is the polysaccharide portion and the most variable component of LPS. It is formed of a branched or linear chain of repeating sugar units and is covalently linked to the core sugars. The variability is due to the number of repeated units and pattern composition can vary across bacterial species and between strains of the same species. Bacterial strains and species can be referred to as either smooth or rough depending on the presence or absence of the O-antigen respectively. In rough bacteria, the O-antigen is often replaced with an extended series of core sugars (113). 23 1.4.1.2 Core sugars The core sugars are formed of two sections being the inner and outer cores. The outer core sugars tend to be highly variable between bacterial species, likely due to environmental pressures. The inner core commonly consists of ketodeoxyoctonic acid (Kdo) and L-glycero-D-manno-heptose (L-D-Hep) sugars (113). 1.4.1.3 Lipid A The lipid A domain is composed of hydrophobic acyl chains anchoring LPS to the outer membrane and hydrophilic head group to which the core sugars are bound. Lipid A structures vary amongst different Gram-negative bacteria but typically consist of a di-glucosamine (GlcN) backbone with four to seven acyl chains attached. Acyl chains vary in length between bacterial species and are attached to the di-GlcN sugars at C2, C3, C2’, and C3’ carbons. The Kdo-core sugar is ligated at the C6’ linkage. Additionally, the backbone typically has phosphate groups at C1 and C4’, and these can be removed or further modified during LPS biogenesis (Fig1.2C,(113, 114)). 24 25 Figure 1.2 Schematic of Gram-negative membrane and General LPS structure. A) Gram-negative bacteria have two distinct membranes. The inner membrane (IM) is a symmetrical phospholipid bilayer imbedded with integral membrane and lipoproteins. The periplasm (PP) separates inner and outer membranes (OM) and contains a thin layer of peptidoglycan (PG) required for cell shape. Lipoproteins covalently bonded to PG are involved in anchoring the OM to the cell wall. The OM itself is an asymmetrical membrane with phospholipids predominantly making up the inner leaflet (periplasm side) and lipopolysaccharide (LPS) being the main constituent of the outer leaflet (cell surface side). B) Schematic of LPS structure. LPS consists of three domains: lipid A (phosphate groups shown as red dots, inner and outer core sugars (represented as green hexagons), and the O-antigen. C) Structure of E. coli lipid A showing a di-glucosamine (GlcN) backbone decorated with 6 acyl chains and phosphate groups. Primary acyl chains are attached to the di-GlcN backbone and secondary chains are attached to a primary acyl chain. Acyl chain length is shown by the number directly below each chain and the number system for di-GlcN carbons is shown in red. (Figure based on reviews by Needham and Trent (2013) (112) and Raetz and Whitfield (2002) (113). 26 1.4.1.4 Biosynthesis and export All components of LPS are initially synthesized on the cytoplasmic side of the inner membrane, before being flipped to the periplasmic side and subsequent transport to the outer membrane. Lipid A biosynthesis occurs via the Raetz pathway (reviewed in (115)), which consists of a step-wise series of reactions catalyzed by Lpx proteins. Within E. coli the pathway consists of LpxABCDHKLM and WaaA. LpxA, LpxC and LpxD are soluble, cytoplasmic proteins and LpxH, LpxB, LpxK, WaaA, LpxL and LpxM are integrated within the inner membrane. These genes and their orthologs are typically essential for bacterial growth, however, lpxM is usually non-essential. The Lpx proteins are highly conserved amongst Gram-negative bacteria, yet additional modifying enzymes in some species can provide alternate lipid A structures (112). Briefly, the Raetz lipid A biosynthesis pathway is as follows: The pathway is initiated by LpxA transferring a β-hydroxymyristoyl chain (C14-OH) to the C3 position of uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) (116). Orthologs of LpxA are able to incorporate acyl chains of different lengths due to the active site acting as an accurate hydrocarbon ruler (117). The acylated UDP-GlcNAc (UDP-3-O-(acyl)-GlcNAc) is then deacylated by LpxC committing the molecule to the pathway. This is followed by the addition of a second C14-OH chain by lpxD (UDP-2,3-diacyl-GlcN), and UDP is cleaved by LpxH, to form 2-3-diacyl-GlcN-1-phosphate (lipid X). LpxB then catalyzes a β-1’-6 linkage between lipid X and UDP-2,3-diacyl-GlcN (115). A phosphate group is added to the 4’ position by LpxK forming Lipid IV A (118), an antagonist of the human innate immune system TLR4/MD-2 complex (explored further in section 1.4.2.2). The addition of Kdo sugars at the 6’ position is then catalyzed by WaaA (formerly KdtA). Secondary laurate and myristate chains are then added by LpxM and LpxL respectively to 27 produce the final hexa-acylated Kdo2-lipid A structure. Bacteria lacking either LpxL or LpxM will generate penta-acyl lipid A if other acyl chain modification enzymes are also absent (115). Additional core sugars are then added to the Kdo2-Lipid A on the cytoplasmic side of the inner membrane (113). MsbA, an ATP-binding cassette (ABC) transporter is responsible for flipping the lipid A-core across the inner membrane to the periplasmic face (119). If present, the O-antigen is ligated to the outer core sugars at this point (113), otherwise, the lipid A-core is transported to the outer membrane via the Lpt system. The Lpt machinery consists of 7 essential proteins, LptA-G. These proteins form two sub-complexes LptB2CFG embedded in the inner membrane with LptDE found in the outer membrane. The periplasmic protein LptA connects the two complexes (120). The current model for lipid A transfer from the inner membrane is as follows: once MsbA flips lipid A to the periplasmic side of the inner membrane, the LptB2FG complex extracts the lipid A and transfers it to LptC which is stably associated with the complex (121). LptC consists of a hydrophobic pocket able to bind LPS and transfer the molecule to LptA. LptA forms oligomers in the presence of LPS, and conformational changes form hydrophobic pockets allowing for binding and transport of LPS across the periplasm (122). LPS is then delivered from LptA to the LptDE complex in the outer membrane. This complex forms a plug-and-barrel assembly in which the lipoprotein LptE is inserted into the C-terminal β-barrel of LptD. LptE directs interactions with LPS, coordinating insertion into the lumen of LptD, which then incorporates LPS directly into the outer leaflet of the outer membrane through an opening in the β-barrel wall (120). 28 1.4.2 The biological function of LPS 1.4.2.1 Membrane integrity LPS forms a structured barrier where it primarily protects the bacterial cell from its environment. The tight packing of acyl chains present on the di-GlcN backbone promotes membrane integrity, and this is further stabilized by negative charges from phosphate groups being bridged by divalent cations (123). Cationic antimicrobial peptides (CAMPs), present in the host and environment, are able to displace cations and interact with negatively charged phosphates to gain entry into the bacterial cell (124). In the absence of divalent cations or the presence of CAMPS, LPS is often modified to mask negative charges to better maintain membrane stability and promote resistance to CAMPs (123). 1.4.2.2 Host-pathogen interactions and Toll-Like Receptor 4 Within the host environment, LPS is a major component involved in host-pathogen interactions. Both the polysaccharide and lipid domains are involved in this dynamic interplay but play several different roles. The highly antigenic O-antigen is involved in protecting the cell from antibiotics, promoting complement evasion, and can also stimulate host cell adhesion in some bacterial species (112, 114). Lipid A is detected by the Toll-Like Receptor 4 (TLR4)/ MD-2 complex, a pattern recognition receptor found in the mammalian innate immune system (125, 126). This receptor is present on many host cell types involved in controlling inflammation reactions such as monocytes, lymphocytes, and endothelial cells (113). LPS is first extracted by LPS binding protein (LBP) before it is transferred to CD14 which presents the molecule to the TLR4/MD-2 complex. Lipid A binding to TLR4/MD-2 leads to dimerization, activation of cytoplasmic TIR domains of 29 TLR4, and initiating downstream signaling cascades. The acyl chains of lipid A sit in a hydrophobic pocket of MD-2, with the di-GlcN backbone atop the protein (Fig. 1.3). The phosphate groups are then involved in interactions with both TLR4 and the incoming, dimerizing TLR4* (125, 127). Signaling cascades stimulated by TLR4 activation are classed by recruitment of myeloid differentiation primary response gene 88 (MyD88). In the MyD88-dependent response, dimerization at the cell surface recruits TIRAP and MyD88 adaptor proteins activating NFκB leading to the production of pro-inflammatory cytokines (125). In the MyD88-independent response, dimerized LPS-TLR4/MD-2 complexes are internalized, resulting in the recruitment of TRIF and TRAM adaptor proteins. This activates IRF3, leading to the production of type 1 interferon and delayed activation of NFκB (128). MyD88-independent activation results in less severe inflammation yet is still required to provide an appropriate immune response to pathogens (113). The unmodified lipid A of E. coli (Fig. 1.2C) is one of the most potent TLR4 agonists and leads to activations of both pathways (125). However, modifications to either the number or position of acyl chains or accessibility of phosphate groups can alter TLR4 activation through skewing preferences of adaptor protein recruitment (123). Indeed, lipid A modification systems are a common mechanism used by Gram-negative pathogens, modulating immune responses through changing the affinity of LPS binding to TLR4 (112). 30 Figure 1.3 Schematic of dimerization of TLR4-MD2 upon lipid A binding Lipid A is first extracted by LPS binding protein (LBP), transferred to CD14, before being presented to MD-2 and TLR4. Once bound to lipid A, dimerization of the TLR4/MD-2 complex occurs and downstream signaling cascades lead to the production of proinflammatory cytokines. Interactions between TLR4 and TLR4* (incoming receptor) are mediated by acyl chain accessibility within the MD-2 binding pocket and phosphate groups present on the di-GlcN backbone. These sites on lipid A are often modified to modulate immune system activation. 31 1.4.2.3 Function and regulation of lipid A modifications Modifications to LPS structures on the bacterial cell surface enable bacteria to adjust outer membrane properties to be better adapted to the surrounding environment (123). As the host environment is composed of many, variable compartments, LPS modification systems play an important role in colonization for Gram-negative pathogens. Each domain of LPS can be modified, yet modifications that influence pathogenesis are often found affecting lipid A (112). Modifications to lipid A can take the form of the addition of moieties to the phosphate groups of the di-glucosamine backbone or changing the number and/ or position of acyl chains. The proteins involved either modify lipid A on the periplasmic side of the inner membrane before transport or at the outer membrane (123). These modifications systems do not modify the whole population of lipid A, leading to heterogeneity of cell surface properties (114). Furthermore, lipid A modification systems are closely linked to other regulatory networks involved in pathogenesis. This provides a means of promoting survival and colonization through the coupled expression of virulence factors and remodeling of the outer membrane to efficiently modulate host-pathogen interactions (112). 1.4.2.3.1 Transcriptional regulation of lipid A modifications At the transcriptional level, two-component systems play a substantial role in the control of outer membrane properties. In Bordetella bronchiseptica, PagP is involved in acylating the C3’ position of lipid A with a C16-OH acyl chain. This activity is controlled by the master regulator of virulence factor production in Bordetella spp., BvgAS, where this modification promotes persistence within the mouse respiratory tract yet doesn’t provide increased protection to CAMPs (129). 32 The PhoPQ two-component system is found in a wide range of bacterial species, including E. coli, Pseudomonas aeruginosa, and Salmonella enterica subsp. enterica serovar typhimurium (130). Phosphorylation activity of PhoQ is responsive to pH, depletion of divalent cations, and the presence of certain antimicrobial peptides. Phosphorylated PhoP acts on a wide range of genes, including those involved in virulence, magnesium uptake, and outer membrane modification systems (131). In S. typhimurium, PhoP activates the transcription of PagP and PagL, enzymes involved in acylating and deacylating lipid A respectively. PagP adds a secondary C16-OH acyl chain to the C2 position, while PagL removes the primary acyl chain at the C3 position (132). Together, the activities of PagP increase resistance to CAMPs through tighter packaging of the outer leaflet, while PagL activity results in reduced immune activation through altering the patterns of interactions between the lipid A acyl chains and the TLR4/MD-2 complex (112, 132). Alongside PhoPQ, PmrAB is another two-component system largely involved in LPS modifications in many Gram-negative pathogens. In Salmonella spp., PhoPQ directly activates PmrAB, yet in E. coli and Pseudomonas spp. the system is activated by Fe3+ ions and low pH (112). PmrA then upregulates genes such as eptA and arnT whose gene products are responsible for the transfer of phosphoethanolamine and 4-amino-4-deoxy-L-arabinose (Ara4N) respectively to one or both phosphate groups on the di-GlcN backbone of lipid A (123, 132). As the phosphate groups interact with key amino acids to promote TLR4 dimerization, modification of these groups provides immune-modulatory properties while also changing the net charge of the outer membrane (125). 33 1.4.2.3.2 Post-transcriptional regulation of lipid A modifications The genes involved in many of the lipid A remodeling systems are controlled by overlapping transcriptional and post-transcriptional regulatory networks. This provides a means of fine-tuning the expression of these genes while also allowing for rapid changes to transcription and translation of the modification machinery (97). Several sRNAs have been identified in the direct or indirect control of lipid A and other LPS modification systems. MicF was one of the first chromosomally encoded sRNAs found within the E. coli genome, shown to inhibit translation of the outer membrane porin OmpF (65, 133). The expression of this 90 nt sRNA has been shown to be tightly regulated by several transcription factors in various stress responses. MicF has also been shown to target the transcript of the lipid A modifying enzyme lpxR (134). This deacetylase removes the secondary acyl chain from the C3’ position in many bacterial pathogens (123). In S. typhimurium and E. coli, lpxR is transcriptionally activated by the virulence regulators HilD and Ler respectively, and deacylation activity is associated with reduced TLR4 activation (135). MicF is a negative regulator of lpxR, where it acts both to inhibit translation by sequestering the ribosome binding site, and also induces RNase E-dependent degradation of the lpxR mRNA (134). MicA is a 70 nt sRNA transcribed by the membrane stress-responsive sigma factor, sigma E (σE) (136). MicA negatively regulates phoP translation, thus impacting the downstream production of several LPS modifying enzymes (137). This negative regulation is dependent on the nuclease activity of RNase III, cleaving an Hfq dependent RNA duplex formed between MicA sRNA and phoP mRNA (90). Importantly, phosphorylation and activation of PhoP initiates the transcription 34 of another sRNA, MgrR. This 98 nt sRNA has been shown to bind several targets including the phosphoethanolamine transferase eptB (138). This lipid A modifying enzyme is transcribed by σE and targets the Kdo2 of the lipid A structure providing moderate resistance to antimicrobial peptides (123). Thus, σE promotes eptB expression through suppressing phoP translation, where in turn PhoP promotes other lipid A modifications while simultaneously suppressing eptB expression(139). Complex regulatory circuits such as these allow for substantial fine-tuning of outer membrane properties within the host environment. This also takes advantage of the heterogeneity of the outer leaflet, in which different populations of modified LPS present on the cell surface provide varying effects in the modulation of the immune system 1.4.3 B. pertussis LPS structure and modification As with other Gram-negative pathogens, LPS is an important feature of B. pertussis pathogenesis. It consists of a penta-acylated di-GlcN backbone, with Kdo2 core sugars, and a distal trisaccharide (Fig. 1.3) (140). As no O-antigen is present, B. pertussis LPS is often referred to as lipo-oligosaccharide (LOS) (141). In the absence of O-antigen, the trisaccharide portion of LOS provides an important physical barrier, protecting against the LPS disrupting surfactant protein A in the host respiratory tract (142). The structure of B. pertussis lipid A consists of three primary C14-OH acyl chains and secondary acyl chains of C10-OH and C14 (140). The phosphate groups on the glucosamine backbone are modified with the addition of glucosamine moieties at either one or both phosphates (143). Thus, the cell surface can consist of unmodified lipid A, a single GlcN sugar at either 1 or 4’ positions or, double modified lipid A (Fig. 1.4). While not as potent as E. coli LPS, B. pertussis LOS is a modest TLR4 agonist with this reduced level of activation resulting in delayed immune responses and clearing. However, B. pertussis 35 LOS is sufficient at inducing the production of proinflammatory cytokines, such as IL-23 and IL-6 which further induce IL-17 production in T-cells (33, 144). 36 A) Unmodified B. pertussis lipid A compared to B) modified lipid A. Both structures show penta-acylated di-GlcN backbones with acyl chain lengths depicted below each chain. Phosphates present on carbons 1 and 4’ are the site of modification where additional GlcN sugars are introduced (143). Figure 1.4 Structures of lipid A from B. pertussis Tohama-1 strain BP338 37 1.4.4 Lipid A glucosamine modification locus The modification of B. pertussis lipid A is under the control of genes found in the lipid A glucosamine modification (lgm) locus (117). Originally annotated in the B. pertussis genome as locus tags BP0399-BP0396, this operon encodes four genes arranged in two overlapping pairs, lgmAB and lgmCD, separated by 188 bp. A recently identified fifth ORF, lgmE, is located within the intergenic region of lgmB and lgmC in the opposite orientation to the other lgm locus genes (Figure 1.5A) (145). LgmA is predicted to encode a GT2 family glycosyltransferase (146), with a cytoplasmic catalytic domain and C-terminal transmembrane domain. LgmA is a homologue of ArnC, a glycosyltransferase involved in Ara4N-modification in Salmonella and E. coli. Structural predictions of LgmA (Fig. 1.5B) are based on the 3D structure of GtrB from Synechocystis sp. PCC6803 which transfers a glucose sugar from uridine di-phosphate (UDP)-Glc to the lipid carrier undecaprenylphosphate (C55P) (147). LgmB is a homologue of ArnT which catalyzes the transfer of Ara4N to lipid A in Salmonella and E. coli. In B. pertussis, lgmB is predicted to encode a GT83 family glycosyltransferase (146), Structural predictions of LgmB are based on ArnT from Cupriavidus metallidurans (148) and show 11 transmembrane helices and a periplasmic soluble domain. Modelling of the LgmB structure also indicates that the protein is lacking two transmembrane helices compared to other ArnT homologs (Fig. 1.5C). 38 LgmC is a YdjC-like protein with proposed deacetylase activity. It is expected to be a soluble, cytoplasmic protein with a conserved N-terminal domain with proposed catalytic activity (145). Structural predictions of LgmC (Fig. 1.5D) are based on TTHB029 from Thermus thermophilus HB8. TTHB029 is a deacetylase closely related to PgdA, a GlcNAc deacetylase for Streptococcus pneumoniae involved in peptidoglycan biosynthesis (149). Overlapping with the stop codon of lgmC is the start codon of lgmD. This ORF encodes a small protein composed of predominantly four transmembrane helices. Originally it was postulated to be part of a flippase, with a similar function to ArnE and ArnF. However, a bacterial transmembrane pair family protein with related function to chlorohexidine efflux transporters was recently identified as an LgmD ortholog (150). Finally, LgmE is also predicted to encode a small protein consisting of four transmembrane helices. It is structurally related to GtrA from Shigella flexneri bacteriophage X, which is involved in LPS remodeling where it is proposed to function as a bactoprenol-linked translocase (151). Even though found in a broad range of bacterial species, little is known about the function of GtrA-like proteins. However, they are commonly found closely associated with the structure and function of other glycosyltransferases (152). Sequential deletion of lgm locus genes identified that LgmABC are the minimum machinery required for the transfer of glucosamine to B. pertussis lipid A, where LgmD is not essential for the modification (117). Due to the overlap with both lgmB and lgmC, lgmE was previously mutated to encode a premature stop codon at a codon which also produced a silent mutation in 39 lgmC. This lgmE mutant strain phenocopied the wildtype strain and it was proposed that lgmE is not required for lipid A glucosamine modification (145). Taking these features into account, the current model for lipid A modifications in B. pertussis proposes that: First, LgmA transfers GlcNAc from UDP-GlcNAc to undecaprenyl phosphate (C55P). This is then deacetylated by LgmC to form C55P-GlcN. A yet to be identified flippase is involved in transferring the sugar-linked lipid carrier from the cytoplasmic side of the inner membrane to the periplasmic side. From here, LgmB then catalyzes the transfer of the glucosamine from C55P to an available phosphate group on lipid A (Fig. 1.5E) (143). 38 39 Figure 1.5 Genetic architecture of the lgm locus and predicted structures and function of gene products. The lgm locus consists of five gene operons arranged into 3 overlapping units. The stop codon of lgmA (1059 bp) overlaps with the start codon of lgmB (1620 bp), similar to lgmC (1104 bp) and lgmD (456 bp). These overlapping pairs are separated by a 188 bp intergenic region located between lgmB and lgmC. In this intergenic region, on the opposite strand, the lgmE open reading frame (393 bp) overlaps with the 5’ end of lgmC (168 bp overlap) and the 3’ end of lgmB (42 bp overlap). B) Predicted structure of LgmA based on the crystal structure of GtrB (147) with predicted catalytic residues shown as blue sticks. C) Predicted structure of LgmB based on the crystal structure of ArnT (148). Conserved predicted catalytic residues for glycosyltransferase function as described for ArnT (153) are shown as blue sticks. Location of absent alpha-helices compared to orthologs are shown as red sticks. D) predicted structure of LgmC based on the crystal structure of TTHB029 (149). Predicted catalytic resides are shown as blue sticks. E) Hypothetical model of lgm locus involvement in the modification of B. pertussis lipid A. Here LgmA catalyzes the transfer of N-acetyl glucosamine (GlcNAc) from uridine-diphosphate to the lipid carrier undecaprenyl phosphate (C55P) to form C55P-GlcNAc. This is then deacetylated by LgmC to produce C55P-GlcN. A yet to be identified flippase then transfers C55P-GlcN from the cytoplasmic side of the inner membrane to the periplasmic side. Here LgmB then transfers the glucosamine sugar to lipid A, prior to lipid A export to the outer membrane. 40 1.4.5 Function and regulation of lgm locus In the context of host-pathogen interactions, the lgm locus appears to play a role in modulating host immune responses. Deletion of the lgm locus is associated with a significant reduction in TLR4 activation, indicating the glucosamine modification of B. pertussis lipid A is a TLR4 agonist. Furthermore, the modification appears to provide protection to outer membrane stress and resistance to antimicrobial peptides, where deletion of the lgm locus sensitizes cells to treatment with CAMPs such as polymyxin B (117, 154). Taken together, the glucosamine modification of B. pertussis lipid A provides stimulation of, yet protection against the innate immune system. This is atypical amongst Gram-negative pathogens as a single lipid A modification produces seemingly juxtaposed effects. Thus, the effects of this modification are likely tightly regulated and fine-tuned within the host to best promote survival and immune modulation. The lgm locus has previously been identified as being regulated by BvgAS, the master regulator of virulence factor production in B. pertussis (143). Transposon mutants of BvgS showed a substantial reduction in transcript abundance of lgmA and lgmB when compared to wild type. However, lgm locus transcripts were still identified in the Bvg- mode, suggesting BvgAS independent activation. Furthermore, strains grown in a chemically defined liquid medium showed greater transcript abundance of lgmA and lgmB compared to strains grown on blood agar plates (143). This implies that the locus may also be regulated by factors based on nutrient availability. Taken together this indicates that there are several regulatory networks involved in the regulation of the modification machinery encoded within the lgm locus. Importantly, previous work identified that lgmE transcripts are also produced from within the lgm locus (145). 41 This evokes the idea that complementary sense and antisense transcripts may be able to base pair, interacting as part of a post-transcriptional regulatory mechanism involved in fine-tuning outer membrane remodeling by means of a cis-encoded asRNA. 1.5 Hypotheses and thesis goals In light of recent outbreaks of whooping cough, the efficacy of the current vaccine has been brought into question. Even though sufficient at protecting immunized individuals from developing whooping cough, the current acellular pertussis vaccine is less effective at preventing asymptomatic colonization (9). As interests turn towards developing the next generation of pertussis vaccine, a deeper understanding of B. pertussis molecular pathogenesis and host-pathogen interactions will be key in mitigating future outbreaks. As with many other Gram-negative pathogens, B. pertussis pathogenesis is a tightly regulated and multi-layered process. The two-component system BvgAS sits at the center of virulence factor production and coordinates the expression of over 500 genes. Recently, additional two-component systems have been shown to play an important role in integrating environmental signals and influencing the expression of genes in the Bvg+ and Bvg- modes (56). Furthermore, the RNA chaperone Hfq has been shown to be strongly involved in the regulation of several key virulence factors (44). However, even with several putative sRNAs identified, the post-transcriptional regulatory networks in B. pertussis gene expression are largely uncharacterized. The work in this thesis aims to explore gene loci potentially regulated at the post-transcriptional level by analyzing the transcriptome of endonucleases commonly involved in the processing of regulatory RNAs. The lgm locus, a gene locus involved in LPS modification (117), is shown to be differentially expressed in both endonuclease mutants and contains an antisense open reading frame potentially 42 involved in the regulation of the locus. The lipid A modification under the control of the enzymes within this locus provides protection from, but activation of the host innate immune system (117, 154). Given these juxtaposed effects, the expression of the modification machinery is likely fine-tuned to balanced properties which promote colonization and survival through immune modulation and maintenance of membrane integrity. Here, I hypothesize that display of lipid A modifications in B. pertussis is a tightly controlled process integrating regulatory signals at the transcriptional, post-transcriptional, and post-translational levels. Furthermore, the antisense ORF, lgmE, functions as a dual function regulatory RNA where both mRNA and protein product further fine-tune lgm locus gene expression. Objective 1 – Regulatory RNAs work in conjunction with RNA chaperones and ribonucleases to modulate the stability of the target mRNA. Two major endonucleases in Gram-negative bacteria are RNase III and RNase E. RNase III specifically cleaves double-stranded RNA and is commonly associated with the processing of cis-encoded asRNA. RNase E is involved in RNA metabolism within the bacterial cell and is closely associated with sRNA turnover due to interactions with Hfq and the RNase E C-terminal domain. To better understand post-transcriptional regulatory networks in B. pertussis, mutant strains of RNase III and RNase E were generated and the impacts on the B. pertussis transcriptome analysis by RNA-Seq. This data set was then utilized to identify metabolic pathways, virulence factors and other gene loci differentially expressed in these mutant backgrounds. Comparing this data set to what is 43 currently known about the B. pertussis transcriptome then allowed for estimation of operons and gene loci likely influenced by regulatory RNAs. Objective 2 – Several regulatory networks appear to play a role in the activation of the lgm locus. Promoter regions for sense and antisense transcripts were mapped using RNA-Seq data and in silico promoter mapping tools. Luciferase reporters were then used to clone predicted lgm locus promoter regions to validate these findings and assess promoter strength under varying growth conditions. To better understand the role of lgmE, new deletion mutants of lgmB and lgmC were generated to not overlap with the open reading frame of lgmE and to verify that previously observed phenotypes of lgmB and lgmC were not due to double deletions with lgmE. The intergenic region between lgmB and lgmC, where lgmE is located, was then deleted to determine the impact of the loss of lgmE on the lipid A modification of B. pertussis LPS. Objective 3 – Transcripts of lgmE have previously been shown to be transcribed from the lgm locus and postulated to function as an asRNA. LgmE is predicted to encode a 131 amino acid, membrane-associated peptide with homology to GtrA-like proteins. Here the potential of lgmE to be translated and act as a dual-function RNA was explored. The lgmE ORF was first recombinantly expressed in E. coli and B. pertussis to determine if a viable protein product is encoded and if it associates with the bacterial membrane. The impact of the over expression and deletion of lgmE on the other lgm locus transcripts was then assessed. Finally, structural features and putative functions of lgmE were then modelled using in silico tools. 44 Chapter 2: RNase III and RNase E influence post-transcriptional regulatory networks involved in virulence factor production, metabolism, and regulatory RNA processing in Bordetella pertussis. 2.1 Synopsis Bordetella pertussis has been shown to encode regulatory RNAs, yet the post-transcriptional regulatory circuits on which they act remain to be fully elucidated. Mutants lacking the endonucleases RNase III and RNase E were generated and assessed for their individual impact on the B. pertussis transcriptome. RNA-Seq analysis showed differential expression of ~25% of the B. pertussis transcriptome in each mutant with only 28% overlap between data sets. Both endonucleases exhibited substantial impact on genes involved in amino acid uptake, e.g. ABC transporters, and in virulence e.g. the type III secretion system, and the autotransporters vag8, tcfA, and brkA. Interestingly, mutations in RNase III and RNase E drove the stability of many transcripts, including those involved in virulence, in opposite directions; a result that was validated by qPCR and immunoblot for tcfA and brkA. Of note, whereas similar mutations to RNase E in E. coli have subtle effects on transcript stability, a striking >20-fold reduction in four gene transcripts, including tcfA and vag8, was observed in B. pertussis. The dataset was further compared to the regulon controlled by the RNA chaperone Hfq to identify B. pertussis loci influenced by regulatory RNAs. This analysis identified ~120 genes and 19 operons potentially regulated at the post-transcriptional level. Thus, these findings revealed how changes in RNase III- and RNase E-mediated RNA turnover influence pathways associated with virulence and cellular homeostasis. Moreover, loci potentially influenced by regulatory RNAs were highlighted, providing insights into the post-transcriptional regulatory networks involved in fine-tuning B. pertussis gene expression. 45 2.2 Introduction Bacterial pathogens must be able to rapidly adapt to the range of environments encountered within the host during infection. Successful colonization requires tightly regulated gene expression balancing stress state responses, resisting specific immune and nutritional challenges, and production of virulence factors (85, 155). Many environmental signals are detected by sensor kinases that coordinate rapid transcriptional changes through the phosphorylation of response regulators (156). However, rapid changes in gene expression can also be coordinated at the post-transcriptional level by regulatory RNAs. In recent years, there has been substantial expansion in the number of identified regulatory RNAs and in the understanding of the wide range of adaptive responses in which they are involved. Regulatory RNAs have been shown to play roles in modulating quorum sensing responses, changing cell surface structures, and fine-tuning bacterial metabolism (63, 64, 85). There are three main classes into which regulatory RNAs are grouped: 1) regulatory elements found in the 5’ untranslated region (UTR), such as riboswitches and RNA thermometers, 2), cis-encoded, antisense RNAs (asRNA) that are transcribed from the opposite strand of the target mRNA, and 3) small non-coding regulatory RNAs (sRNA), 50-400 nucleotide trans-encoded transcripts that require an RNA chaperone, such as Hfq, to catalyze interactions with the target transcript (73). Interactions between regulatory RNAs and the mRNA targets often lead to changes in the secondary structure of the target mRNA. These changes can modulate rates of translation by altering accessibility to ribosome binding sites or leading to cleavage of the target mRNAs. Cleavage events can result in either the degradation or stabilization of the target mRNA, whereby the regulatory RNAs are associated with directing the activity of one or several ribonucleases that are involved in the cleavage of their target RNA(s) (157). 46 Ribonucleases involved in regulatory RNA turnover can digest from the terminal ends of an RNA transcript (exonuclease) or cleave at an internal site (endonuclease; (94)). Two key endonucleases involved in regulatory RNA processing in bacterial pathogens are RNase III and RNase E. RNase III is an endoribonuclease with specific activity for double-stranded RNA. It acts as a homodimer with a key role in the maturation of ribosomal RNAs (rRNAs) and tRNAs (93, 158). RNase III has been shown to influence Staphylococcus aureus virulence through cleavage of the RNAIII transcript (91, 92) and has an important role in mediating changes of metabolic state in Escherichia coli (158). Due to its specificity for double-stranded RNA, RNase III has also been implicated in processing asRNAs and their targets (159). RNase E is an essential endoribonuclease that forms a homotetramer associated with the bacterial inner membrane. The N-terminal ribonuclease domain containing the catalytic site is required for the maturation of rRNAs but is also involved in the degradation of many cellular transcripts. Three other proteins - RhlB, enolase, and PNPase - complex with RNase E through its C-terminal half forming a multicomponent structure known as the degradosome (94, 95). The C-terminal half has been shown to be required for interactions with the RNA-chaperone Hfq, with the sRNA and mRNA molecules bound to the proximal and distal faces of Hfq respectively (79, 160). Through these interactions with Hfq and its role in sRNA turnover, RNase E has been shown to regulate the expression of virulence factors and mediate several stress responses at the post-transcriptional level (161-163). In Bordetella pertussis, the causative agent of whooping cough, virulence factor production is centrally controlled by the BvgAS two-component system (19). The system coordinates two 47 main transcriptional states known as the Bvg+ and Bvg- modes. When the cells are grown at 35-37°C, B. pertussis enters the Bvg+ phase in which the sensor kinase BvgS phosphorylates the response regulator BvgA. BvgA~P is then able to promote the expression of virulence-associated genes (vags) and suppress the expression of virulence repressed genes (vrgs; (14, 19, 164)). BvgS can be modulated by lowering the growth temperature, or by adding millimolar concentrations of nicotinic acid or MgSO4 to growth media in vitro. This Bvg- mode is further characterized by the repression of vags and the expression of vrgs. Finally, a third gene expression profile can be produced by the BvgAS system. Known as the Bvg intermediate mode (Bvgi), it is characterized by the expression of vrgs, vags not requiring full BvgA~P activation, and Bvgi-phase specific polypeptides (Bips) of which BipA is the most widely studied (165, 166). Indeed, the regulation of genes within the virulence regulon has many complexities, since several overlapping regulatory systems have been shown to modulate the expression of genes in the Bvg+ or Bvg- modes (57, 58). Interestingly, Hfq has been shown to be required for the expression of many virulence factors (108). Additionally, recent studies have also identified potential virulence-associated sRNAs, suggesting the involvement of regulatory RNAs in pathogenesis (44, 106, 108, 167). Even though regulatory RNAs have been identified, there are few examples in B. pertussis in which the targets for these regulatory RNAs have been identified. To understand more about the contribution of regulatory RNAs to post-transcriptional gene regulation in Bordetella, mutant alleles of both the endonucleases RNase III and RNase E were generated and analyzed for their 48 impact on the B. pertussis global transcriptome using RNA-Seq. Here, it is shown that both endonucleases are involved in processing approximately 25% of the B. pertussis transcriptome including transcripts associated with bacterial metabolism and virulence factor production. In this regard, autotransporter proteins involved in serum resistance and host colonization – brkA and tcfA (54, 168) - were further examined as these were representative of both the subtle and more dramatic differential expression patterns observed within our data set. Validating the robustness of the RNA-Seq analysis, it was shown that changes in transcript abundance for both autotransporters is mirrored at the protein level. Through comparisons of our RNA-Seq dataset to published studies (44, 106), more than 100 gene loci were determined as potentially influenced by regulatory RNAs. This study thus probes the post-transcriptional landscape of B. pertussis in showing how altered RNA processing impacts the B. pertussis transcriptome, whilst also identifying gene loci potentially influenced by asRNA and sRNA. 49 2.3 Materials and Methods 2.3.1 Bacterial strains, plasmids, and growth conditions. All strains used in this study are detailed in Table 2. Lysogeny broth (LB; Tryptone 10 g/L, yeast extract 5g/L, NaCl 10g/L) or LB plus agar was used for the growth of Escherichia coli strains at 37°C. Bordetella strains were grown either on Bordet-Gengou (BG; BD DifcoTm) agar plates supplemented with 15% defibrinated sheep’s blood (Dalynn Biologicals) or in complete Stainer-Scholte medium (SSM) (169) supplemented with 0.15% bovine serum albumin (BSA; Sigma) (170). B. pertussis strains were cultured in either 5 or 15 mlSSM and grown at 37°C at 200rpm. As needed, media were supplemented with nalidixic acid (30 µg/ mL), gentamycin (15 µg/ mL), and/or diaminopimelic acid (DAP) (250 µg/ mL). 50 Table 2.1 Strain and primer list. Bacterial Strain or Plasmid Description Reference/Source E. coli Strains DH5α Molecular cloning strain RHO3 Conjugation strain; kans Δasd ΔaphA, DAP auxotroph (171) B. pertussis Strains BP338 (WT) Wild-type B. pertussis Tohama-1 strain; nalr (172) BP338rncD45A B. pertussis containing catalytically inactive RNase III allele, rncD45A; nalr This Study BP338rneΔCT B. pertussis containing truncation of the C-terminal domain of RNase E, rne1-578; nalr This Study Plasmids pSS4894 Suicide vector containing I-SceI restriction enzyme and cognate restriction site, used for allelic exchange; gmr (173) pIG-rncD45A pSS4894 with up- and downstream fragments required for mutagenesis of rnc gene in B. pertussis; gmr This Study pIG-rneΔCT pSS4894 with up- and downstream fragments required for in-frame deletion of rne C-terminus in B. pertussis; gmr This Study pBBR2-pcpn Broad host range, low copy number plasmid, consists of cpn10 heatshock promoter adjacent to multiple cloning site; kanr (117) pBBR2-rnc pBBR2-pcpn with wild type B. pertussis rnc; kanr This Study pBBR2-rne pBBR2-pcpn with wild type B. pertussis rne; kanr This Study Primer name Sequence 5'-3' Description rncD45A 1_fw aaaaaaACGCGTGTACATCAACGGAAAATTGGTGCC Used in construction of pGI-rncD45A rncD45A 1_rev aaaaaaGCTAGCGCCGAGAAACTCCAGCCGCTCGTT rncD45A 2_fw aaaaaaGCTAGCGTGCTGAACTTCGTCGTCGCGGCG rncD45A 2_rev aaaaaaGGATCCGGCGGCGCGGCGGCTGGCGCCCGG rne-cterm_1 fw aaaaaaGGATCCCGAAGGCTCGCACATCACCTGCCC Used in construction of pGI-rneΔCT rne-cterm 1_rev aaaaaaGAATTCTTCGGTCTTGGCCGGCTCGGCGCT rne-cterm 2_fw aaaaaaGAATTCCGCACAAAGCGGGTTGCGGCAGCG rne-cterm 2_rev aaaaaaGTCGACCCAGGTGATGACCAACTCGCCGAC Rnc_fw aaaaaGTCGACATTGACCGCTATGTCCCTTGCCACG Used in construction of pBBR2-rnc Rnc_rev aaaaaaGGATCCTCATTTAACCTCTTGGGCCACTGC RNase E_fw aaaaGAATTCATGAAGCGCATGCTGTTTAATGCGAC Used in construction of pBBR2-rne RNaseE_rev aaaaaaGGATCCTCAGTGACGCGTTTCGACTTGCAC brkA - qpcr_fw CGCAGGAGTTCAAAAGCACG Used in qPCR of BrkA brkA - qpcr_rev GAATGTTGATCCGGTCGCC tcfA - qpcr_fw GGTATGTGGACACTTTCTC Used in qPCR of TcfA tcfA - qpcr_rev GCTTGAAATCCTCCAGAGACA RpoB - qpcr_fw GCTGGGACCCGAGGAAAT Used in qPCR of RpoB, housekeeping gene RpoB - qpcr_rev CGCCAATGTAGACGATGCC 51 2.3.2 Construction of endoribonuclease mutants in B. pertussis To generate the point mutation in the rnc gene on the B. pertussis chromosome, ~500 bp upstream and downstream of the rncD45 codon were amplified using primers detailed in Table 2. Fragments were designed to be joined to each other using a NheI site. The position of the NheI site, when incorporated into the B. pertussis genome, would provide the desired D45A mutation, whilst not influencing the adjacent serine codon. This ~1000 bp fragment was then cloned into pSS4894 (173) between MluI sites and BamHI restriction sites to generate pIG-rncD45A. This construct was first transformed into E. coli RHO3 before conjugation into B. pertussis. The truncation of the rne C-terminal half was generated by first amplifying ~500bp regions flanking the 1398 bp of the rne C-terminal half which was to be removed. Flanking regions were ligated together at EcoRI sites and introduced to pSS4894 at SalI and BamHI restriction sites. This construct, pIG-rneΔCT, was transformed into RHO3 for conjugation. Allelic exchange plasmids were delivered to B. pertussis by conjugation with minor alterations to the published procedure (173). Briefly, recipient BP338 was grown on BG agar in the presence of 50 mM MgSO4 and nalidixic acid for 3 days. These cells were then harvested from plates using a sterile swab and suspended in 1.5 mL of SS broth supplemented with DAP. The OD600nm of the resuspended BP338 was then measured. The donor RHO3 strains were grown overnight and OD600nm measured. BP338 and donor RHO3 strains were then mixed in a ratio of 2:1 and spotted onto SS-agar plates containing SS-supplements, BSA (0.15%), and 50 mM MgSO4. Conjugation mixtures were incubated for 6h at 37oC before being swabbed and re-swabbed onto 52 fresh BG plates with nalidixic acid (30 µg/ mL), gentamycin (100 µg/ mL), and 50 mM MgSO4. After 4 days incubation at 37oC single colonies were re-streaked onto BG plates with nalidixic acid to allow activation of the BvgAS system and I-SceI expression driven by the pertussis toxin promoter. These plates were grown until distinct colonies could be observed. From this, single colonies were then screened by PCR for the presence of the mutant allele. PCR-confirmed mutants were then grown for ~72 h in complete SSM and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) as per the manufacturer’s instructions. Gene fragments including up and downstream regions used for allelic exchange were then amplified and mutations confirmed by Sanger sequencing (Genewiz). 2.3.3 RNA-Seq and bioinformatic analyses Wild type and mutant cells were grown to an OD600nm 0.6-0.7 in SSM and 2 mL of cells were harvested into 4 mL RNA Protect Bacteria (Qiagen). RNA was extracted from cell pellets using an RNAqueous RNA extraction kit (Ambion) following kit instructions, and DNA was removed using the TurboDNAse kit. RNA concentrations were measured by nanodrop spectrophotometer and DNA removal verified by PCR. Total RNA quality was assessed using BioAnalyzer 2100, with each sample producing an RNA integrity number greater than nine. Ribosomal RNA depletion was completed using the RiboZero Bacteria Kit (Illumina). The KAPA Stranded Total RNA Kit (KAPA Biosystems) was used for library construction (five replicates per genotype), and sequencing was done by the University of British Columbia’s Sequencing and Bioinformatics Consortium on an Illumina HiSeq2500. Single-end 100bp reads 53 were checked for quality using FastQC v0.11.7 (174) and aligned to the B. pertussis sp. Tohama I reference genome from NCBI (NCBI:txid257313, (175)) using the alignment program STAR v2.6.1a (176). Counts of aligned reads were generated using HTSeq’s count function v0.9.1 (177). Raw library sizes for all samples had a minimum of 1,032,961, median of 4,355,770, and maximum of 8,888,916 reads after removing low count genes (fewer than 10 counts across the number of biological replicates). Differentially expressed genes between each of the mutants and wild type bacteria were determined using the Wald statistical test implemented in the Bioconductor package DESeq2 v1.24.0, R version 3.5 (178). Results were corrected for multiple testing using the Benjamini-Hochberg method and were filtered for genes with an adjusted p-value ≤ 0.05 and an absolute fold change ≥ 1.5. Functional enrichment of these differentially expressed genes was performed using an overrepresentation analysis via the R package Gage v2.34.0 (179), testing for enriched pathways from the KEGG database for B. pertussis sp. Tohama I. Pathways resulting from this analysis were considered significant based on a false discovery rate (FDR)-corrected q-value threshold of ≤ 0.1. To generate counts of mapped reads for each sample and genotype along a defined region of interest, IGVtools’ count function v2.4.14 (180) was used. Volcano plots were generated by plotting log2 fold change against -log10 of the p-value for all genes detected in our RNA-Seq analysis. Genome coordinates of putative sRNAs (105) present in the Bordetella genome were added prior to DESeq2 analysis to determine any differential expression amongst these potential regulatory RNAs. Subsets of differentially expressed genes 54 were generated by filtering locus tags present in other transcriptomic analysis data sets of Bordetella pertussis (14, 44, 106). 2.3.4 qPCR Wild type and mutant cells were grown in SSM at 37°C. Cultures were harvested at mid-log phase to late log phase and stationary phase. Cell pellets were resuspended in 100 µl Tris-EDTA pH 7.0 with 1 mg/ mL lysozyme. RNA was then extracted following the RNAqueous RNA extraction kit protocol (Ambion) and treated for DNA contamination using Turbo DNase I (Ambion). RNA concentrations were measured by nanodrop™ spectrophotometer and DNA removal verified by PCR. To verify gene transcript abundance changes, cDNA was made from 250 ng of each RNA sample using I-script cDNA synthesis kit (BioRad) following kit instructions. All primers used are detailed in Table 2.1 and were designed to have an annealing temperature of 60°C. Quantitative PCR (qPCR) reactions were set up using SsoAdvanced SYBR green master mix and measured on a Bio-Rad CFX96 instrument. Each sample was run in duplicate with each 10 µl reaction containing 500 nM of each primer pair and 12.5 ng cDNA. The thermal cycling program consisted of an initial melt step of 98°C for 3 min, then 95°C for 15 secs and 60°C for 30 secs for 40 cycles, followed by melt curve analysis. Results were normalized using rpoB as a housekeeping gene and fold changes calculated using the 2-ΔΔCt method (181). 2.3.5 Immunoblots B. pertussis BP338 wild type and endonuclease mutant strains were grown in complete Stainer-Scholte -medium to the desired growth phase, mid-log (OD600nm 0.5-0.6) late log (OD600nm 0.8-55 0.9), and stationary phase (time point = 108 h) or from plates at 72 h. For each time point, 1 mL of culture was harvested by centrifugation, and cell pellets resuspended to an OD600nm 5.0 in SS salts (154) to adjust for equal loading. An equal volume of 2x sample buffer was added and the samples heated to 95-100°C for 5 mins. Each time point sample was run on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Membranes were probed with polyclonal antibodies raised against BrkA (182), TcfA (55), and monoclonal antibodies to CyaA (Sigma). Primary antibodies were visualized using HRP-conjugated secondary antibodies. The band intensities were then measured using ImageJ (183) to estimate fold changes in protein expression. 2.3.6 Data availability Sequence and count files are available at the Gene Expression Omnibus under accession number GSE164312. (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164312) 56 2.4 Results 2.4.1 Generation and growth characteristics of the RNase III mutant, BPrncD45A Cis-encoded asRNAs are transcribed from the opposite strand from the target RNA and can interact with the target through complementary base pairing (73). Since RNase III specially cleaves RNA-RNA duplexes, it has been implicated in several asRNA control mechanisms. In B. pertussis, the rnc gene encoding RNase III is part of a 5 gene operon consisting of lepA, lep, rnc, era, and recO (175). Both lep and era have been identified as essential genes in B. pertussis (35) and overlap with the start and stop codon of the rnc gene respectively. Repeated attempts to delete rnc were unsuccessful, likely due to potential polar effects. Previous in vitro studies of Escherichia coli RNase III have shown that mutations at the active site aspartic acid (D45) resulted in a ⁓30,000-fold reduction in ribonuclease activity (184). Also, approaches in Streptomyces coelicolor to diminish RNase III nuclease activity while maintaining other important biological functions targeted the active site aspartic acid (185). Furthermore, the importance of this residue in endonuclease activity was also modelled using the structure of RNase III from Aquifex aeolicus (186). Amino acid sequences of the rnc genes from B. pertussis, E. coli, S. coelicolor, and A. aeolicus were aligned (Fig. 2.1A) and residues in B. pertussis RNase III corresponding to known active site motifs were mapped. As shown in Fig. 2.1A, the D45 residue shown to be important for catalytic activity (184-186) is highly conserved in B. pertussis. This aspartic acid residue was substituted for an alanine on the chromosomal copy of the rnc gene using allelic exchange (173) and assessed the impact of this mutation on asRNA regulation in B. pertussis. When grown on Bordet-Gengou (BG) agar, the B. pertussis RNase III mutant, BPrncD45A, 57 produced haemolytic colonies which appeared to be smaller than wild type. This growth defect was further observed when the strain was grown in complete Stainer-Scholte media (SSM; Fig. 2.1C), with the RNase III mutant strain showing an extended lag phase yet reaching comparable optical densities to wild type over the course of the growth curve. This growth defect was reversed through complementation with the wild type copy of the gene constitutively expressed from a low-copy number plasmid (Fig. 2.2A). 58 59 Figure 2.1 Design and growth characteristics of B. pertussis endonuclease mutants. A) Alignment of the predicted amino acid sequences of A. aeolicus, E. coli, and S. coelicolor, and B. pertussis rnc. The mutated residue at D45 is indicated by a black arrow. A schematic of the mutation introduced to the rnc gene is shown below the alignment. B) Alignment of the predicted amino acid sequence of the B. pertussis rne gene with E. coli and S. typhimurium rne. The black arrow indicates the last amino acid within the open reading frame of the C-terminal truncation. This residue maps to the rne-105 mutation in E. coli, in which a frame shift mutation results in a premature stop (187). A schematic of the mutation introduced into the rne gene on the B. pertussis chromosome is shown below the alignment. Alignments for both endonucleases and homologues were run using Clustal Omega (188) and visualized using ESPript3 (189). C) Comparison of the growth kinetics of the wild type (♦), BPrncD45A (■) and BPrneΔCT (▲) shown as linear co-ordinates of OD readings at 600nm. Inset: Growth curve plotted in semi-log coordinates. Growth rates were consistent over 4 independent experiments. A representative experiment is shown. BPrncD45A and BPrneΔCT refer to the RNAse III and RNase E mutant, respectively. 60 2.4.2 Generation and growth characteristics of the RNase E mutant, BPrneΔCT The turnover of mRNAs targeted by sRNAs is efficiently catalyzed by the endonuclease RNase E. Through interactions with the RNase E C-terminal half, the RNA chaperone Hfq bound to an sRNA is proposed to assist in directing the endonuclease to target transcripts for degradation (190). RNase E is an essential ribonuclease required for the maturation of ribosomal RNAs and tRNA precursors (94). Despite the essentiality of RNase E, investigators working with E. coli and Salmonella have established viable RNase E mutations, allowing studies of the enzyme in vivo (191, 192). Indeed, mutations resulting in the truncation of the RNase E C-terminal domain have been shown to reduce RNase E function without negatively impacting the enzyme’s role in rRNA maturation (187). Importantly, the RNase E C-terminal half is required for mRNA targeting mediated by Hfq and small RNAs (160). To understand more about the role RNase E plays in transcript stability and turnover in B. pertussis, 465 amino acids were deleted from the C-terminal half of RNase E to generate the strain BPrneΔCT (Fig. 2.1B). This mutation was designed to mimic the rne-105 mutation in E. coli (187). When grown in SSM, the mutant strain grew to an optical density greater than that of the wild type strain and aggregated less in liquid media (Fig. 2.1C). When grown on BG agar, the colonies of the RNase E mutant were similar in size to those of the wild type strain from which it was derived; however, the RNase E strain was non-haemolytic. The lack of hemolysis was attributed to either loss or impaired activity of adenylate cyclase toxin (CyaA) which encodes a C-terminal RTX domain required for haemolysis. Immunoblots of CyaA expression in the RNase E mutant showed a substantial decrease compared to wild type. Haemolysis and CyaA expression were reverted to wild-type levels when BPrneΔCT was complemented with the full-61 length rne gene (Fig. 2.2B and 2.2C). 62 63 Figure 2.2 Complementation of endonuclease mutants. A) Growth kinetics of rncD45A complemented with B. pertussis rnc in pBBR2pcpn () compared to BP338 wild type (♦) and BPrncD45A (■). B) Hemolytic activity of BP338 wild type (top left), BPrneΔCT (bottom left), BPrneΔCT complemented with empty vector (bottom right), and BPrneΔCT complemented with B. pertussis rne in pBBR2pcpn (top right). C) Complementation of CyaA expression in BPrneΔCT by immunoblot analysis. Samples were taken at growth phases indicated above each lane (L = log-phase, S = Stationary phase). Monoclonal antibodies (Sigma) were used to probe for the expression of CyaA. 64 2.4.3 Impact of mutations in RNase III and RNase E on the B. pertussis transcriptome The mutations generated in the B. pertussis rnc and rne genes were designed to alter RNA turnover in the cell. Overall, this would impact RNA metabolism as well as many systems associated with post-transcriptional regulatory circuits. To determine the changes to the regulation of the B. pertussis transcriptome, the wild type, and mutant strains were grown to mid-log phase (OD600nm = 0.6-0.7) and RNA was extracted for transcriptomic analysis by RNA-Seq. Sequencing reads were aligned to the Tohama I genome (NCBI:txid257313, (175)) and gene expression changes compared to wild type using DESeq2 (178). Differentially expressed genes were defined as having a fold change of ≥ 1.5 compared to wild type and an adjusted P-value ≤ 0.05 across five biological replicates. In the RNase III mutant, there were 1062 genes differentially expressed when compared to the wild type (Fig. 2.3A). As shown in volcano plots (Fig. 2.3B) mapping the spread of differentially expressed genes, the expression of 585 of these genes were negatively affected by the loss of RNase III activity, while 477 were upregulated. Compared to wild type, ptlEFGH were amongst the most downregulated genes in this data set, while the type three secretion system (T3SS) chaperone protein, BP2265, was amongst the most upregulated (Table 2.2, Table A.1). Specific pathways, identified using Generally Applicable Gene-set Enrichment (GAGE) (179), were shown to be dysregulated in the RNase III mutant (Fig. 2.3C). This included pathways associated with quorum sensing (bpe02024), ABC transporters (bpe02010), and tRNA biosynthesis (bpe00970). The RNase E mutant showed 956 genes differentially expressed when compared to wild type 65 (Fig. 2.3A). Like the RNase III mutant, more than 580 genes were downregulated by the mutation to RNase E (Fig. 2.3B). Comparing the two data sets, 444 genes were commonly differentially expressed, and of these 131 genes (Table A.2) showed opposing patterns in fold-change in the two mutant backgrounds. Here, the autotransporters vag8 and tcfA were the most downregulated genes showing a >25-fold reduction when compared to wild type (Table A.1). Interestingly, the rne gene itself was amongst the most upregulated genes suggesting a potential impact on RNase E autoregulation (Table 2.2) (94). The RNase E mutant also shows enrichment in pathways associated with B. pertussis pathogenesis (bpe05133), bacterial secretion (bpe03070), and metabolic pathways (bpe01100) (Fig. 2.3C). RNase E is involved in the processing of sRNAs in various bacterial pathogens, yet RNase III can also play a role in sRNA turnover due to RNA duplex formation with the target mRNA (193). Several putative targeted sRNAs have been previously identified by in silico analysis of the B. pertussis genome (105). Using the predicted genome coordinates of these 17 sRNAs, it was examined whether the endonuclease mutations altered the expression of this subset of putative regulatory RNAs in B. pertussis. Even though sRNAs are typically processed by RNase E, it was surprising that only 2 of the predicted sRNAs (bprK and bprM’) were differentially expressed in the RNase E mutant, although 4 sRNAs (bprB, bprJ, bprM, bprM’), were found to be differentially expressed in the RNase III mutant. One of these sRNA species, bprM’, was differentially expressed in both mutants, but it was upregulated in the RNase III mutant and down regulated in the RNase E mutant (Table A.3). Taken together, the endonuclease mutations each affected approximately one-quarter of the B. 66 pertussis transcriptome. Even though each mutation ultimately impairs endonuclease function, only approximately half of the differentially expressed transcripts appeared to become less abundant through the loss of full RNase III or RNase E activity. These data suggest that these endonucleases may contribute to stabilizing many mRNAs within the B. pertussis transcriptome. Another outstanding observation was the substantial variation, and moderate overlap between the impacts of these two endonucleases, implying novelty with a degree of redundancy. 67 68 Figure 2.3 Differential gene expression in the RNase III (BPrncD45A) and RNase E (BPrneΔCT) mutants compared to wild type as determined by RNA-Seq. A) UpSet plot showing the total number of genes differentially expressed in each mutant strain compared to wild type. The total number of differentially expressed genes in BPrncD45A (1062) and BPrneΔCT (956) are indicated on the scale at the bottom left of the plot. Genes unique to each set are indicated by a filled circle and genes shared in differential expression are indicated by filled circles connected by a bar. The number of genes expressed is indicated above each histogram. B) Volcano plots illustrating the range of differentially expressed genes from transcriptomic analysis of BPrncD45A mutant (left) and the BPrneΔCT mutant (right). Labeled genes represent 5 topmost, and 5 bottom most differentially expressed genes from each data set. Differential expression cut-offs showing fold change of ≤ or ≥ 1.5x compared to wild type and a P-value ≤ 0.05 are indicated by the grey lines. All genes identified in the RNA-Seq analysis are plotted. The total number of genes showing a significant positive or negative fold change (FC) relative to wild type in each mutant strain is indicated below the plots. C) GAGE analysis determining KEGG pathway enrichment in endonuclease mutants. Enriched pathways are indicated by a filled circle indicating downregulation (purple) or upregulation (orange) of the associated KEGG pathway. 69 Table 2.2 Top ranking differentially expressed genes in transcriptomic analysis of endonuclease mutants in B. pertussis. Lists are sorted by fold change showing the five top- and bottom-most altered transcript abundances compared to wild type. BPrncD45A vs wild type BPrneΔCT vs wild type Locus Tag Gene FC Locus Tag Gene FC BP3795 ptlG -13.20 BP2315 vag8 -48.22 BP3794 PtlF -13.15 BP1201 tcfA -27.69 BP3796 ptlH -13.15 BP2236 bscW -27.15 BP3793 ptlE -13.02 BP2927 Putative oxidoreductase -21.74 BP2638 putative secreted protein -12.29 BP1487 smoM -16.18 BP3302 hypothetical protein +6.85 BP2722 Putative membrane transport ATPase +6.29 BP3409 hypothetical protein +6.69 BP0398 lgmB +5.83 BP2265 TTSS (secretion chaperone) +6.68 BP0475 rne +5.81 BP1579 putative cytochrome C +5.04 BP0397 lgmC +5.69 BP3455 Putative acetolactate synthase +4.91 BP0399 lgmA +5.68 70 2.4.4 Validation of RNA-Seq data set To verify the gene fold-changes reported in the RNA-Seq data set, the differential expression of two genes in the mutant backgrounds was experimentally characterized. Specifically, the expression changes of the autotransporters BrkA and TcfA, virulence factors that were also shown to be differentially expressed in the Δhfq mutant (44), were examined. Fig. 2.4A demonstrates the normalized counts of both brkA and tcfA and shows a near to 2-fold increase in transcript abundance in the RNase III mutant. A stronger phenotype was observed in the RNase E mutant where there was a 4-fold reduced abundance of brkA transcripts, and strikingly a normalized count for tcfA approaching zero, which translated to a 28-fold reduction in transcript abundance compared to wild type (Table A.1). To verify these data at the RNA and protein level, fresh cultures were inoculated into SSM, and samples for qPCR were taken at late log phase for RNA analysis; for protein expression, samples for immunoblots were taken across a time course at log phase and stationary phase. The results from qPCR showed an approximately 2-fold increase in brkA and tcfA transcripts in the RNase III mutant compared to wild type. In the RNase E mutant, qPCR showed a 2-fold and 20-fold reduction in brkA and tcfA respectively (Fig. 2.4B). Differences in protein abundance were estimated by analyzing the density of bands corresponding to the expression of BrkA and TcfA in immunoblots. Using ImageJ (183) for quantification, the RNase III mutant showed approximately a 1.5-fold increase in BrkA and TcfA levels when compared to wild type. In the RNase E mutant, protein expression of BrkA is reduced by approximately 2-fold whereas TcfA protein expression was almost non-detectable across the time course (Fig. 2.4C). These data thus corroborated the RNA-Seq analysis by showing gene and protein expression patterns that 71 mirrored what was seen in the transcriptome analysis of the two mutants. Notably, phenotypes for each endonuclease mutant at both the RNA (qPCR) and protein (immunoblot) levels were returned to wild type levels upon complementation with the wild type copy of the appropriate gene (Fig. 2.4B and Fig. 2.4C). As RNA binding motifs were removed in the BPrneΔCT strain, a second mutant strain was developed to confirm the role of RNase E in the stability of BrkA and TcfA. The temperature-sensitive mutant BPrneG66S was generated by allelic exchange (Section A.1, Fig. A.1A) and preliminary results showed similar impacts on TcfA and BrkA expression when grown at 37°C (Fig. A.1B). This provides further evidence that fully functional RNase E targets these transcripts for processing. 72 Figure 2.4 Validation of the RNA-Seq data sets using brkA and tcfA. A) Normalized counts of brkA and tcfA transcripts as reported from transcriptome analysis of wild type B. pertussis and BPrncD45A and BPrneΔCT. Box plots indicate the number of reads for each gene across five biological replicates. B) Validation of the RNA-Seq data by qPCR. RNA was extracted from wild type and endonuclease mutants in late log phase. Bar graph shows the mean of the relative expression of brkA and tcfA in endonuclease mutants and complement strains compared to wild type plotted on a log2 Y-axis. Error bars show the standard deviation of three technical replicates. A representative of three experiments is shown. C) Validation of RNA-Seq data by immunoblots. Samples were taken at growth phases indicated above each lane (L = log-phase, S = Stationary phase). Expression of BrkA and TcfA was probed for using antisera raised against each protein. Reduction and absence of TcfA in stationary 73 phase for the three strains is due to the protein being released from the cell surface (55). 2.4.5 Mutations in RNase III and RNase E drove virulence factor stability in opposite directions. The BvgAS two-component regulatory system sits at the center of virulence gene expression in Bordetella. In the Bvg+ mode, more than 500 genes are regulated by phosphorylated BvgA (Table 2.3; (6)), including several genes associated with virulence. Since both RNase III and RNase E are implicated in bacterial virulence factor production and pathogenesis (86), they might influence or be influenced by the BvgAS regulon. Comparison between the RNA-Seq data set collected here and the recent transcriptomic analysis of the BvgAS regulon (14) showed that approximately 50% of the genes in the BvgAS regulon were differentially expressed in the RNase III and RNase E mutant strains (Table 2.3). 74 Table 2.3. Comparison of B. pertussis transcriptome analyses The number of genes found to be differentially expressed in BPrncD45A and BPrneΔCT which are also found to be differentially expressed in transcriptomic analyses of the BvgAS regulon (14) and the hfq regulon (44). Data sets from these studies were chosen due to similarities in growth conditions of wild type and mutant strains. No. of genes differentially regulated Data set BvgAS or Hfq regulon BPrncD45A BPrneΔCT BvgAS regulon BvgAS Up regulated 237 111 144 BvgAS Down regulated 322 155 145 BvgAS associated metabolism genes 119 69 64 Well-characterised VAGS 71 36 66 Hfq Regulon Hfq Up regulated 156 96 93 Hfq Down regulated 212 86 88 75 B. pertussis expresses several virulence factors in the Bvg+ mode, with many characterized through in vitro and in vivo infection models (194). As part of the BvgAS regulon, these well-characterized virulence factors number approximately 70 genes and comprise those encoding toxins, secretion systems, and colonization factors. To determine whether the mutations in RNase III and RNase E impacted the stability of these virulence gene transcripts, the fold changes of these transcripts were identified within our RNA-Seq data set. Figure 2.5 and Table A.1 detail the fold changes of the 71 well-characterized virulence genes in the two endonuclease mutants. Interestingly, 66 of these virulence factor transcripts were found to be less abundant in the RNase E mutant including the cya operon which is likely responsible for the non-haemolytic phenotype observed with this mutant. Autotransporters tcfA and vag8, and the putative type 3 secretion chaperone, bscW, were more than 25-fold downregulated when compared to wild type. In the RNase III mutant, 36 of these 71 virulence factors were significantly differentially expressed. Of these virulence factors, 10 were found to be more abundant when compared to wild type and shown to be less abundant in the RNase E mutant; although it should be noted that the ptl locus was downregulated in both mutant backgrounds. The BvgAS system can also produce a third gene expression profile intermediary of Bvg+ and Bvg- known as the Bvg intermediate mode (Bvgi) (165). The marker protein of the Bvgi phase, bipA, showed a similar pattern in transcript abundance wherein its transcript abundance was reduced in the RNase III mutant yet increased in the RNase E mutant. Finally, although the Tohama-1 strain of B. pertussis does not have a functional T3SS when grown in vitro (i.e. in the absence of host cells) (44, 195), transcripts associated with the T3SS locus were less abundant in both mutants, with the tip complex protein, bsp22, being more abundant in the RNase III mutant. 76 Taken together, these data identify an interesting relationship in which RNase III and RNase E have opposing activities on select virulence factor transcripts. This association suggests these endonucleases may be involved in modulating transcript stability during pathogenesis as a potential means of fine-tuning virulence factor expression within the host. 77 78 Figure 2.5 RNase III and RNase E have opposing activities on virulence factor transcripts. Depiction of volcano plots filtered to show differential expression of well-characterized virulence factors (14) in BPrncD45A (top) and BPrneΔCT (bottom). In both plots, significantly differentially expressed genes of the type three secretion locus and pertussis toxin liberation operon are indicated by triangles (blue) and squares (orange) respectively. Plots map 69 of the 71 genes in the subset; BP1880, and BP2260 were not detected from our RNA-Seq analysis. FC refers to fold change. 79 2.4.6 RNase III and RNase E processed transcripts associated with metabolism in the Bvg+ mode During the Bvg+ mode, phosphorylated BvgA alters the expression of many genes associated with metabolic and catabolic pathways. This suggests that B. pertussis coordinates substantial changes to cell homeostasis between Bvg+ and Bvg- modes. Since both RNase E and RNase III are associated with controlling metabolic gene expression in E. coli (158), differentially expressed metabolism genes associated with the Bvg+ mode (14) were identified. This comparison showed that 69/119 of the Bvg-associated metabolism genes were also affected by the mutation to RNase III. Of this subset, all genes associated with the RNase III mutation were less abundant (Fig. 2.6). The same analysis was performed on the RNase E mutant and showed that 64/119 of the Bvg-associated metabolism genes were differentially regulated. Of these 64 differentially expressed genes, 50 were shared between the RNase E and RNase III mutants and the transcripts associated with many members of this gene subset were less abundant compared to wild type (Fig. 2.6). Further analysis showed that 23 of these 50 genes were ABC transporters associated with amino acid uptake (Fig. 2.6) found in the GAGE pathway bpe02010 (Fig. 2.3C). Taken together, these data suggest that both RNase III and RNase E play a role in the maintenance of many metabolism transcripts in the Bvg+ mode. Since many of the genes involved appeared to be associated with resource uptake, the data suggests that these endonucleases may be somehow involved in coordinating the transition in cell homeostasis between Bvg+ and Bvg- modes. 80 Heatmap shows the average fold changes of the 50 genes associated with B. pertussis metabolism in the Bvg+ phase differentially expressed in both the BPrncD45A mutant (left) and the BPrneΔCT mutant (right). Genes names shown in red indicate ABC transporters associated with the KEGG pathway bpe02010. Figure 2.6 Impact of endonuclease mutations on Bvg regulated metabolism genes. 81 2.4.7 Comparative analysis of B. pertussis transcriptomic data provided insights into regulatory RNA networks Previous work in B. pertussis has identified several regulatory RNAs, yet not much is known about the target transcripts on which they act. As endonucleases are often involved in the processing of the target mRNA coupled with regulatory RNAs, the findings from the transcriptomic analysis of the two endonuclease mutants were compared to previously published studies depicting the B. pertussis primary transcriptome (106) and the hfq regulon (44). This was initiated to identify putative gene loci which might be regulated by either asRNAs or sRNAs. As recently detailed in the report of the B. pertussis primary transcriptome (106), a majority of the asRNA transcripts are produced from internal promoters found within the large number of insertion (IS) elements present within the B. pertussis genome. Transcription from promoters within the insertion elements has been shown to run into the adjacent genes. In some cases, these are found in antisense to the adjacent gene and can influence transcript stability (196). Since RNase III is involved in cleaving double-stranded RNA, whether RNase III was involved in the resolution or degradation of transcripts at IS antisense junctions was investigated. The B. pertussis primary transcriptome identified 192 genes adjacent to IS elements and influenced by an antisense transcript (106). Of these, 39 genes (20%) were found to be differentially expressed in the RNase III mutant (Table A.4.). Genes such as fim2, metX, and recR as well as other genes in this subset were less abundant when compared to wild type, suggesting that endonuclease RNase III may be required to stabilize the interacting transcripts produced from these loci. 82 Data from the two endonuclease mutants was also compared to the regulon controlled by the RNA chaperone, Hfq, as this protein plays a substantial role in mediating the interactions, stability, and processing of sRNAs with their mRNA targets (44, 108). This analysis established that approximately 50% of the genes in the hfq regulon were differentially expressed in both endonuclease mutants (Table 2.3). This overlap of genes included many ABC transporters and virulence factors, including the autotransporters, brkA, tcfA, and vag8. Moreover, many genes required for the Type III secretion system were down regulated in all three mutant backgrounds. Given this, a search was undertaken for additional operons that may be influenced in a similar way. This analysis identified 19 unique operons (Table A.5), including several ABC transporters, a quinol oxidase biosynthesis cluster, and the ptl operon required for pertussis toxin secretion (197). Thus, using the known relationship between RNase E and Hfq and the specific activity for double-stranded RNA by RNase III, this work identified a number of gene loci that were previously not known to be regulated at the post-transcriptional level. 83 2.5 Discussion Regulatory RNAs provide bacterial pathogens with a mechanism to rapidly modify gene expression profiles while under stress. Often processed by one of several ribonucleases, cleavage of the target mRNA can substantially alter the stability of the transcript and/or modulate rates of translation. RNase III and RNase E are endonucleases involved in processing of cis-encoded asRNA and trans-encoded sRNAs respectively. Here, mutants of both RNase III and RNase E were generated by allelic exchange, and the impact of these mutations on the B. pertussis transcriptome was assessed by RNA-Seq. The mutations to RNase III and RNase E resulted in the differential expression of approximately 1000 genes, representative of about 25% of the B. pertussis genome. Fig. 2.3B shows that many of the transcripts found to be differentially expressed in the two mutants were less abundant when compared to wild type. With the impaired activity in both endonuclease mutant backgrounds, increases in transcript abundance were expected. However, it was surprising to see more than half of the differentially expressed transcripts were reduced in abundance in the mutant strains. In the case of the RNase III mutation, this reduction in transcript abundance might have been, in part, a consequence of the slow growth phenotype associated with the mutation (198). For example, much of the lipid A biosynthesis pathway genes, lpxABDHK were approximately 2-fold reduced when compared to wild type, corresponding to the approximately 2-fold reduction in the doubling rate of the mutant cells. However, there was no growth defect associated with the C-terminal truncation of RNase E, and yet a similar proportion of transcripts were reduced in the BPrneΔCT mutant strain. Interestingly, the lgm locus (locus tags BP0397-BP0399) which is involved in the modification of lipid A (117), was increased almost 6-fold in 84 the RNase E mutant (Fig. 2.3B). Changes in transcript abundance may be associated with several mechanisms involved in RNA half-life such as changes in rates of translation and a loss of cleavage events mediated by these two endonucleases resulting in alternate RNA secondary structures (199). Differential processing of regulatory RNAs may also contribute to the stability of these transcripts (200). RNase E is associated with the turnover of sRNAs in bacteria through the C-terminal domain coordinating interactions with the RNA chaperone Hfq (201). RNase III is also involved in regulatory RNA circuits by cleaving RNA-RNA duplexes such as those formed from asRNA binding to its mRNA target (63, 82). The RNA-Seq data set was compared to other published studies to establish associations between the endonuclease mutants and known sRNAs and asRNAs. Using this approach, 5 previously identified sRNAs (105) were shown to be differentially expressed in the endonuclease data set. Of these, 4 of the sRNAs were unique to the RNase III mutant. Even though RNase III is typically associated with cleaving asRNAs, this endonuclease also processes trans-encoded regulatory RNAs (90-92, 202). Furthermore, as the RNase III mutation influenced many metabolic processes, this might have resulted in several stress responses contributing to the production of sRNAs enriched under these conditions (64, 85). It is interesting that RNase III also played a role in regulating many genes found to be in an antisense orientation to an adjacent insertion element. Many of these genes were down regulated in the mutant strain, suggesting that the full catalytic function of RNase III is potentially required for the activation of many genes near insertion elements. One of the most striking phenotypes of the RNase E mutant was the loss of haemolysis (Fig. 85 2.2B) and a marked reduction of the expression of CyaA (Fig. 2.2C). Haemolysis in Bordetella pertussis is due to the activity of adenylate cyclase toxin, CyaA, which is produced and exported by the genes found in the cya operon (27). The RNA-Seq analysis here showed that in the RNase E mutant, the cya operon was approximately 2-fold reduced compared to wild type, a reduction which was further confirmed by immunoblot. This would suggest a decrease in haemolysis, although a greater than 2-fold reduction was observed. Interestingly, diminished adenylate cyclase activity was also observed in the Δhfq strain (108). Of note, qPCR analysis of cyaA in this hfq mutant showed that transcript abundance was similar to the wild type strain, implying that the cyaA mRNA requires post-transcriptional modification for optimal translation. Taken together, this suggests that RNase E is likely involved in processing of the cyaA transcript in an Hfq-dependent manner. Hfq is known to catalyze the interactions with a small RNA (79, 160); however, the associated regulatory RNA involved with modulating cyaA translation is yet to be identified. Additionally, the reduction in the cya operon transcript abundance in the RNase E mutant may also suggest that this endonuclease is involved in stabilizing the mRNA of the entire locus. Overall, this provides several layers of complexity around the regulation of adenylate cyclase toxin expression in B. pertussis, although the importance of this regulation in Bordetella pathogenesis is yet to be determined. To identify loci potentially regulated by regulatory RNAs, the transcriptomes of each endonuclease mutant were compared to the hfq regulon (44). This revealed a substantial overlap with genes that were also differentially expressed in the mutant hfq background. From this, it was observed that much of the T3SS was downregulated in both the RNase III and RNase E mutant strains as well as in the Δhfq strain, further implying a role for regulatory RNAs in the 86 function of the secretion system. In many bacteria, the control of the T3SS tends to be complex and both regulatory RNAs and endonucleases have been implicated in this process (203). In Yersinia, both RNase E (96) and Hfq (204, 205) were shown to play a role in the function of the secretion system. Amongst the Bordetella spp. regulation of T3SS consists of the extra cytoplasmic sigma factor BtrS, a cognate anti-sigma factor, BtrA, and partner-switching modules BtrVW (41). In both the endonuclease mutants here, these regulatory proteins were differentially expressed compared to wild type under these conditions. However, there were no significant changes in transcript abundance of btrASVW in the Δhfq background (44). These data add an additional layer of complexity to the regulation of the T3SS, potentially involving factors outside the T3SS locus. Further investigation would be required to determine a mechanism by which this may work. Alongside the T3SS, more than 90% of widely studied virulence factors involved in B. pertussis pathogenesis (14) were decreased in abundance in the RNase E mutant, with more than half of these well-characterized virulence factors being more than 4-fold reduced, and some more than 20-fold reduced in abundance. This was quite a striking finding since similar mutations in E. coli are often associated with a more subtle impact on transcript stability (187). Surprisingly, many of these virulence factors were upregulated in the RNase III mutant. Of the 444 genes that shared differential expression in both mutants, 131 of these genes had opposing fold changes in the two mutant backgrounds. The opposite effects on transcript stability produced by these two endonucleases have been observed previously; however, the reason for this remains unclear (200). Gene transcripts showing opposite stabilities in the mutant backgrounds include the response regulator bvgA and autotransporters vag8, tcfA, and brkA. It is intriguing that the 87 mutations in RNase III and RNase E exerted such a drastic impact on the transcripts for tcfA and vag8 since both these autotransporters were also strongly down regulated in the Δhfq strain of B. pertussis (44, 108). Taken together these data suggest that the abundance of these autotransporter transcripts could be determined by regulatory RNAs. Complex regulatory interactions involving RNase III, RNase E, and Hfq have been characterized previously (84, 90, 200). For example, the inactivation of the sodB transcript in E. coli under iron-limited conditions requires RNase III, RNase E, and the sRNA RyhB in complex with Hfq (202). However, the regulatory RNA influencing the expression of vag8 and tcfA in B. pertussis and the mechanism by which it interacts with these genes is yet to be elucidated. Currently, there are few well-defined mechanisms by which regulatory RNAs work in B. pertussis. The work here highlights additional regulation of metabolism and key virulence factors involved in B. pertussis pathogenesis. Furthermore, by comparing the endonuclease RNA-Seq data to the currently available data on the B. pertussis transcriptome, gene loci potentially influenced by regulatory RNAs have been highlighted. Even though there are other ribonucleases present in B. pertussis, the mutant strains generated here provide details on the post-transcriptional landscape of B. pertussis while also contributing another layer of understanding of the complex regulatory circuits responsible for Bordetella pathogenesis. 88 Chapter 3: Regulation of Lipid A modifications in Bordetella pertussis involves several overlapping networks modulating expression at transcriptional, post-transcriptional, and translational levels 3.1 Synopsis The lgm locus is a 5 gene operon in B. pertussis involved in modifying lipid A with glucosamine. The locus consists of 4 genes, lgmA-D, in one orientation and the 5th open reading frame, lgmE, overlapping in the opposite orientation. Recent transcriptomic analysis of endonuclease mutants involved in processing regulatory RNAs identified the lgm locus as strongly differentially expressed compared to wild type. Given that an antisense open reading frame is present in the lgm locus, the work in this chapter aims to elucidate whether the regulation of lipid A modifications in B. pertussis may be fine-tuned by a cis-encoded asRNA. Also, given that lipid A remodeling systems in Gram-negative pathogens are commonly part of complex regulatory networks, additional mechanisms of regulation within the lgm locus are also identified. Luciferase reporter assays of lgm locus promoters indicate that the locus is responsive to Bvg mode, nutrient limitation, pH, and CO2%. Furthermore, promoters for lgmA and lgmE are reciprocally activated at low pH in early log phase. Additionally, the mutations to RNase III and RNase E have significant impacts on TLR4 activation albeit without the loss of the glucosamine sugar from lipid A, suggesting that lipid A modification may be regulated by these endonucleases. Lastly, co-translation of LgmA and LgmB appear to be required for functionality. Overall, it is suggested that the lgm locus forms a non-contiguous operon, in which gene expression is modulated at the transcriptional, post-transcriptional, and potentially translational levels. 89 3.2 Introduction Lipopolysaccharide (LPS) is the outer layer of the asymmetrical Gram-negative outer membrane. It is a complex, multicomponent structure protecting the bacterial cell from the surrounding environment (112, 206). LPS itself is formed of 3 key domains: Lipid A, the core sugars, and the O-antigen. Lipid A is the hydrophobic anchor to which the inner core sugars (Kdo) are attached (207). The O-antigen is ligated to the outer core sugars; however, the O-antigen is absent in some bacterial species (208). As the bacterial outer membrane physically interacts with the environment, LPS is often the site of modification and remodeling enabling the bacterial cell to adapt and change in response to current conditions (112). LPS remodeling is a key mechanism of successful host colonization for many Gram-negative pathogens, as the host consists of many, commonly hostile, environments. Each portion of the LPS molecule can be targeted for modification, changing surface properties to provide increased stability of the outer membrane, and resistance to antimicrobial peptides (123). These modifications are also used to modulate immune responses. The TLR4/MD-2 complex of the mammalian innate immune system is able to produce proinflammatory cytokines once activated upon LPS binding, specifically through interactions with lipid A (Fig. 1.3) (112, 123, 125). Due to this bioactivity, Gram-negative pathogens have several enzymatic processes which can remodel the lipid A portion of LPS. These modifications can change the number of acyl chains and/or add or remove moieties present on lipid A to alter the binding of the molecule to the TLR4/MD-2 complex (112, 123). The modifications systems can often be energetically expensive and require tight regulation outside and within the host environment (112). 90 In this context, changes to the bacterial outer membrane are often coordinated with other pathways of pathogenesis to promote colonization of the invading organism. This requires several, overlapping regulatory systems to ensure the remodeling machinery is expressed at the right time and to modulate effects of outer membrane properties throughout different compartments in the host. Importantly, the expression and activity of lipid A modifications systems are typically fine-tuned by inputs from both transcriptional and translational regulation systems (112). Two-component systems are a core feature of transcriptional regulation in bacterial pathogens. These signal transduction systems allow the bacterial cell to respond to environmental changes and dictate relevant gene expression changes (15, 17). They are formed of a membrane-bound sensor kinase, which is activated in response to environmental signals. This results in the phosphorylation of a response regulator which, in its phosphorylated state, can bind specific sequences found in promoter regions of specific genes to modify RNA polymerase behavior (15, 209). The modulation of lipid A modifications by two-component systems is well studied in various Gram-negative pathogens, such as S. typhimurium, P. aeruginosa, and E. coli, where such modifications provide protection and survivability against the host environment (112). PhoPQ, is a two-component system activated through low pH and restricted availability of divalent cations Mg2+ and Ca2+ (210, 211). Phosphorylated PhoP upregulates many genes in response to this environment. This includes pagP and pagL, lipid A modification genes which acylate and deacylate lipid A respectively (112, 212). This remodeling provides outer membrane stability and increased resistance to antimicrobial peptides (123). In addition to the transcriptional regulation of lipid A modifying enzymes, PhoPQ also has been shown to produce 91 several non-coding RNAs involved in the modulation of outer membrane modifications (97, 112, 213). Post-transcriptional regulation mechanisms require the target mRNA to interact with a regulatory, non-coding small RNA (sRNA). These interactions are often catalyzed by the RNA chaperone Hfq, and the mRNA-sRNA complex being cleaved by a ribonuclease such as RNase III or RNase E (73). Both mgrR and micF are PhoQ transcribed sRNAs which downregulate EptB and LpxR expression respectively in an Hfq/RNase E dependent manner (134, 138). LpxR is a lipid A deacylase involved in immune system evasion in Yersinia enterocolitica (112). Alongside the post-transcriptional regulation of this outer membrane enzyme, LpxR is further post-translationally inhibited by Ara4N modified lipid A (214). This multifaceted way in which lipid A modifications are regulated allows for rapid, fine-tuned response to the host environment. However, these regulatory circuits of outer membrane remodeling are still being elucidated in some important Gram-negative pathogens. In B. pertussis, the two-component system BvgAS is regarded as the master regulator of virulence factor production. At physiological temperatures (35-37°C) cells enter the Bvg+ mode in which BvgS phosphorylates BvgA which then activates virulence-associated gene loci (vags) and down regulates virulence repressed gene loci (vrgs). At temperatures below 35°C, or if cells are grown in the presence of millimolar concentration of MgSO4 or nicotinic acid, B. pertussis enters its virulence off state, the Bvg- mode, in which vrgs are expressed and vags are repressed. Alongside the cohort of classical virulence factors such as toxins and adhesins, BvgAS has been shown to regulate genes associated with cell physiology and metabolisms(14). Importantly, BvgAS has been shown to play a role in the control of a lipid A modification system in B. pertussis (143). 92 The lipid A of B. pertussis forms a penta-acylated structure which is further modified with GlcN on the 1- and 4’- phosphates of the di-GlcN backbone (144). This modification is under the control of enzymes encoded within the lipid A glucosamine modification (lgm) locus (117). The genes, lgmA-E, form two overlapping pairs (lgmAB and lgmCD) which are separated by a 188 bp intergenic region. The 5th open reading frame, lgmE, is found overlapping lgmB and lgmC in this intergenic region and in the opposite orientation (Fig. 1.5A). As shown by mutational analysis of genes in the locus, LgmABC are the minimum machinery required for the addition of a glucosamine sugar to the lipid A backbone (117, 145). The presence of the GlcN modification can be identified by analysis of lipid A fractions using mass spectrometry. The presence of the sugar is indicated by peaks at 1720 m/z for a single modification and 1881 m/z for double modified lipid A. Deletion of the lgm locus, resulting in the absence of the glucosamine sugar, is shown as a major peak at 1559 m/z, representative of the penta-acylated, unmodified lipid A (117, 144). Functionally speaking, the lipid A modification in B. pertussis has been shown to provide the bacterial cell with protection against exposure to cationic antimicrobial peptides, such as Polymyxin B (154). In comparison to E. coli lipid A, a potent TLR4 activator, modified B. pertussis lipid A is a moderate TLR4 agonist (215). Furthermore, when the lgm locus is deleted to generate an unmodified lipid A, these strains show a significant reduction in TLR4 activation when compared to wild type (117). Taken together, this provides a seemingly juxtaposed effect of a single lipid A modification. The addition of the glucosamine sugar provides stimulation of, but also protection against the innate immune system. This implies the lipid A modification in B. pertussis must be tightly regulated in order to ensure the properties of the outer membrane are correctly balanced and honed within the host. Additionally, the antisense 93 open reading frame present within the lgm locus suggests the potential of an antisense RNA regulatory mechanism. The lgm locus has previously been shown to be controlled by the master regulator of virulence factor production, BvgAS. As measured by RT-PCR, transcript abundance from the lgm locus was substantially reduced in a BvgS transposon mutant (143). Additionally, recent transcriptomic analysis of the BvgAS regulon identified lgmABC as genes positively regulated by BvgA (14). Also, the lgm locus may be regulated at the post-transcriptional level as transcriptomic analyses of Δhfq (44) and mutants of the ribonuclease RNase III and RNase E in B. pertussis were also shown to have an impact on lgm locus activity (Fig. 2.3) (216). This implies that much like other Gram-negative pathogens, outer membrane remodeling in B. pertussis involves substantial crosstalk between regulatory networks, yet the underlying mechanisms are still unknown. Here, we provide further insight into the regulatory systems contributing to the control of lipid A modifications in B. pertussis. Nutrient limitation and environmental pH are identified as conditions modulating the transcription of the lgm locus genes. Furthermore, at the post-transcriptional level, endoribonucleases involved in processing regulatory RNAs are shown to have a significant impact on the stability of lgm locus transcripts and outer membrane properties of B. pertussis. Also, it is proposed that the lgm locus forms a noncontiguous operon in which this rare genetic architecture plays a role in ensuring the correct expression of the modification machinery. 94 3.3 Materials and methods 3.3.1 Bacterial strains, plasmids, and growth conditions. All strains used in this study are detailed in Table 3.1. Lysogeny broth (LB; Tryptone 10 g/L, yeast extract 5g/L, NaCl 10g/L) or LB plus agar was used for the growth of Escherichia coli strains. Bordetella strains were grown either on Bordet-Gengou (BG; BD DifcoTm) agar plates supplemented with 15% defibrinated sheep’s blood (Dalynn Biologicals) or in complete Stainer-Scholte (SS) medium (pH 7.6) (169) supplemented with 0.15% bovine serum albumin (BSA; Sigma) (170). Growth of bacterial strains was performed at 37°C. As needed, media were supplemented with nalidixic acid (30 µg/ mL), gentamycin (15 µg/mL), and/or diaminopimelic acid (DAP) (250 µg/ mL). 95 Table 3.1 Strain and primer list Bacterial Strain or Plasmid Description Reference/Source E. coli strains DH5α Molecular cloning strain RHO3 Conjugation strain; kans Δasd ΔaphA, DAP auxotroph (171) B. pertussis strains BP338 Wild-type B. pertussis Tohama-1 strain; nalr (172) BP338rncD45A B. pertussis containing catalytically inactive RNase III allele, rncD45A; nalr BP338rneΔCT B. pertussis containing truncation of the C-terminal domain of RNase E, rne1-578; nalr BP338ΔlgmA-D Deletion of BP0399-BP0396 (lgmABCD containing lgmE); nalr (145) BPM2859 Tn5lac insertion into BP0398 (lgmB); nalr kanr (217) BP338lgmBΔ6-520 Deletion of BP0398 (lgmB) where deletion does not overlap with 3’ end of lgmE; nalr This work BP338lgmCΔ110-367 Deletion of BP0397 (lgmC) where deletion does not overlap with 5’ lgmE and maintains predicted promoter; nalr This work BP338ΔlgmE Deletion of the 188 bp intergenic region between lgmB and lgmC where lgmE is located; nalr This work Plasmids pB4 Broad host range vector derived from pBBR2pcpn, kanr This work pB4-LgmB pB4 with wild type copy of lgmB, kanr This work pB4-LgmA+LgmB pB4 with lgmA and lgmB in non-overlapping orientation with each gene with its own RBS, kanr This work pB4-285LgmAB pB4 with last 200 bp of lgmA full-length lgmB in native overlapping orientation, kanr This work pBluescriptIIKS- Multifunctional cloning vector, ampr Stratagene pIG02 Derived from pSS4894 Suicide vector containing I-SceI restriction enzyme and cognate restriction site, used for allelic exchange; gmr (173) pIG02-ΔlgmB2 pIG02 with flanking regions of lgmB for clean deletion not overlapping with lgmE, gmr This work pIG02-ΔlgmC2 pIG02 with flanking regions of lgmC for clean deletion not overlapping with lgmE, gmr This work pIG02-ΔlgmE pIG02 with flanking regions of the intergenic region between lgmB and lgmC, gmr This work pSS3967 Ectopic luxCDABE promoter assay vector, ampr gmr (218) pIG04 Derived from pSS3967, rrnBT2 terminator inserted upstream of multiple cloning site, ampr gmr This work pIG04-pLgmA115 pIG04 with 115 bp fragment upstream of lgmA, ampr gmr This work pIG04-pLgmA180 pIG04 with 180 bp fragment upstream of lgmA, ampr gmr This work pIG04-pLgmA425 pIG04 with 425 bp fragment upstream of lgmA, ampr gmr This work pIG04-pLgmE900 pIG04 with 900 bp fragment upstream of lgmE, ampr gmr This work pQC1794-pLgmE900 In situ luciferase reporter with 900 bp fragment upstream of lgmE, ampr gmr This work pUC18T-mini-Tn7T-Km-FRT Mobilizable transposition vector; ampr kanr (219) pTNS2 Tn7 transposase vector for expression of tnsABCD, ampr (219) pUC18-miniTn7T-lgmAB Tn7transposon containing 425 bp upstream of lgmA and wild type copies of lgmA and lgmB in native overlapping orientation, ampr kanr This work 96 Primers Sequence 5’-3’ Description nLgmBKO_UPfw aaaaaaACGCGTCAGCGCGACGACGAGCCGTGGTTC Used in generation of BP338lgmBΔ6-520 nLgmBKO_UPrev aaaaaGTCGACTCGGGTAGCGAGAGTCATTGGGAAC nLgmBKO_DOWNfw aaaaaaGTCGACGTGCTGGTGGACGAAGCCTTCAAG nLgmBKO_DOWNrev aaaaGAATTCCCGGCGCATCGGCGGCGAACGCCGGC nLgmCKO_UPfw aaaaaGTCGACTGGGCGAGCGGCTGCTGGTCTCGGC Used in generation of BP338lgmCΔ110-367 nLgmCKO_UPrev aaaaaaGAATTCGGGAGACACCTCTTCGACATGCGC nlgmCKO_DOWNfw aaaaaaGAATTCGCCTGGCAAGCCGTTTATGAGTTC nLgmCKO_DOWNrev aaaaaaGGATCCCGATCCGCAGCGGACGCATCTATG LgmB-Cdel1fw aaaaaaACGCGTCGAACTGGCTGGCCAGCCGCGCAT Used in generation of BP338ΔLgmE LgmB-Cdel1rev aaaaaaGAATTCATGACCAGTGAACGATACGACGAA LgmB-Cdel2fw aaaaaaGAATTCCGCTAGCCGCCTGTGGGCGTTCCG LgmB-Cdel2rev aaaaaaGGATCCTGCGCGACCCGGCGGTGGCGCGGG pLgmA115fw aaaaaaGAATTCCCGAAGCCCGGCGAACGCTCCCAA for cloning region 115 bp upstream of lgmA pLgmA180fw aaaaaaGAATTCGCATTGTTCGCACAATGTAACTTT for cloning region 180 bp upstream of lgmA pLgmA425fw aaaaaaGAATTCCGCAATCATGGACGAACCAGGTAT for cloning region 425 bp upstream of lgmA pLgmArev aaaaaaGTCGACTCGGTATACATGAACACACCACGG For cloning of lgmA promoter regions pLgmE900fw aaaaaaaGAATTCAATGCGGAACATTCCGGACCTTACC For cloning region 900 bp upstream of lgmE pLgmErev aaaaaaCTCGAGGCAATGAACCAGGCAATCTGTCGCAG For cloning of lgmE promoter regions LgmA-D285+LgmBfw aaaaaGGATCCGACTATCTCATCAGCGGCAACGCGG For pB4-285LgmAB of LgmA-D285+LgmBrev aaaaaaAAGCTTCTAGCCGCCTGTGGGCGTTCCGTC LgmEantisense_ RTprobe GAAGCCTTCAAGGGGCGTGTCTGTGACGGAACGCCC Gene specific primer for lgmE antisense transcript LgmBsense_RTprobe GCCAACGTGGTGGGCTGGCTGTTGGCCTTCGTCGTA Gene specific primer for lgmB sense transcript LgmBC-IRfw CTCACTTTCCGCCACCTTGCGGCA For amplification of intergenic region between lgmB and lgmC; Used in qPCR of lgmE LgmBC-IRrev GAAGGCCAGCCCGATCAACACCAG rpoB_fw GCTGGGACCCGAGGAAAT Used in qPCR of RpoB, housekeeping gene rpoB_rev CGCCAATGTAGACGATGCC lgmAqpcrfw TACCTGGTGGCCGACTAT Used in qPCR of lgmA lgmAqpcrrev CAGCGAAATCAGGTTGATGC LgmBqPCRfw GCATGCCGTATTTCCACAAG Used in qPCR of lgmB LgmBqPCRrev ATTCGTTGACGCCGAACA LgmCqPCRfw GCATGTCGAAGAGGTGTCTC Used in qPCR of lgmC LgmCqPCRrev CATCGATGGCCTCGTTCAT LgmDqPCRfw TTCCTGCACGCGTTCTT Used in qPCR of lgmD LgmDqPCRrev ACGCCCATGTCGAACAG 97 3.3.2 Construction of deletion mutants To generate BP338lgmBΔ6-520, primers were designed so that the subsequent deletion would leave at least a 6 amino acid margin off the 3’ ends of lgmA and lgmE. A similar approach was taken for BP338lgmCΔ110-367, where primers were designed to leave a 6 amino acid margin of the 5’ end of lgmD, with approximately 200 bp left upstream of lgmE to not remove the predicted promoter. For the deletion of lgmE, primers were designed to delete the full 188 bp region between lgmB and lgmC. Up- and downstream fragments of approximately 600 bp in size flanking these regions were then amplified using Q5 polymerase (New England Biolabs) and ligated into the allelic exchange plasmid, pIG02, at appropriate restriction sites. Allelic exchange plasmids were then delivered to B. pertussis by conjugation with minor alterations to the published procedure (173) as described in section 2.3.2. PCR-confirmed mutants were then grown for ~72 h in complete SSM and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) as per the manufacturer’s instructions. Gene fragments including up and downstream regions used for allelic exchange were then amplified and mutations confirmed by Sanger sequencing (Genewiz). 3.3.3 Preparation of bacterial cells for TLR4-activation assays B. pertussis strains were grown on BG agar at 37 °C for 4 days, harvested by sterile swab into SS salts, and used to inoculate 5-15 mL complete SSM, with the appropriate antibiotics, to a starting OD600nm of 0.01. Cultures were grown at 200 rpm at 37 °C for approximately 40 hrs or until an OD600nm of 0.5 to 0.7 had been reached. Bacterial cells were harvested by centrifugation and resuspended into phosphate-buffered saline (PBS) pH 7.4 to an OD600nm of 5.0 and heat-98 inactivated by incubation at 56 °C for 1 hr. To confirm heat-killing, 50 μL of each cell suspension was spotted onto BG agar supplemented with no antibiotics and incubated at 37 °C for 5 days. Heat-killed cells were stored at -20 °C. 3.3.4 Growth and maintenance of HEK-Blue cell lines HEK-blue cell lines hTLR4 and Null2 (InvivoGen) were maintained in Dulbeccos’ modified eagle medium (DMEM) containing, 1 mM pyruvate, 2 mM Glutamax, 100 µg/ mL Normocin, 100 units/ mL penicillin, and 100 µg/ mL streptomycin (Gibco, ThermoFisher Scientific). Cells were grown in humid air at 37°C and 5% CO2 Null2 and human TLR4 (hTLR4) cell lines express secreted alkaline phosphatase under control of an NFκB promoter, where the hTLR4 is also stably transfected with hTLR4, MD-2, and CD14 co-receptor genes. With this cell line, hTLR4 activation drives transcription from NFκB promoters, resulting in the expression of secreted alkaline phosphatase. Maintenance of reporter and transfected genes required the hTLR4 cell line to be grown in the presence of 100 µg/mL Zeocin, 200 µg/mL Hygrogold, and 30 µg/mL Blasticidin (InvivoGen) and Null 2 cell lines were grown in the presence of 100 µg/ mL Zeocin (InvivoGen). 3.3.5 TLR4-NFκB activation assays HEK-Blue cell lines were assayed for hTLR4 activation by heat-killed bacterial samples as per the manufacturer’s guidelines (InvivoGen). Briefly, HEK-Blue hTLR4 and Null2 in complete DMEM and appropriate antibiotics were seeded in 96-well plates 50, 000 cells/ well, 100 μL/ well. HEK-Blue cells were incubated at 37°C (as described above) for 24 hrs. Fresh medium (100 μL) was added to each well, and the cells were incubated for another 24 hrs. HEK-Blue 99 cells were then washed with 100 μL of medium and stimulated with heat-killed B. pertussis cells at a 1/100 or 1/1000 dilution. In each assay, the Null2 cell line, unstimulated hTLR4, and no seeded cells were used as negative controls. After 20 hrs of incubation, the culture supernatants were assayed for secreted alkaline phosphatase activity by mixing 20 μl of supernatants with 180 μL of QUANTI-Blue (InvivoGen) reagent in a fresh 96-well plate. This mixture was then incubated at 37°C in the dark for 15 mins, 30 mins, and 60 mins, to allow for colour change. Absorbance at 650 nm was measured by plate reader and used as a reporter of alkaline phosphatase activity, indicating NFκB activation via hTLR4. 3.3.6 Polymyxin B assay B. pertussis strains were grown on BG agar with 30 µg/mL nalidixic acid at 37°C for 4 days. These cells were harvested by sterile swab into SS salts and used to inoculate 5-15 mL complete SSM with nalidixic acid at an initial OD600nm of 0.01. Cultures were grown at 37°C while shaking at 200 rpm for 20-24 hrs. Cultures were then diluted into SS salts to an OD600nm of 0.002 and serially diluted to 10-4. A 1 mg/ mL stock of Polymyxin B (PmB) in H2O was serially diluted into SS salts to the desired concentrations and 50 μL added to polypropylene 96-well plates. To this, 50 μl of the diluted bacterial cultures were mixed with each dilution of PmB and incubated at 37°C for 2 hrs. After incubation, 2 μl of each dilution was spotted onto BG agar plates with no antibiotics and incubated at 37°C for at least 72 hrs to visualize growth. 3.3.7 Extraction of lipid A and mass spectrometry analysis Lipid A modifications were studied by mass spectrometry analysis using Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF). Cells were grown to stationary phase (>72 100 hrs) in complete SSM and supplemented with appropriate antibiotics. Cultures were centrifuged (4000×g), washed twice with phosphate buffer (10 mM Na2HPO4, 1.7 mM KH2PO4), and freeze-dried overnight. Lipid A was extracted from lyophilized cells and de-salted as described (220). Ten milligrams of the lyophilized bacterial cells were resuspended in 400 μL of a mixture of isobutyric acid: 1 M ammonium hydroxide (5:3, v/v) and kept on a heat block for 2 hrs at 100°C with vortexing every 15 min, then centrifuged at 2000×g for 15 min and the supernatant was mixed with a double volume of water and lyophilized overnight. Samples were then washed with methanol and lipid A was solubilized in 80 μL of chloroform:methanol:water (3:1.5:0.25, v/v). Lipid A suspension was desalted with a few grains of ion-exchange resin (Dowex 50W-X8; H+). Two microliters aliquot of lipid A suspension was loaded onto a polished steel target, air-dried, and covered by 1 μL of 2,5-dihydroxybenzoic acid matrix (Sigma) dissolved in 0.1 M citric acid solution in chloroform:methanol:water (3:1.5:0.25, v/v) and allowed to air dry. The target was inserted in an Applied Biosystems MALDI-ToF spectrometer. Data acquisition and analysis were performed using the Data Explorer software. 3.3.8 Luciferase reporter assays. The luciferase reporter plasmid pIG04 is a suicide vector which possesses a 1.8 kb region that directs homologous recombination of the vector in the B. pertussis genome at BP0771-BP0773. It was derived from the luciferase reporter, pSS3967 (218), in which the rrnB T2 terminator sequence was inserted 24 bp upstream of the multiple cloning site, where insertion was shown to reduce background activity (data not shown). This is referred to as an “ectopic” reporter within this work as it is designed for the measurement of the activity of short fragments away from the original genome location. Additionally, the luciferase reporter plasmid pQC1794 (a gift from 101 Drs. Qing Chen and Scott Stibitz, FDA) is referred to as an in situ reporter as cloned fragments guide homologous recombination of the vector at the locus of interest. Gene fragments upstream of lgmA of 115 bp, 180 bp, and 425 bp were amplified and cloned into pIG04 between EcoRI and SalI sites. Gene fragments upstream of lgmE of approximately 900 bp were amplified and cloned into pQC1794 and pGI04 at EcoR1 and XhoI. All reporter plasmids were transformed into RHO3 for conjugation into the BP388 wild type strain and selected for on BG plates with 30 µg/ mL nalidixic acid and 15 µg/ mL gentamycin. Single colonies were then screened by PCR, and positive clones re-streaked onto fresh BG plates. For promoter activation studies, reporter strains were grown for 3 days on BG plates with 30 µg/ mL nalidixic acid and 15 µg/ mL gentamycin. Cells were harvested into SS salts and used to inoculate 5 mL complete SSMwith the appropriate antibiotics, to a starting OD600nm of 0.01. Strains were grown at 200 rpm at 37°C for 48 hrs unless otherwise stated. Cultures were then added to white-walled 96-well plates (200 µL) with at least 4 technical replicates, and luciferase activity was enumerated by plate reader. Alternative growth conditions were accomplished by omitting FeSO4∙7H2O (iron limitation) or MgCl2 and CaCl2 (divalent cation limitation) from SSM preparation. Also, modulation into the Bvg- mode was achieved by adding 50 mM MgSO4. Media pH was adjusted to the desired value prior to autoclaving and samples were taken to measure values after. Cultures growth at 5% CO2 was performed in 15 mL centrifuge tubes (Falcon) with 0.2 µM filtered vented caps (Corning) on an angled tube roller to ensure correct aeration in this environment. 102 3.3.9 Beta-galactosidase assays B. pertussis strains containing Tn5 lac insertions in ptxA, lgmB, and dnt were grown on BG-agar plus 75 µg/ mL kanamycin. Strains were then harvested in SS-salts and used to inoculate 10 mL complete SSM (with previously described alterations where appropriate) to a starting OD600nm of 0.01. Cultures were then grown at 37°C at 200 rpm for 44-48 hrs before 5 mL of each culture was harvested by centrifugation. Cell pellets were then resuspended in 1 mL Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCL, 1 mM MgSO4∙7H2O, 50 mM β-mercaptoethanol) and OD600nm measured. Samples were then lysed by adding 100 µL 0.003% SDS and 25 µL chloroform and mixed thoroughly by vortexing. Cell lysates were then transferred to a 96-well plate in 180 µl aliquots (6 technical replicates). To each well, 20 µL of 4 mg/ mL ortho-Nitrophenyl-ß-galactoside (ONPG) was added and incubated for 10 mins at room temperature before reactions were stopped by adding 50 µL, 1 M Na2CO3. Plates were then read at Abs420 and Abs550 and beta-galactosidase activity calculated using the following formula: 600550420 )75.1(10001AbsvtAbsAbsUnitMiller×××−×= 3.3.10 Reverse transcription PCR Cell cultures were inoculated to a starting OD600nm of 0.01 and grown to an OD600nm of 0.6 - 0.8. From this, 1 to 2 mL of each culture was harvested into twice the volume of RNAprotect Bacteria Reagent (Qiagen). RNA stabilized samples were then harvested by centrifugation, supernatants discarded, and cell pellets stored at -80°C until use. For RNA extraction, cell pellets were resuspended in 100 µL Tris-EDTA pH 7.0 with 1 mg/ mL lysozyme. RNA was then extracted following the RNAqueous RNA extraction kit protocol (Ambion) and treated for DNA 103 contamination using Turbo DNase I (Ambion). DNA removal was verified by PCR and RNA concentrations were measured on a nanodrop™ spectrophotometer. For determining sense and antisense transcript production from the lgm locus, primers were designed to anneal to lgmB (sense) and lgmE (antisense) in regions flanking the 188 bp intergenic region within the lgm locus. Reactions consisted of 200 ng wild type RNA, 1 mM of either gene-specific primer or for assessment of total RNA, and 15 mM random primers (Invitrogen). Reverse transcription was performed with SuperScript™ III (Invitrogen) as per the manufacturer’s instructions. Subsequent PCR reactions were performed with GoTaq™ G2 green master mix (Promega), using primers amplifying a 168 bp fragment nested in the lgmB to lgmC intergenic region (lgmB-C). For quantitative PCR (qPCR) reactions, cDNA was made from 250 ng of each RNA sample using I-script cDNA synthesis kit (BioRad) following kit instructions. All primers used, detailed in Table 3.1, were designed to have an annealing temperature of 60°C using the OligoAnalyzer tool (IDT). PCR reactions were set up using SsoAdvanced SYBR green master mix and measured on a Bio-Rad CFX96 instrument. Each sample was run in duplicate with each 10 µL reaction containing 500 nM of each primer pair and 12.5 ng cDNA. The thermal cycling program consisted of an initial melt step of 98°C for 3 min, then 95°C for 15 secs and 60°C for 30 secs for 40 cycles, followed by melt curve analysis. Results were normalized using rpoB as a housekeeping gene and fold changes calculated using the 2-ΔΔCt method (105). 104 3.3.11 Mapping read counts and lgm locus promoter predictions Read counts were first mapped to each base position with the lgm locus using the “count” tool from the Integrative Genomics Viewer (180). Data were then combined into a single table and averaged by genotype. The average read count at each base was then plotted using R. Regions displaying a peak of reads were then analyzed using the BacPP webserver (221). Sequences were analyzed as overlapping 80 bp fragments which were individually assessed for the probability of sigma factor binding. Fragments with the highest probability of sigma factor binding were then mapped to the locations within or upstream of the lgm locus. 105 3.4 Results 3.4.1 Endonucleases RNase III and RNase E significantly impact B. pertussis TLR4 activation RNA-Seq analysis of mutations to the endoribonucleases RNaseIII and RNase E in B. pertussis identified approximately one-quarter of the pertussis transcriptome was differentially expressed compared to wild type. Both mutants showed an impact on virulence factor expression and ABC transporters involved in amino acid uptake (216). Interestingly in the RNase E mutant, a gene locus involved in outer membrane modification was amongst the top 5 most “up-regulated” genes (Table 2.2). The genes lgmA (BP0399), lgmB (BP0398), and lgmC (BP0397) are involved in modifying B. pertussis lipid A with glucosamine, similar to the aminoarabinose modification of lipid A seen in other Gram-negative pathogens (112). Differential expression analysis of the RNA-Seq data set showed that lgmA-C had a near 6-fold induction within the RNase E mutant strain, BPrneΔCT, compared to wild type (Table 2.2). The reported fold changes of all lgm locus transcripts from RNA-Seq analysis of the endonuclease mutants are reported in Fig. 3.1A. Alongside the impact on lgmABC, lgmD and lgmE were also shown to have a 2-fold increase in the RNase E mutant when compared to wild type. Interestingly, in the RNase III mutant strain, BPrncD45A, a much more subtle effect on this locus was observed as only lgmB and lgmC showed a 2-fold reduction in transcript abundance. The endonuclease mutants were then tested to determine if changes to transcript abundance impacted the properties of the outer membrane. Both mutant strains were assayed for TLR4-NFκB activation and sensitivity to polymyxin B to ascertain changes to lipid A and to assess outer membrane stability. For TLR4 activation, heat-inactivated bacterial cells previously grown 106 to mid-log phase were used to stimulate HEK293 cells expressing human TLR4 for 20 hrs. Secreted alkaline phosphatase, measured in a colorimetric assay, was used as a reporter of NFκB activation. Fig. 3.1B details TLR4 activation by the ribonuclease mutants compared to wild type and an lgm locus deletion mutant. The data shows that in the RNase E mutant, the increased lgm locus transcript abundance corresponded to a significant increase in TLR4 activation. A significant reduction of TLR4 activation by the BPrncD45A mutant is also seen, yet this is not as substantial as the deletion of the full lgm locus. A similar result was observed with outer membrane stability, in which a consistent 2-fold increase in sensitivity to polymyxin B treatment was observed by the BPrncD45A mutant (Fig. 3.1B). Interestingly, the BPrneΔCT strain was as susceptible as wild type in this assay despite the increased lgm locus transcript stability and TLR4 activation observed from this strain. Given the phenotypes observed within these assays, it was postulated that there may be a structural difference in the lipid A domain of the LPS in the endonuclease mutants. To verify this, lipid A was extracted from stationary phase cultures and analyzed by MALDI-ToF. As shown in Fig. 3.1C, the lipid A of both endonuclease mutants appeared to be structurally similar to wild type and showed the presence of the glucosamine modification as represented by the peak at 1720 m/z. 107 108 Figure 3.1 Impact of endonuclease mutants on lipid A modification in B. pertussis. A) Bar graph representation of fold-changes of lgm locus transcripts as reported from transcriptomic analysis of BPrncD45A and BPrneΔCT compared to wild type (216). Fold-change values are reported at the end of each bar. Compared to wild type, grey dotted line indicated 1.5x difference from wild type levels, which was the cut-off for differential expression analysis. B) hTLR4 activation by BPrncD45A and BPrneΔCT compared to WT and BP388ΔlgmA-D. Data are representative of three experiments: five technical replicates per experiment. Strains were compared to the wild type strain using a one-way ANOVA with Bonferroni post-hoc test, p-value < 0.0001 (****). C) polymyxin B survival assay. The concentration of polymyxin B is indicated above each lane and serial dilution of B. pertussis strains (10-1 to 10-4) are indicated beside each image. The image displayed is representative of 4 experiments. D) MALDI-ToF analysis of lipid A extracted from wild type, BPrncD45A, and BPrneΔCT. Major peaks are identified with numbers and the presence of GlcN modification is indicated by the black arrow. 109 3.4.2 Transcriptional profiling of the lgm locus in endonuclease mutant backgrounds The five genes within the lgm locus consist of two overlapping pairs of genes, lgmAB, and lgmCD, which are separated by 188 bp. A fifth open reading frame, lgmE, is found in the opposite orientation, overlapping with lgmB and lgmC and encompassing the intergenic region (Fig. 1.5A) (145). Lipid A modification systems are often part of complex regulatory pathways to ensure outer membrane stability with changes in environment (112). Given this, it was hypothesized that additional and overlapping regulatory elements are involved in lgm locus gene expression. The presence of both sense and antisense ORFs in the lgm locus and the role of endoribonucleases in lgm locus transcript stability strongly suggested that post-transcriptional regulation may be involved in outer membrane modification in B. pertussis. To further understand the role that RNase III and RNase E may play in outer membrane remodeling, read counts from transcriptomic analysis of the endonuclease mutants were mapped to the lgm locus (Fig. 3.2A). Maps of this nature can be used to estimate the presence and location of transcript features. The troughs in the maps, where read counts are near zero can indicate terminators or potential ribonuclease cleavage sites. Peaks in read count are indicative of highly expressed or more stable transcripts, such as promoters and potential ribosome binding sites (RBS) as ribosome binding also can influence transcript half-life (222). Several peaks are present within the mapped reads of the lgm locus, but it was interesting that such a large peak was seen in the region between lgmB and lgmC (lgmB-C), where lgmE is located. The read counts shown here are independent of strand direction so there are several possibilities for this stable region, one of which is that it is simply the combined read counts of lgmB-C and lgmE 110 transcripts. To confirm this observation, the presence of sense and antisense transcripts being produced simultaneously from the lgm locus was validated by RT-PCR. Gene-specific primers were designed to probe the lgmB sense strand and the lgmE antisense strand for first-strand cDNA synthesis using superscript III reverse transcriptase. The second set of primers was used to amplify a ~170 bp fragment from the intergenic region of lgmB and lgmC. The RT-PCR results are shown in Fig. 3.2B. The data shows that a PCR product of the expected size can be generated from a cDNA pool synthesized from gene-specific RT-primers of lgmB and lgmE, strongly suggesting that both sense and antisense transcripts are produced at the same time. 111 112 Figure 3.2 Mapped reads counts of lgm locus transcripts and presence of sense and antisense transcripts. A) Read counts of lgm locus transcripts as reported from RNA-Seq analysis of wild type, BPrncD45A, and BPrneΔCT. Traces are representative of the mean read count from 5 biological replicates at a given genome coordinate. Black arrows indicate troughs, areas of low or 0 reads. Red arrows indicate peaks, regions with a spike in read count. A schematic of lgm locus genes is shown below the x-axis indicating the position of each open reading frame. Purple arrows adjacent to lgmE and lgmA are indicative of putative promoters elucidated from in silico promoter prediction. B). RT-PCR showing the nested PCR product of the lgmB and lgmC intergenic region (lgmB-C). Antisense and sense transcripts were derived from gene-specific primers for lgmE and lgmB respectively. 113 3.4.3 LgmA promoter is responsive to Bvg phase, iron, and divalent cation availability Using the mapped reads of the lgm locus, several other peaks suggested the presence of promoters in proximity to lgm locus genes. Regions spanning approximately 500 and 300 bp upstream of lgmA and lgmE respectively, consisted of stable regions which could be representative of either a promoter or sequences protected by ribosome binding (Fig. 3.2A). These regions were then analyzed using the prokaryotic promoter prediction webserver, BacPP, to identify sigma factor binding sites (Table B.1) (221). This approach predicted several motifs likely bound by housekeeping and alternative sigma factors featured within 120 bp upstream of lgmA and lgmE (Fig. 3.3). This data was then validated using luciferase reporter assays to measure promoter activity of these upstream regions. Additionally, promoter strength under varying growth conditions were also examined. 114 Figure 3.3 regions upstream of lgmA and lgmE with predicted promoter elements. A) lgmA promoter region showing predicted binding motifs. BacPP predicted sigma factor binding region shows the probability of a given sigma factor to bind in this region. BvgA binding site was identified in Ref. (143), and a Fur binding region was estimated through alignments with the E. coli Fur consensus sequence. B) lgmE promoter region showing BacPP predicted sigma factor binding sites and probability of binding. 115 Regions 115 bp, 180 bp, and 425 bp upstream of lgmA were cloned into the luciferase reporter plasmid pIG04. This was to capture predicted promoter sequences and the peak identified from the mapped reads upstream of the lgm locus. This lux reporter is a suicide vector that incorporates into the B. pertussis genome at BP0772, allowing for measurement of ectopic promoter activity. The activity of each promoter fragment from wild type B. pertussis is shown in Fig. 3.4A. It was surprising to see that the increasing length of promoter sequence corresponded to increased luciferase activity since analysis by BacPP suggested no sigma factor binding sites outside of the first 200 bp upstream of lgmA (Table B.1). However, the small peak in the mapped reads around 400 bp upstream of lgmA may, in part, explain the observed activity (Fig. 3.2A). Conditions which resulted in varied promoter activity were then determined. Modulating B. pertussis into the Bvg- phase by growing cells in the presence of MgSO4 (50 mM) decreased lgmA promoter (plgmA) activity (Fig. 3.4A), corroborating previous findings that the lgm locus is BvgA regulated. Cells were then grown in media deficient of divalent cations, as this condition is commonly found to be involved in activating lipid A modifications. Interestingly, these growth conditions corresponded to a reduction in activity from each plgmA reporter. Additionally, growing cells in low iron, mimicking a nutrient limited environment, provided a strong induction in plgmA activity (Fig. 3.4A). To confirm these observations, use of a second reporter strain was used to verify lgm locus activation. The BP338 derived mutant strain, BPM2859 consists of a Tn5 lac insertion within lgmB (217). Using this construct, in comparison to some well-characterized vags, a similar pattern of β-galactosidase activity was observed under modulating and low iron conditions (Fig. 3.4B). Taken together, this confirms previous observations that the lgm locus is regulated by Bvg mode but also suggests an iron-116 responsive element may be involved in promoter activity. As a Fur binding motif for B. pertussis has not been well characterized, the E. coli Fur binding consensus sequence was aligned to the 425 bp sequence upstream of lgmA. This identified a sequence with ~50% similarity to the E. coli Fur binding motif, which overlaps with a predicted high-affinity BvgA binding motif (Fig. 3.3A) (143) implying an interesting arrangement of transcription factor binding at this promoter. 117 118 Figure 3.4 lgm locus reporter assays A) Schematic of lgm locus regions captured for reporter assays. The lgmA promoter fragments (-115, -180, and -425 bp) are encompassed by the orange box, and the lgmE promoter fragment (-900 bp) is encompassed by the blue box. Tn5lac insertion point in strain BPM2859 is indicated by yellow arrow B)Luciferase activity of lgmA and lgmE (pQC1974-pLgmE900) promoters. Readings were taken of late-log phase cultures (44-48 hrs) from liquid cultures with the given change in media composition. Error bars show the standard deviation of four technical replicates. Graphed data are representative of three experiments. C) β-galactosidase activity of Tn5 lac mutant under varying growth conditions. Controls were performed from strains grown on BG-agar. Test conditions were performed in complete SSM with the changes in media composition stated below each set of bars. Dnt and ptx mutant strains were chosen for activity lesser than and greater than the lgmB tn5 lac mutant as reported in Ref. (217). 119 3.4.4 Ectopic plgmE reporter shows differential activation signals when compared to in situ plgmE reporter In silico approaches predicted the lgmE promoter to be located 117 bp upstream of the lgmE start codon, within the annotated open reading frame of lgmC. Initial attempts to identify promoter activity from this region by cloning fragments up to 330 bp in length into pIG04, did not provide any measurable lux activity. To address this, a in situ lux reporter, pQC1794, was used. For this approach, a ~900 bp region upstream of lgmE was cloned into this suicide vector, to provide a homologous sequence to guide the incorporation of the lux operon into the lgm locus. This construct allowed for the direct measure of lgmE promoter activity under varying conditions. This strain was then grown under the same conditions as the plgmA reporter strains, where it was shown that the plgmE promoter was of similar strength to the plgmA-115 reporter, but notably less active than the longer plgmA reporters (Fig. 3.4A). Additionally, the plgmA-115 promoter saw a 6.8-fold increase in luciferase activity under iron-limited conditions yet the lgmE promoter experienced a 1.5-fold increase, implying a difference in responsiveness under different growth conditions (Fig. 3.4A). Interestingly, taking this promoter fragment and cloning it into pIG04, which inserts into the chromosome at a different locus, luciferase activity of similar intensity to the in situ reporter was also seen, suggesting the promoter may be some distance upstream of lgmE. Additionally, this ectopic reporter for lgmE did appear to be down regulated by treatments with MgSO4 whereas the in situ reporter was not (Fig. 3.5), suggesting promoter location relative to the sense lgm transcript may be important for lgmE regulation. 120 Figure 3.5 Comparison of in situ and ectopic luciferase reporters of the lgmE upstream region. The same ~900 bp fragment was cloned into both pQC1794 (in situ) and pIG04 (ectopic) reporters. Luciferase activity was measured at 44 - 48 hrs after growth in complete SSM with the given change in media composition. Graphed data are representative of three experiments and error bars represent the standard deviation of three technical replicates. 121 3.4.5 Low pH and CO2 % induce reciprocal activation of plgmA and plgmE. Induction of the lgmE promoter was observed, yet this was modest compared to that of the lgmA promoter. As lipid A modification systems are often activated when bacterial cells are under stress, conditions simulating the host environment were investigated to assess the impact on lgm locus activation. The human respiratory tract has been assessed to range between pH 6.8 and 7.0 when healthy, yet this is variable during infection (223). As a respiratory pathogen, B. pertussis has to adapt to these lower pH conditions in the respiratory tract while also acclimatizing to increased CO2 % (56). Given this, the growth of wild type B. pertussis was assessed at a range of pH values. As shown in Fig. 3.6A adjustments to the pH of the growth media from the standard pH 7.6 showed that B. pertussis growth is permissive within a relatively narrow range (pH 6.9 - 8.3). When comparing the wild type to the ΔlgmA-D (full lgm locus deletion) an increased impairment of growth was observed at pH 7.0 (Fig. 3.6B). Since the differences in growth rate between the two strains became most divergent between 24 and 48 hours, the lgm locus may play a role in promoting growth at low pH in a growth phase-dependent manner. Given this, the activity of lgmA and lgmE promoters was assessed under low pH and high CO2 % conditions. Also, as the activation of these diametrically opposed promoters may be coordinated by growth phase, the activation of each promoter was measured at an early log phase time point (24 hrs) and again at the standard, late log-phase time point (48 hrs). Under standard growth conditions at 24 hrs, the in situ plgmE reporter strain showed greater activation than the plgmA425 reporter (Fig. 3.6C). However, this diminished by 48 hrs (Fig. 3.6D) to near the previously observed activation levels at this time point (Fig. 3.4A). Furthermore, as pH is lowered either through manual adjustment of the growth media or growth 122 in 5% CO2, a strong induction of plgmA was observed which simultaneously corresponded to a reduction in plgmE activity (Fig. 3.6C), indicating an inverse relationship of their activation. This relationship is not only growth phase-dependent but also responsive to pH and CO2 concentration. Given that the ectopic lgmE reporter (pIG04-plgmE-900) showed a similar activation pattern as the in situ reporter, this implies that environment responsive transcription factors may be involved in coordinating this reciprocal activation. As no transcription factor binding motifs were found upstream of lgmE, the elements involved, and the biological relevance of this promoter dynamic are yet to be elucidated 123 Figure 3.6 Impact of pH and % CO2 on B. pertussis growth and lgm locus activation. A) Growth curve of B. pertussis wild type grown at pH 6.6 - 8.3, reported values found in the human respiratory tract (223). No growth is observed at pH 6.6. Plotted data are representative of three separate experiments. B) Growth rate comparison of wild type and BP338ΔlgmA-D strains in standard, complete SSM at pH 7.6 (dotted lines) and SSM adjusted to pH 7.0 (solid lines) Plotted data are representative of four separate experiments. C) Luciferase activity from reporter strains plgmA and plgmE at pH 7.0 and 5% CO2 at early log phase (24 hrs) and D) late log phase (48 hrs). The same reporter strains were grown in complete SSM (pH7.6) at the same time. Error bars are representative of the standard deviation of 4 technical replicates. Plotted data are representative of two separate experiments. 124 3.4.6 Deletion of lgmE impacts TLR4 activation without loss of glucosamine modification Previous studies of the lgm locus made single gene deletions within the locus to determine the impact on the glucosamine modification of lipid A in B. pertussis. This work showed that lgmABC is the minimum machinery required for the modification of lipid A (117). The antisense ORF, lgmE, was not originally annotated in the B. pertussis genome and was discovered through alignments with other Bordetella spp. (224) and recently sequenced B. pertussis outbreak strains. Taking this into account, previous deletions of lgmB and lgmC overlapped with the coding region for lgmE. As lgmE promoter activity suggests a potential role in the regulation of the lgm locus, new deletions strains of lgmB and lgmC were generated to confirm whether previously observed phenotypes were not due to “double deletions” with lgmE (i.e ΔlgmBE and ΔlgmCE). Schematics of the new lgm locus mutants are shown in Fig. 3.7A. The new lgmB deletion strain, lgmBΔ6-520, was designed to leave at least a six amino acid “margins” off the 3’ end of lgmA and lgmE. The lgmC mutation, lgmCΔ110-367, aimed to delete conserved catalytic residues (145) while leaving the putative lgmE promoter sequence intact. Amongst this cohort of lgm locus mutations, a deletion of lgmE consisting of the 188bp between lgmB and lgmC which did not overlap with either coding sequence was also constructed. Fig. 3.7B shows that both lgmBΔ6-520 and lgmCΔ110-367 presented a significant reduction in TLR4 activation compared to wild type. This is further supported by mass spectrometry analysis of lipid A, which showed the absence of the glucosamine modification (Fig. 3.7D). The lgmE mutation was also associated with a significant reduction in TLR4 activation (Fig. 3.7B). Analysis of lipid A structures by mass spectrometry determined the presence of a small peak corresponding to the correct size of the glucosamine modification. This method of mass 125 spectrometry analysis is not quantitative, suggesting that even though small, this peak connotes the presence of the lipid A modification within a population of these cells. As lgmE overlaps with the intergenic region between lgmB and lgmC, the transcript itself may be involved in regulation or transcript stability. To elucidate if the lgmE deletion led to changes in transcript abundance, the relative expression of lgm locus genes was assessed by qPCR. Fig. 3.7C shows the fold changes of lgm locus genes in the ΔlgmE mutant compared to wild type. Here, a substantial reduction in lgmE corresponding to the gene deletion was observed, as well as a modest reduction (2-fold) in lgmB transcript abundance. All other lgm locus genes were unaffected by the deletion of lgmE (Fig. 3.7C). The work in this section aimed to identify if clean deletion of lgmB and lgmC had unintended polar effects via interference on the previously unannotated lgmE ORF. As shown, newly-designed, markerless deletions of lgmB and lgmC phenocopy the previous deletion mutants which overlapped with lgmE validating the requirement of lgmB and lgmC on the glucosamine modification. (145). As the deletion of lgmE also had a significant impact on TLR4 activation (Fig. 3.7B), lgmE itself may play a role in the glucosamine modification of B. pertussis lipid A, potentially though influencing the transcript stability of lgmB. 126 127 Figure 3.7 Assessment of new lgm locus mutants on glucosamine modification of B. pertussis lipid A A) Schematic of lgm locus mutants developed to determine the role of the lgmE ORF. B) hTLR4 activation by lgmBΔ6-520, lgmCΔ110-367, and ΔlgmE strains compared to wild type and BP388ΔlgmA-D. Data are representative of four separate experiments: four replicates per experiment. Strains were compared to the wild type strain using a one-way ANOVA with Bonferroni post-hoc test, p-value- < 0.0001 (****). C) qPCR of ΔlgmE strain compared to wild type BP338. Fold change is presented on a Log2 scale. Grey dotted line is equal to a 2-fold reduction compared to wild type. Error bars are the standard deviation of two technical replicates. Plotted data is representative of two separate experiments. D) Mass spectra of MALDI-ToF analysis of lgmBΔ6-520, lgmCΔ110-367, and ΔlgmE strains. Major peaks are labeled with numbers and GlcN modification is indicated by black the arrow. 128 3.4.7 Co-translation of LgmAB is required to complement lgmB mutation LgmABC has previously been shown to be the minimum machinery required for the modification of B. pertussis lipid A (117). Recent mutational analysis of LgmA and LgmC identified putative active site residues required for glycosyltransferase and deacetylase activity respectively, however, a similar analysis of lgmB was not performed (145). LgmB is a GT83 family glycosyltransferase with structural homology to ArnT where both LgmB and ArnT are involved in the glycosylation of lipid A with glucosamine and aminoarabinose, respectively. It had previously been observed that expression of lgmB alone was insufficient to complement the ΔlgmB mutant strains. Furthermore, it was shown that both lgmA and lgmB are required for the full restoration of the glucosamine modification to these ΔlgmB mutants (117). To investigate this observation and ascertain if this phenotype is associated with previous lgmB deletions overlapping with lgmE, several constructs were built in collaboration with Dr. Daniel Jun, a post-doc in the Fernandez lab at the University of British Columbia, to test the requirement for both lgmA and lgmB in the complementation of lgmBΔ6-520. Fig. 3.8A details schematics of the constructs used to complement the lgmBΔ6-520 mutant strain. Previous complementation attempts used the broad host range vector plasmid pMMB67HE (117). As B. pertussis encodes a β-lactamase, concerns had been raised over the concentration of ampicillin required to maintain selection on the plasmid and whether this was interfering with membrane integrity and lgm locus activation. To address this, the first construct was cloned into the mini-Tn7 transposon system for chromosomal integration at a defined location (225). This construct, Tn7LgmAB, contained the wild type copy of lgmAB containing an upstream region of approximately 500 bp shown to likely include the native promoter for lgmA (Fig. 3.4). Other 129 constructs utilized plasmid-mediated approaches consisting of lgmB alone (pB4-lgmB) and lgmA and lgmB in a non-overlapping orientation (pB4-lgmA+lgmB) (Fig3.8A). LgmA encodes an N-terminal catalytic domain with a C-terminal transmembrane (TM) domain containing two TM helices. As the predicted topology of lgmB lacks two TM helices when compared to the structure of ArnT (approximately 60 amino acids between L459 and L460) (Fig. 1.5C), it was hypothesized that the membrane domain of LgmA may be contributing to LgmB function. To test if these work in a complex, the last construct, pB4-lgmA285-endB, was built to capture and translate the transmembrane domain of lgmA, with the full-length lgmB downstream in its wild type orientation (Fig3.8A). TLR4 activation and lipid A analysis are shown in Fig. 3.8B and Fig. 3.8C. As previously observed, the construct with lgmB alone was insufficient to restore the glucosamine modification to B. pertussis lipid A. This was also the case for, pB4-lgmA285-endB, the construct encoding the C-terminal transmembrane helices of LgmA. Interestingly even though pB4-lgmA+lgmB, carrying lgmA and lgmB with their own ribosome binding sites did display increased TLR4 activation compared to the lgmBΔ6-520 strain, it was not returned to wild type level, nor sufficient to restore the glucosamine modification. Lastly, Tn7LgmAB was the only construct that recovered the glucosamine modification to the lipid A domain of the lgmBΔ6-520 mutant strain. In tandem with previous findings, the native orientation of lgmA and lgmB may be required for function. Additionally, this activity appears to be independent of markerless deletions of lgmB which also removed the 3’ end of lgmE. 130 Taken together, these results suggest there may be some dependency on the co-translation of LgmA and LgmB in the function of the lgm locus. This provides evidence that alongside transcriptional regulation within the lgm locus, there may be additional translation/post-translational control. As gene regulation in bacteria is indeed multifaceted, it is not surprising that the genetic organization within an operon can also be a tool in modulating gene expression. 131 132 Figure 3.8 Complementation of BP338lgmBΔ6-520 requires co-translation of LgmAB A) Schematic representation of plasmid constructs used to assess complementation of BP338lgmBΔ6-520. B) hTLR4 activation by lgmBΔ6-520, and test complementation constructs compared to wild type and BP388ΔlgmA-D. Data is representative of three experiments: four replicates per experiment. Strains were compared to the wild type strain using a one-way ANOVA with Bonferroni post-hoc test, p-value < 0.0001 (****). C) Mass spectra of MALDI-ToF analysis of lgmB and test complementation constructs. Major peaks are labeled with red numbers, and GlcN modification is indicated by the black arrow 133 3.5 Discussion The regulation of the lipid A modifications in Gram-negative pathogens is often complex and multifaceted allowing for rapid and fine-tune remodeling of the bacterial cell surface (112). The lgm locus in Bordetella spp. appears to not be an exception to this characteristic complexity. The glucosamine modification in B. pertussis lipid A is coordinated by genes found in the lgm locus. This is a five gene locus, formed of two overlapping pairs lgmAB and lgmCD, separated by a short intergenic region. The fifth ORF, lgmE, is found in this intergenic region and antisense to the other lgm locus genes (Fig. 1.5A). This operon structure is a rarer architecture, yet several examples have been identified in the genomes of bacteriophages, Gram-positive and Gram-negative bacteria (76). Operons typically link the activity of functionally-related genes in response to an environmental stimulus (226). Even though the function of lgmE is currently unknown (explored further in Chapter 4), TLR4 activation by the ΔlgmE strain was significantly reduced compared to wild type, indicating a functional role. Recently, a gene locus in S. aureus, involved in menaquinone biosynthesis was also shown to consist of a five gene operon with an antisense open reading frame overlapping an intergenic region on the sense strand (227). Saenz-Lahoya et al. (2019) define this as a “noncontiguous operon”, in which overlapping transcription is used as a mechanism of regulation. From this, it is proposed that the lgm locus forms a noncontiguous operon in which the genetic architecture is a tool to regulate outer membrane remodeling. As the glucosamine modification of lipid A in B. pertussis provides protection from, but also stimulates the innate immune system, the noncontiguous operon may function as a means to fine-tune the expression of the modification machinery. The data presented here provide insight into the transcriptional and post-transcriptional regulatory mechanisms integrated into modulating lgm locus activity. 134 3.5.1 Post-transcriptional regulation The ribonucleases RNase III and RNase E are intricately involved in the processing and activity of regulatory RNAs (73). Transcriptomic analysis of mutant strains of the nucleases identified the lgm locus as moderately downregulated (~2-fold) in the RNase III mutant but strongly upregulated (~6-fold) in the RNase E mutant (Table 3.2, Fig. 3.1A). To further investigate the impact of these mutations on outer membrane stability, the mutant strains were characterized by assessing TLR4 activation, sensitivity to antimicrobial peptides, and analyzing the lipid A structures by mass spectrometry. The data in Fig. 3.1 show that the RNase III and RNase E mutations significantly impacted TLR4 activation, yet these phenotypes were not due to the loss of the glucosamine sugar on lipid A. These data indicate that these ribonucleases are likely involved in regulating the timing or quantity of the glucosamine presented on the cell surface, potentially through post-transcriptional regulation of lgm locus transcript abundance. The post-transcriptional regulation of lipid A modification in Gram-negative pathogens has been well described in E. coli, S. typhimurium, and P. aeruginosa (97, 112). The sRNAs MgrR, MicA, and MicF are amongst a few characterized regulatory RNAs involved at various points during LPS biogenesis and remodeling (134, 139). These sRNAs serve as negative regulators of their target mRNAs which requires the RNA chaperone, Hfq, to catalyze interactions and the subsequent cleavage by RNase E. The strong increase in lgm locus transcript stability seen in the RNase E mutant implies that RNase E likely targets these mRNAs for degradation. Furthermore, given that the RNase E mutant lacks a full C-terminal domain, and that microarray analysis of a Δhfq B. pertussis strains have shown significant differences in lgm locus transcript abundance 135 compared to wild type (44), a putative sRNA may be involved in promoting degradation of lgm locus transcripts. The presence of an antiparallel ORF in the lgm locus, suggests these ribonucleases may be involved in resolving sense mRNA-asRNA interactions. Mapping read counts from RNA-Seq analysis of endonuclease mutants in B. pertussis identified an abundance of reads from the genomic coordinates of lgmE (Fig. 3.2A). This may be the result that both the lgmB-C intergenic region and lgmE are highly expressed and thus more abundant, or that this region has greater stability, potentially protected from nuclease degradation (200). Using strand-specific primers for cDNA synthesis, the transcription of lgmB and lgmE were shown to occur simultaneously (Fig. 3.2B), however, this method could not be used to accurately quantify transcript abundance. To address this, the mapped reads and in silico predictions of promoter elements were used to identify and assay the activity of promoters for lgmA and lgmE. Luciferase reporter assays (Fig. 3.4) showed low promoter activities from the lgmE promoter region compared to the lgmA promoter under the same growth conditions and timepoint. In this context, the abundance of reads mapped to the lgmB-C intergenic region consisted of a low proportion of lgmE transcripts. This suggests that the lgmE transcript may be contributing to the stability of the lgmB-C transcript, potentially by masking RNase E cleavage sites to prevent degradation, or through recruitment of RNase III (74). Sense mRNA-asRNA complexes are often processed by RNase III, due to its specific activity for double-stranded RNA (73). In E. coli, the asRNA, gadY, binds the sense strand transcript gadXW to promote RNase III mediated cleavage. The cleavage product of the sense strand is more stable than the uncleaved gadXW mRNA (75). Here, RNase III may be involved in a similar role as the reduced catalytic activity of the RNase III mutant 136 leads to a modest reduction in the abundance of lgmB and lgmC transcripts (Fig. 3.1A). As there is also a significant reduction in TLR4 activation in the RNase III mutant, processing of this RNA-RNA duplex may be an essential step to promote translation of the sense mRNA. Several additional factors could be involved during the post-transcriptional regulation of the lgm locus, including sRNAs to target RNase E, and potentially RNA polymerase interference from diametrically opposed promoters. The findings presented in this chapter demonstrate the activity of at least two endonucleases determine the stability of the lgm locus mRNA and reveal an added layer of complexity in the regulation of outer membrane modifications in B. pertussis. 3.5.2 Transcriptional regulation The lgm locus has previously been shown to be a gene operon activated by BvgA~P, yet still active in Bvg- mode locked mutants (143). This suggests that the lgm locus is likely activated and controlled by additional regulatory networks. To provide further context of the post-transcriptional regulation that was previously discussed in section 3.5.1 and offer a more comprehensive understanding of environmental signals involved, luciferase reporters were used to determine the impact of various growth conditions on the modulation and activation of the lgm locus transcription. Promoter regions in the lgm locus were estimated by using in silico promoter prediction and mapped lgm locus transcripts reads from RNA-Seq analysis of the ribonuclease mutants (Fig. 3.2A, Fig. 3.3.). This identified regions upstream of lgmA and lgmE which may contain promoters. These regions were cloned into luciferase reporters to determine the activity of each promoter region. For the region upstream of lgmA, three promoter fragments of increasing length were cloned. This captured the peak of mapped sequence reads upstream of 137 lgmA spanning approximately 500 bp, and isolated predicted promoter elements featured within the first 200bp. Assays of the lgmA luciferase reporter strains indicated that luciferase activity corresponds to the fragment length (Fig. 3.4A). This suggests that each fragment could potentially be capturing elements assisting with promoter activation. Furthermore, assays of plgmA reporter strains showed that growing cells on solid media, limiting divalent cation availability, and modulation of Bvg mode using MgSO4, reduced lgm locus activation. Limiting iron also led to a substantial increase in plgmA activity. These data suggest that integrated signal transduction plays a substantial role in lgm locus activation. Analysis of the ~430 bp upstream of lgmA identified binding sites for several sigma factors, including RpoE, RpoH, and RpoS, suggesting the global regulatory environment within B. pertussis directly influences lgm locus activation (Fig. 3.3A). Previously, a putative high-affinity BvgA binding site was identified upstream of lgmA (143), and here, a binding site with weak homology to the E.coli Fur motif (47.4%) was also identified, supporting the observations from the luciferase assays. Interestingly these two binding sites overlap. Overlapping transcription factor binding sites commonly occur in bacterial promoters, yet the reasons for this configuration are not well understood. It is postulated that this is an evolutionary mechanism allowing for efficient signal integration of different regulatory networks, where binding affinities are important factors determining promoter activity (228). Interestingly, lgmA was recently identified as part of the regulon of a two-component system required for maximal expression of genes while in the Bvg- mode (58), suggesting additional transcription factor binding motifs are likely present upstream of lgmA. Distinct promoter elements for plgmA are still being elucidated, but as lipid A modifications are an integral part of mediating host-pathogen interactions, this level of signal integration is likely associated with fine-tuning of LPS remodeling during infection. 138 For assessment of the lgmE promoter, an in situ luciferase reporter was integrated into the B. pertussis chromosome within the lgm locus. These assays showed that lgmE promoter activation was a fraction of that of the lgmA promoter (Fig. 3.4A). Furthermore, cells modulated into the Bvg- mode with 50 mM MgSO4 or limiting availability to iron and divalent cations drastically change lgmA promoter activity. A weak induction is observed under these conditions suggesting different signals are involved in activating the lgmE promoter. Similar in silico analysis of the lgmE promoter predicted that RpoH and FliA are the only sigma factors likely to bind within the cloned promoter region. Interestingly, the use of a lux reporter to measure the activity upstream of lgmE from an ectopic site did seem responsive to modulation to the Bvg- mode using MgSO4 (Fig. 3.5). This suggests that the lgmE promoter is potentially influenced by a different cohort of transcription factors to lgmA, and the location within the lgm locus may have an important role in the activity of this promoter. To investigate this relationship between the two promoters further, reporter strains were placed under conditions found in the human respiratory tract (high % CO2 and low pH) (223), and growth phase dependent features on lgm locus activation were evaluated (Figure 3.6A). During early log-phase (24 hrs), the plgmA reporter strains showed greater luciferase production when strains were grown at pH 7.0 and further activated at 5% CO2 when compared to control conditions (complete SSM, pH 7.6; Fig. 3.6C). At this same time point, conditions yielding an increase in plgmA activity correspond to a proportional decrease in plgmE activity. This impact on divergent promoters is also observed within the noncontiguous operon in S. aureus. By mutating promoter elements, increased abundance of the sense transcript resulted in a reduction in the antisense transcript and vice versa (227). It was proposed that RNA polymerase 139 interference is likely the explanation for this relationship. In contrast, evaluation of the ectopic lgmE reporter showed a similar response to growth conditions as the in situ lgmE reporter (though reduced in comparison), suggesting other transcription factors may be involved in this regulation. Of interest, a two-component system responsive to CO2 has been identified in B. pertussis. PlrRS has been shown to enhance the expression vags, such as CyaA and FhaB, at elevated CO2% (229, 230). In the context of promoter activities presented here, PlrRS may be involved in the activation of plgmA and the repression of plgmE. These data suggest an intriguing mechanism by which transcript production within the lgm locus is being mediated. This reciprocal promoter activity may act as a passive mechanism of control allowing for tightly regulated expression of modification machinery while adapting to outer membrane stress. At the late log-phase (48 hrs), plgmE activation was markedly lower than plgmA and was not impacted by the environmental growth conditions, as was seen at 24 hrs (Fig. 3.6D). The growth phase-dependency of plgmE may, in part, explain the patterns of activation seen in Fig. 3.4A, where modulating conditions and nutrient limitations also do not influence plgmE activity at the 48 hrs timepoint. Growth phase-dependent expression of regulatory RNA systems is regularly seen, even to the point of facilitating coordinating shifts between growth phases (85). This is also seen in the gadY antisense transcript which is highly expressed during stationary phase in E. coli (231). Overall, this may suggest that lgmE is expressed early in the B. pertussis growth phase to modulate lgm locus activity, followed by downregulation towards log and stationary phases. Currently, it is not known if lgmE has a positive or negative effect on lgm locus gene expression, or how this changes throughout different growth phases. 140 3.5.3 Genetic architecture of the lgm locus Overlapping ORFs are a common gene arrangement for bacterial operons, ensuring similar expression levels of related genes in response to environmental stimuli (232-235). The 3’ end of lgmA overlaps with the 5’ end of lgmB, forming a fusion of the start and stop codons (i.e., ATGA). Fig. 3.7 shows that deletions of lgmB result in an unmodified lipid A and a significant reduction in TLR4 activation. The introduction of a wild type copy of lgmB expressed on a plasmid was insufficient to restore the glucosamine modification (Fig. 3.8). Furthermore, lgmA and lgmB expressed on the same plasmid in a non-overlapping orientation or encoding the transmembrane domain of lgmA with the full-length lgmB were also unable to complement the loss of modification seen in the lgmB mutant. In fact, lgmA and lgmB in the native orientation, with overlapping ORFs, is necessary for function (Fig. 3.8). Taken together, it is likely that LgmA and LgmB need to be co-translated for proper function. The mechanism behind the translation overlapping ORFs is not well characterized in prokaryotes, however, it has been postulated that a “programmed shift in open reading frame” (236) allows for translational termination of the upstream gene but allows the same ribosome (or only the 30s subunit) to slip back in frame with the downstream gene to initiate its translation (237). Genes found this orientation may be part of a complex or have closely related functions. Thus, the coupled translation is likely to ensure the correct molar production of each protein involved (232, 238). Overall, it is proposed that LgmA and LgmB are translationally coupled to ensure functional levels of each protein in the glucosamine modification of lipid A. The co-translation may act as an additional regulatory mechanism, ensuring the glycosyltransferase activity of these proteins is closely linked temporally, allowing for rapid changes to outer membrane properties in response to environmental stimuli. 141 This chapter discusses the complex regulatory network involved in B. pertussis outer membrane modifications. The lgm locus, responsible for the glucosamine modification of lipid A, has been shown to be influenced at the transcriptional level by growth phase, nutrient limitation, and environmental pH. Additionally, RNase III and RNase E appear to play a role in post-transcriptional regulation through a potential asRNA mechanism involved as part of a noncontiguous operon. Finally, LgmA and LgmB appear to be co-translated, a requirement for correct functionality of the modification machinery and potential regulation molar levels of these gene products. The work presented here provides evidence that lipid A modification in B. pertussis is modulated at transcriptional and post-transcriptional levels and suggests potential translational regulation through the integration of signals from several different regulatory systems. Given that the outer membrane sits at the interface of host-pathogen interactions the data here provide insight into the mechanisms by which B. pertussis responds to the host environment. This multi-faceted approach to regulating and refining the lipid A modification machinery provides a means to balance the maintenance of cell surface integrity and host immune system modulation. Furthermore, it provides controls on the timing and abundance of modified LPS at the cell surface in order to promote colonization. 142 Chapter 4: Characterization of LgmE, a putative antisense protein involved in the regulation of lgm locus 4.1 Synopsis LgmE is located within the lgm locus (BP0399-BP0396) overlapping and antisense to the annotated ORFs for lgmB and lgmC. LgmE was not originally annotated in the B. pertussis Tohama I genome but was identified through alignments with other Bordetella spp. and genomes of more recent outbreak strains of B. pertussis. The lgmE ORF is 396 bp encoding a 131 amino acid protein predicted to be in the GtrA protein family. Proteins in this family are largely uncharacterized membrane proteins but appear to be involved in the modification of membrane components in various bacteria. Previous work has shown that lgmE transcripts are produced at the same time as other lgm locus genes, yet, whether the ORF encodes a functional protein is still to be determined. Using both in vitro and in silico tools we show that LgmE is a membrane-associated protein with some influence on the outer membrane properties in B. pertussis. Furthermore, bioinformatic analyses suggest several GtrA-like protein family members are either covalently linked to or interact with glycosyltransferases. Taking this into account, protein-protein interactions are modeled, suggesting a potential role of LgmE as a membrane-bound anchor for LgmB. Overall, the work in this chapter provides insight into the role of a putative dual function RNA involved in regulating lgm locus activity. 143 4.2 Introduction There are many mechanisms by which regulatory RNAs can modulate the activity of a target mRNA. Transcribed from the same genetic locus (cis-encoded) or a different genomic coordinate (trans-encoded), regulatory RNAs bind target mRNAs to modify the properties of these transcripts (73, 239). Target transcript stability can be influenced by promoting or mitigating ribonuclease cleavage or in modulating translation of the target mRNA through changing access to RBSs via manipulating RNA (73). Over the past three decades of study, regulatory RNAs have typically been regarded as non-coding entities, however, it has recently been observed that several may also encode functional proteins (240, 241). Primarily discovered with trans-encoded sRNAs, meta-analyses of bacterial transcriptomic data have identified that many annotated non-coding sRNAs in fact contained small ORFs (242). These small proteins are often associated with modulating the function of the protein produced from the target mRNA, providing an added level of control in bacterial gene regulation (240, 241). It has also been postulated that cis-encoded antisense RNAs may also have dual functions (243). Proteomic analyses of Pseudomonas putida and S. typhimurium have indicated that several previously annotated non-coding asRNAs contained ORFs embedded within the sequences, however, the function of these proteins is not known (244, 245). In the obligate human pathogen B. pertussis, a gene locus involved in lipid A modifications has been shown to contain an antisense ORF. The lgm locus consists of five genes, lgmA-E. It forms an operon of two overlapping pairs, lgmAB and lgmCD separated by 188 bp. The fifth open reading frame, lgmE is found in the intergenic region overlapping with, and in the opposite orientation to lgmB and lgmC (Fig. 1.5). LgmE itself was not originally annotated in B. pertussis 144 Tomaha I genome and was identified through alignments with other B. pertussis outbreak strains and Bordetella spp. (145). The lgmE ORF contains all the hallmark features of a translated protein. It is bound by a start codon and stop codon with an adjacent RBS upstream of the gene and is predicted to encode a 131 amino acid peptide approximately 14.4 kDa in size. This protein product is predicted to form four transmembrane helices with the N- and C-termini located within the cytoplasm. Furthermore, LgmE is related to the GtrA-like protein family. GtrA is a small (104 amino acid) inner membrane protein involved with O-antigen modification in Shigella flexneri bacteriophage X, where it is proposed to function as a bactoprenol linked translocase (151). Broadly speaking, GtrA-like proteins have not been well characterized, yet they are commonly associated with other enzymes and glycosyltransferases involved in the glycosylation of several bacterial moieties (152). A site-directed mutant introducing a premature stop codon to lgmE on an lgm locus complementation plasmid was shown to have no impact on the glycosylation of B. pertussis lipid A. As the protein product appeared to not be essential for the glucosamine modification, it was originally postulated that lgmE may operate only as a regulatory RNA (145). However, as shown in the previous chapter (Section 3.4.6), deletion of the 188 bp between lgmB and lgmC on the chromosome, where lgmE is located, was shown to significantly reduce TLR4 activation while having a modest impact on the abundance of the lgmB transcripts (Fig. 3.7). In contrast to previous findings, these data suggest that the protein product encoded by the lgmE ORF may be involved in the function of the lgm locus, as well as having a role as a regulatory RNA. 145 The work presented here aimed to determine if lgmE may act as a potential dual function asRNA, in which the encoded protein is involved in the function of the lgm locus. It is shown that the lgmE ORF encodes a genuine protein, which localizes with membranes when expressed in E. coli and B. pertussis. Also, overexpression of lgmE in B. pertussis significantly reduces TLR4 activation. Additionally, a putative function for LgmE is hypothesized through analysis of protein family members and modelling of structural properties. 146 4.3 Materials and Methods 4.3.1 Bacterial strains, plasmids, and growth conditions. All strains used in this study are detailed in Table 4.1. Lysogeny broth (LB; Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) or LB plus agar was used for the growth of E. coli strains. Bordetella strains were grown either on Bordet-Gengou (BG; BD DifcoTm) agar plates supplemented with 15% defibrinated sheep’s blood (Dalynn Biologicals) or in complete SS medium (SSM) (169) supplemented with 0.15% bovine serum albumin (BSA; Sigma) (170). Growth of bacterial strains was performed at 37°C unless otherwise stated. As needed, media were supplemented with nalidixic acid (30 µg/mL), gentamycin (15 µg/mL), and/or diaminopimelic acid (DAP) (250 µg/mL). 147 Table 4.1 Strain and primer list Bacterial Strain or Plasmid Description Reference/Source E. coli strains DH5α Molecular cloning strain RHO3 Conjugation strain; Kms Δasd ΔaphA, DAP auxotroph (171) BL21 (DE3) General purpose protein expression strain; B F– ompT gal dcm lon hsdSB (rB–mB–) λ (DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λs) (246) C41 (DE3) Derivative of BL21with improved membrane protein expression characteristics; F– ompT gal dcm hsdSB (rB- mB-) (DE3) (247) C43 (DE3) Derivative of BL21with improved membrane protein expression characteristics; F– ompT gal dcm hsdSB (rB- mB-) (DE3) (247) B. pertussis strains BP338 Wild-type B. pertussis Tohama-1 strain; Nalr (172) BP338lgmCΔ110-367 Deletion of BP0397 (lgmC) where deletion does not overlap with 5’ lgmE and maintains predicted promoter; Nalr BP338ΔlgmE Deletion of the 188 bp intergenic region between lgmB and lgmC where lgmE is located; Nalr BP338NhislgmE In frame insertion of 6×His tag and 3× glycine linker to N-terminal end of the chromosomal copy of lgmE Plasmids pXR1 pBAD24 derivative with N-terminal 6×His tag and S-tag and C-terminal 10×His tag sequences, Ampr (248) pXR1-LgmE pXR1 containing lgmE ORF in frame with N-terminal tag sequences (no C-terminal tag), Ampr This work pGEM-tetR10.oriApra TetR encoding gene expression system for use in Mycobacterium smegmatis; referred to as pT10 in text pIG10 Tetracycline inducible gene expression vector, derived from pT10 and optimized for use in B. pertussis. Incorporates into B. pertussis genome, Ampr Gmr This work PIG10-xr1LgmE pIG10 with lgmE ORF with N-terminal sequences excised from pXR1, Ampr Gmr This work pIG10-LgmEChis pIG10 with lgmE ORF in frame with C-terminal 6×His tag encoded on plasmid Ampr Gmr This work pIG10-sfGFP pIG10 containing superfolder green fluorescent protein (sfGFP), Ampr Gmr This work pIG-LgmEsfGFP In frame translational fusion of LgmE and sfGFP in pIG10 vector backbone without TetR sequences and associated promoters, Ampr Gmr This work pBBR2-LgmE Broad host range vector containing lgmE ORF, Kanr This work pB4 Broad host range vector derived from pBBR2pcpn, Kanr This work PB4-LgmC pB4 with wild type copy of LgmC, Kanr This work pB4-L63MLgmC pB4 with “truncated” lgmC with methionine start codon replacing 63rd amino acid, as seen in the BPD420 strain, Kanr This work pIG02-NhisLgmE Allelic exchange vector for insertion of N-terminal 6×His tag to chromosomal copy of LgmE, Gmr This work pBluescriptIIKS- Multifunctional cloning vector, Ampr Stratagene 148 Primers Sequence 5’-3’ Description LgmE_pXR1-fw aaaaaaGAATTCATGCGCGGCCTGCTGCGACAGATT For cloning of lgmE ORF into pXR1 LgmE_pXR1-rev aaaaaaGTCGACAGGGGCGTGTCTGTGACGGAACGC LgmE_N-his_RFfw2 TCAAGGGAGACACCTCTTCGACATGCATCATCAC CATCACCACGGTG For insertion of N-terminal 6×His sequence to the chromosomal copy of lgmE LgmE_N-his_Rfrev2 ATCTGTCGCAGCAGGCCGCGACCACCACCGTGG TGATGGTGA LgmE_KI_Fw aaaaCTCGAGCGCCGCGCCGTGGACCTGCGGCCAGC For cloning of flanking regions of lgmE start codon for N-terminal mutagenesis LgmE_KI_Rev aaaaGAATTCCTGCGGCCGGACGACACGCTGGTGGC sfGFP_fw aaaaaaGAATTCATGTCGAAGGGCGAGGAGCTCTTC For cloning of sfGFP into pIG10 sfGFP_rev aaaaaaGTCGACCTTATACAGCTCGTCCATCCCGTG lgmE-gfp_fw aaaaaaTCTAGAAATGCGGAACATTCCGGACCTTACC For generation of lgmEsfGFP fusion protein lgmE-gfp_rev aaaaaaATGCATAGGGGCGTGTCTGTGACGGAACGC TetR sys fw aaaaaaGAATTCAGACTCCCAGAGCCTATCTATCAC For amplification of TetR genes and promoters from pT10 TetR sys rev aaaaaaTCTAGAGTCATGAGGTCACCATGGTCATTA pCpnTetR_fw ATCCTCGTGGCATGCCAGAACTTCCCTATCAGTGAT AGAGTTGAACCTCCCTATCAGT For insertion of pCpn -35 and -10 promoter sequences in Pmyc1tetO pCpnTetR_rev CGCAAACGTCACTAGACTCCCAGATTATATCTATCA CTGATAGGGAGGTTCAACTCT 09tetR1_rf_fw CCCACCCGGGGTGGGCGAGTTTCTCTTGCGTGTGCT CGGTCGA For insertion of pTetR1 -35 and -10 promoter sequences in Pimyc 09tetR1_rf_rev CGTACACACGGGTACTCCCAGATTTTACTCGACCGA GCACACG 09tcpn_rf_fw CCCACCCGGGGTGGGCGAGTTTGAACTGCGTGTGCT CGGTCGA For insertion of pCpn -35 and -10 promoter sequences in Pimyc 09tcpn_rf_rev CGTACACACGGGTACTCCCAGATTATACTCGACCGA GCACACG tetRpromo_fw aaaaaaGAATTCAATGCCGACCAGGCACCACCGCAC For amplification of tetracycline responsive promoters into pIG04 tetRpromo_rev aaaaaaGTCGACCTAGACTCCCAGAGCCTATCTATC LgmE fw_SalI aaaaaaGTCGACATGCGCGGCCTGCTGCGACAGATT For cloning of lgmE into pBBR2pcpn or pIG10 with a C-terminal 6×His tag LgmE rev_BamHI aaaaaaGGATCCTCAAGGGGCGTGTCTGTGACGGAA LgmE rev2_BamHI aaaaaaGGATCCAGGGGCGTGTCTGTGACGGAACGC LgmBC-IRfw CTCACTTTCCGCCACCTTGCGGCA Used in qPCR of lgmE LgmBC-IRrev GAAGGCCAGCCCGATCAACACCAG rpoB_fw GCTGGGACCCGAGGAAAT Used in qPCR of RpoB, housekeeping gene rpoB_rev CGCCAATGTAGACGATGCC lgmAqpcrfw TACCTGGTGGCCGACTAT Used in qPCR of lgmA lgmAqpcrrev CAGCGAAATCAGGTTGATGC LgmBqPCRfw GCATGCCGTATTTCCACAAG Used in qPCR of lgmB LgmBqPCRrev ATTCGTTGACGCCGAACA LgmCqPCRfw GCATGTCGAAGAGGTGTCTC Used in qPCR of lgmC LgmCqPCRrev CATCGATGGCCTCGTTCAT LgmDqPCRfw TTCCTGCACGCGTTCTT Used in qPCR of lgmD LgmDqPCRrev ACGCCCATGTCGAACAG 149 4.3.2 Construction of pIG10 gene expression system. Tetracycline responsive promoter systems are classed as either Tet-On or Tet-Off in which the addition of tetracycline results in induction or silencing of the desired promoter. The plasmid pGEM-tetR10.oriApra (referred to as pT10 here) obtained from Dr. Jan Burian in the laboratory of Dr. Charles Thompson at the University of British Columbia, encodes a Tet-On, TetR gene driven by the constitutive mycobacterial promoter Pimyc (Fig. 4.1A) (249). Adjacent to the TetR promoter but in the opposite direction, the promoter Pmyc1tetO (250) drives the expression of the gene of interest. This promoter consists of two Tet operator (tetO) sites, upstream of the -35 and -10 motifs (Fig. 4.1A), to which the TetR protein binds to suppress gene expression in the absence of tetracycline. As promoters involved were optimized for use in mycobacteria, promoter regions were exchanged for promoters known to be active in B. pertussis. Additionally, a third tetO motif was inserted downstream and adjacent to the -10 motif to assist in suppressing promoter activity. The TetR system from pT10 was first amplified and subcloned into the small cloning vector pBluescriptIIKS-(pBSII). The 19 bp tetO consensus sequence (251) and alternate promoter sequences (Fig. 4.1A) were added using a modified overlap extension PCR protocol. Using rf-cloning.org (252), primers were designed so that they can self-anneal to produce the desired insert with complementary regions flanking the insertion site. In the first round of PCR, 1 µL of each primer 0.5 µM (approximately 5 ng) was added to a 20 µL reaction with Phusion polymerase. The thermal cycling program was as follows: after initial denaturing at 98°C for 30 seconds, samples go through 5 cycles of 98°C for 8 secs, 55°C for 20 secs and 72°C for 5 secs, and a final extension at 72°C for 30 secs. To each reaction, 50 ng of the target plasmid was then added. For the second round of PCR, a 2-step thermal cycling program was used in which after 150 denaturing at 98°C for 30 secs, samples cycle 15 rounds of 98°C for 8 secs and 72°C for 3 mins 30 secs, and a final extension of 72°C for 5 mins. Reactions were then treated with DpnI for at least 2 hrs at 37°C. Chemically competent DH5α were then transformed with 2 µL of each PCR reaction. Transformants were first screened by PCR for the presence of the desired plasmid and positive clones confirmed by sequencing. Promoters selected to express the tetR gene or drive the expression of the gene of interest included, pTetR1 (251) from the Tn10 transposon, and the promoter of a heat shock gene, pCpn from B. pertussis (253). To determine which combinations of promoters, provide the largest dynamic range of expression, desired inserts were then amplified, digested, and ligated into pIG04 to screen activity using luciferase reporter assays (Fig. 4.1). These reporter plasmids were conjugated into wild type BP338 and positive exconjugants were confirmed by PCR. Reporter assays were performed as previously described (Section 3.3.8) and increasing concentrations of anhydrotetracycline (aTc) were added to induce promoter activity. Fig. 4.1B shows the measured luciferase activity of some of the tested promoter combinations compared to the original pT10 sequences. The promoter combination with the largest fold-change of activity between uninduced (0 ng/ mL aTc) and maximally induced (100 ng/ mL aTc) was identified as v2.3 (Fig. 4.1A). This sequence was then amplified and ligated into the RFP reporter pQC2241 plasmid, in which the RFP gene was removed, to produce the plasmid pIG10. This plasmid inserts into the B. pertussis genome in single copy to allow for tightly controlled, tetracycline responsive gene expression. The protein expression capabilities of pIG10 were first confirmed using RFP before lgmE constructs were cloned into the vector. 151 Figure 4.1 Development of pIG10, tetracycline responsive gene expression system. A) Schematic of promoters and tet operator binding site combinations screened in testing the TetR system in B. pertussis. All three constructs shown use the B. pertussis Cpn10 promoter (pCpn) for the promoter driving the expression of the gene of interest, in this case, the luciferase operon of pIG04. TetR promoters and tetO motifs were added to sequences using overlap extension PCR. B) luciferase activity of TetR constructs from A) cloned into pIG04 and induced with 5-100 ng/ mL anhydrotetracycline (aTc). The original system, pT10, is shown as a comparison of dynamic range of activation in B. pertussis. Above each bar is the fold-induction compared to the uninduced (0 ng/mL aTc) sample. Error bars show the standard deviation of four technical replicates. Plotted data are representative of three experiments. 152 4.3.3 LgmE protein expression The lgmE open reading frame was cloned into pXR1, a derivative of pBAD24, at EcoRI and SalI sites. The resultant vector, pXR1-LgmE, was then transformed into electrocompetent E. coli strains BL21, C41, and C43. Single colonies were grown overnight at 37°C in LB with 150 µg/ mL ampicillin. Overnight cultures were used to inoculate 25 mL of terrific broth (24 g/ L yeast extract, 20 g/ L tryptone, 4 mL/ L glycerol, 0.017 M KH2PO4, 0.027 M K2HPO4) at a 1/100 dilution. Cultures were grown at 37°C at 200 rpm until an OD600nm of 0.8 had been reached (approximately 2.5 hrs). Each 25 mL culture was then split into 4 fresh culture tubes of 5 mL. Arabinose was then added to each set of 4 culture tubes to a final concentration of either 0.2%, 0.02%, 0.002%, or 0% to induce LgmE expression. Culture tubes were grown overnight at room temperature at 180 rpm. Cultures were then harvested by centrifugation and pellets stored at -20°C until ready for use. For B. pertussis, the pXR1-LgmE plasmid was digested at XbaI and XhoI sites to excise the lgmE open reading frame and N-terminal tag sequences. This was then inserted to the pIG10 multiple cloning site at NheI (compatible with XbaI sticky ends) and XhoI. This construct, pIG10-xr1LgmE, was then transformed into RHO3 for conjugation into both wild type BP338 and BP338ΔlgmE. Exconjugants were first screened by PCR to confirm the presence of the desired construct before being restreaked onto fresh BG with 30 µg/ mL nalidixic acid and 15 µg/ mL gentamycin. Strains were grown for 3 days at 37°C before being harvested by sterile swab into SS salts. This was then used to inoculate 5 mL complete SSM to a starting OD600nm of 0.01. To each culture, 100 ng/ mL of aTc was added to induce LgmE expression. Strains were grown for 153 40-44 hrs at 37°C at 200 rpm before being harvested by centrifugation. Cell pellets were stored at -20 until ready for use. 4.3.4 Generation of constructs to detect native expression of lgmE Insertion of the N-terminal 6×His tag sequence to the chromosomal copy of lgmE was completed using allelic exchange. For this, primers were designed to amplify a 1.5 kb region centered on the LgmE start codon (750bp up- and downstream). This fragment was ligated into the cloning vector pBSII for mutagenesis. A similar PCR-based approach for changing promoters in the development of pIG10 (Section 4.3.2) was used here to introduce a 6×His tag to the lgmE flanking regions in pBSII. Introduced sequences increased the lgmE open reading frame by 27 bp. Colonies were screened by PCR and reactions run on a 2% agarose gel to identify the band shift compared to the wild type PCR product. Successful clones were sequenced, and flanking regions amplified and ligated into pIG02. The allelic exchange protocol was then performed as previously described (Section 2.3.2). For detection of native LgmE expression from translational fusion constructs, codon-optimized superfolder green fluorescent protein (sfGFP) was first cloned into pIG10 at EcoRI and SalI restriction sites. The vector backbone was then amplified omitting sequences encoding the tetR gene and associated promoters. This also introduced three bases, CAT, downstream of the ATG start codon to form the NsiI restriction site. The lgmE ORF and the 900 bp upstream region previously identified to contain lgmE promoter activity (Fig. 3.5) were then amplified and ligated into the pIG10-sfGFP PCR product at XbaI and NsiI sites. The new plasmid pIG-LgmEsfGFP 154 was then transformed into RHO3 prior to conjugation into wild type BP338 and BP338ΔlgmE strains. 4.3.5 Membrane preparations for immunoblots For both E. coli and B. pertussis strains expressing LgmE, cell pellets were first thawed on ice then resuspended in 500 µL PBS pH 7.4. Cell suspensions were then lysed by sonication; this was performed in a biosafety cabinet for B. pertussis strains to contain aerosols. Cell debris was separated by centrifugation at 15,000×g for 10 mins at 4°C and supernatants transferred to ultracentrifugation microtubes. PBS was used to adjust volumes and balance samples. Supernatants were then pelleted by ultracentrifugation at 60,000×g for 30 mins. Supernatants (soluble fraction) were transferred to new tubes and membranes resuspended in 100 µL PBS (membrane fraction). The pelleted cell debris (insoluble fraction) was resuspended in 100 µL 1% SDS and incubated overnight at room temperature. 4.3.6 Immunoblots Strains carrying chromosomally encoded NhisLgmE and LgmEsfGFP translational fusion constructs were first grown on BG plates with appropriate antibiotics for >72 hrs at 37°C. Strains were then harvested into SS-salts and used to inoculate 5 mL complete SSM to a starting OD600nm of 0.01. Cells were then grown for either 24 hrs or 48 hrs. For each time point, 1 mL of culture was harvested by centrifugation, and cell pellets resuspended to an OD600nm of 5.0 in SS salts, and an equal volume of 2× sample buffer was added. For samples obtained from membrane preps of LgmE-overexpression strains, an appropriate volume of 5× SDS sample buffer was 155 added to each sample. All samples were boiled for 5 mins and briefly centrifuged prior to loading onto 12% SDS-polyacrylamide gels and transferring to PVDF membranes. The presence of 6×His tags was detected using a polyclonal anti 6×His tag primary antibody (Santa Cruz Biotechnology) and an HRP-conjugated secondary antibody. Chemiluminescence was detected using the MF-ChemiBIS bioimaging system (DNR) and analyzed in ImageJ. 4.3.7 TLR4-NFκB activation assays Strains assayed for complementation of the lgmCΔ110-367 mutant were grown on BG plates for 3 days, before being harvested into SS-salts, and used to inoculate complete SSM to a starting OD600nm of 0.01. Cultures were grown at 37°C at 200 rpm for 40-44 hrs before being harvested by centrifugation. Cell pellets were resuspended in PBS pH 7.4 to an OD600nm of 5.0 and heat killed at 56°C for 1 hr. For complementation, ΔlgmE and strains overexpressing lgmE were similarly cultured, and heat killed. The maintenance and growth of HEK-Blue cell lines and subsequent hTLR4 activation assays by heat-killed bacterial samples were performed as described in sections 3.3.4 and 3.3.5. 4.3.8 Bright-field and fluorescence microscopy For bright-field microscopy, strains carrying translational fusions were taken at desired time points and 1-2 µL pipetted onto 1.0% agarose pads and overlaid with a coverslip. Cells were imaged with a Nikon TE 2000-U microscope equipped with an argon-ion laser (EXFO X-Cite), a 100× oil immersion objective, and a Hamamatsu charge-coupled device (CCD) camera controlled by NIS Elements (Nikon, Melville, NY). Fluorescence of sfGFP expressing bacteria was then detected using an excitation filter for GFP at 485 nm and emission at 520 nm. All images were then analyzed and processed in ImageJ (183). 156 4.3.9 Protein structure and interaction modelling Protein structure predictions of LgmB and LgmE were generated using the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) webserver (254). The highest confidence model predictions were then analyzed using PyMol (Schrödinger), and chain identities designated. Models were then uploaded to PyDock (255), for structural modeling of rigid body protein-protein interactions. The 100 lowest energy interaction models are provided as .pdb files. These were manually screened in PyMol to determine the conformation of protein interactions which may be feasible given predicted locations of N- and C-terminal ends. 157 4.4 Results 4.4.1 Recombinant expression of the antisense open reading frame, lgmE Even though the lgmE ORF is predicted to encode a 131 amino acid membrane protein within the GtrA-like protein family, it was still uncertain if this previously unannotated ORF was truly protein coding. To address this, the lgmE coding sequence was cloned into the pBAD24 derived vector, pXR1 (248) to test if expression in E. coli produced a membrane associated protein. This construct, pXR1-LgmE, consists of lgmE with an N-terminal 6×His tag, thrombin cleavage site, and S-tag, and has a predicted mass of 20.7 kDa. This construct was transformed into the E. coli strains BL21, C41, and C43 for expression trials. The strains C41 and C43 carry mutations that assist in the expression of difficult templates which are either toxic to the bacteria or otherwise impair cell growth, such as the overexpression of membrane proteins (247). Construct carrying strains were grown to mid-log phase in terrific broth at 37°C and induced with 0.2%, 0.02%, and 0.002% final concentrations of arabinose, overnight, at room temperature. Cell cultures were harvested, lysed by sonication, and cell debris pelleted. Cell lysates were then spun at 60,000×g to separate cell membranes. Each fraction was then analyzed by immunoblot to determine the presence of the LgmE construct carrying the N-terminal 6×His tag. Fig. 4.2 shows immunoblots of the crude membrane fraction from arabinose-induced strains. All three E. coli strains were shown to successfully express LgmE from pXR1 as shown by a band of the expected size. Most of the protein is found in the pellet, likely in inclusion bodies, with some protein detected from soluble fractions; however, this may be related to the efficiency of membranes being pelleted during centrifugation (Fig. C.1). Final OD600nm measurements suggest that expression tests did not impair the growth of either strain, however, this may not have been observed due to overnight induction (data not shown). Overall, recombinant expression in E. coli suggests that 158 the lgmE ORF could encode a bona fide protein that associates with the membrane. The overexpression of this protein did not appear to have an impact on the growth of these strains suggesting that the protein product may not be interfering with cellular processes within E. coli. This construct then was moved to B. pertussis to test expression within the native organism. 159 Figure 4.2 Immunoblot of LgmE expression in E. coli membrane fractions. The LgmE construct was expressed from pXR1 (pBAD24 derivative) in E. coli strains BL21, C41, and C43, and induced with arabinose to the final concentration indicated below each lane. LgmE band is indicated by the black arrow. 160 To investigate the expression in B. pertussis, a plasmid system was constructed to allow for the expression of the N-terminally tagged LgmE construct in B. pertussis. Several expression vectors for regulated recombinant gene expression are available for E. coli, but many of these genetic tools do not exist in B. pertussis. Several inducible promoter systems were tested, and a tetracycline responsive system was identified as being the most promising for regulated gene expression in Bordetella. The vector, pIG10 was assembled from a tetracycline-inducible gene expression system, pT10 built for Mycobacterium smegmatis (249), and the vector backbone of the RFP reporter pQC2241 (256) for use in B. pertussis. Promoters for TetR and recombinant gene expression were exchanged for the strong B. pertussis promoter pCpn (Fig. 4.1) (253). The plasmid, pIG10, inserts into the B. pertussis chromosome in single copy and this allowed for tightly controlled gene expression in B. pertussis using ng/mL concentrations of anhydrotetracycline (aTc) as an inducer (Fig. 4.1). The pXR1-LgmE was digested with XbaI and SalI and ligated into the vector pIG10. This fragment captured the pBAD ribosome binding site, N-terminal sequences including the 6×His tag, and the full lgmE sequence. The construct, pIG10-xr1LgmE, was then transformed into both wild type B. pertussis and the BP338ΔlgmE strain. Here, test strains were inoculated to a starting OD600nm of 0.01 and immediately induced using 100 ng/ mL aTc for 40~44 hrs at 37°C at 200 rpm. Similar to the expression trials in E. coli, cells were then lysed by sonication and insoluble, and soluble and membrane fractions were separated by centrifugation and ultracentrifugation. Immunoblotting for the 6×His tagged protein product of LgmE showed that a majority of the protein was found in the insoluble and membrane fractions (Fig. 4.3). Even though a strong signal was found by immunoblot analysis, there was no band indicative of LgmE protein expression visible by Coomassie staining on SDS-PAGE. 161 Additionally, over-expression of this membrane protein did not appear to impair the growth of either B. pertussis strain used here. Overall, the ectopic expression of the lgmE ORF produced a membrane-associated protein product. This provides further evidence that lgmE may be translated, where it could potentially play a role in the function of the lgm locus in B. pertussis. 162 Figure 4.3 Immunoblot of LgmE expression in B. pertussis. N-terminal 6×His tag was probed for in samples induced with 100 ng/ mL aTc for 40 - 44 hr. Membrane, soluble and insoluble fractions are indicated below lanes, and strain identification is shown above. LgmE band is indicated by the black arrow. 163 4.4.2 Alternative start codon for LgmC As mentioned previously, lgmE was originally identified through sequence alignments with recent outbreak strains of B. pertussis and other Bordetella spp. One observation from this is that recent annotations of lgmC also have an alternate start site compared to the original Tohama I annotation (175). This alternate start site would produce a seemingly truncated version of LgmC lacking the initial 63 amino acids. In addition to differences between other Bordetella species, alignments with YdjC-like protein homologues show that the N-terminal end of the Tohama I LgmC is substantially longer than other family members (Fig. 4.4A). Comparing the lgm locus sequences of B. pertussis D420 (257) with Tohama I, the start of lgmC and the lgmE homologue, annotated as GtrA, do not overlap. In fact, the 18 bp between each start codon also provides a potential RBS for each gene (Fig. 4.4B). This led to the hypothesis that a “truncated” form of lgmC may indeed be translated with no overlap of the lgmE and lgmC ORFs. If so, this would allow for subsequent genetic analysis of the 5’ end of lgmE while potentially diminishing polar effects on lgmC. In collaboration with Dr. Daniel Jun, a postdoctoral fellow in the Fernandez lab at UBC, plasmids were constructed to test if “truncated” lgmC is sufficient to complement the lgmCΔ110-367 mutant strain. These plasmids consisted of one construct with the wild type copy of lgmC from Tohama I (pB4-LgmC) and a second, “truncated” gene L63MLgmC which served as the annotated copy of lgmC in BP-D420 (pB4-L63MLgmC; Fig. 4.4B). As seen in Fig. 4.4, both these constructs were able to complement the lgmCΔ110-367 mutant in both TLR4 activation assays (Fig. 4.4C) and the restoration of the glucosamine modification on lipid A (Fig. 4.4D). Taken together these data suggest that the extended N-terminal end of the Tohama I LgmC may not be required for LgmC function and that the original sequence of lgmC may have been misannotated. 164 However, given that a putative promoter for lgmE still overlaps with lgmC there may be some level of interference since complementary transcripts are being produced. 165 166 Figure 4.4 Identification of alternative start for LgmC. A) Alignments of the N-terminal end of TohamaI LgmC, with the annotated starts of LgmC from BPD420, Bordetella avium 197N, and structural homologue TTHB029 from T. thermophilus HB8. B) Schematic of annotated gene arrangement of lgmC and lgmE in B. pertussis D420 and Tohama I strains. Constructs built to complement lgmCΔ110-367 were derived from the Tohama I lgmC and consist of full-length lgmC (green) and “truncated” L63MlgmC (red). C) hTLR4 activation by lgmCΔ110-367, complemented strains, wild type, and BP388ΔlgmA-D. Data are representative of three experiments: four technical replicates per experiment. Strains were compared to the wild type strain using a one-way ANOVA with Bonferroni post-hoc test, p-value < 0.0001 (****). D) Mass spectra of MALDI-ToF analysis of lgmCΔ110-367 and the strains tested in the TLR4 activation assay. Major peaks are labeled as red numbers, and GlcN modification is indicated by the black arrow 167 4.4.3 Native expression of LgmE in B. pertussis Recombinant expression in E. coli and B. pertussis suggests that the lgmE ORF encodes a membrane associated protein product. However, protein detection was achieved using strong, inducible promoters, a high-affinity RBS, and the ORF was expressed with a substantial N-terminal tag sequence. Given this, several constructs were built to determine if the native expression of lgmE can be detected in B. pertussis. With evidence that the 5’ end of lgmC does not appear to overlap with the 5’ end of lgmE, a 6×His tag was introduced to the chromosomal sequence of lgmE to identify native expression and translation of lgmE by immunoblot. The mutagenesis was designed to introduce an N-terminal 6×His tag and a short 3× glycine linker in frame with, and immediately after the native start codon. This tagged chromosomal construct was predicted to produce a protein of approximately 15.1 kDa in size. The tag was introduced via allelic exchange and confirmed by PCR and sequencing. Fig. 4.5 indicates that even though expression of the pIG10-xr1LgmE was detectable, the native expression of lgmE was not detected when immunoblotting for the 6×His tag present in this strain. 168 Figure 4.5 Immunoblot of N-terminal 6×His tag inserted into chromosomal copy of lgmE. Lane of BP338NhislgmE is highlighted by a blue box, where no detected LgmE expression is observed. Expression trial was run adjacent to pIG10-xr1LgmE (20.7 kDa, black arrow) and pIG10-LgmEChis (14.1 kDa, red arrow) as controls of expression and detection conditions. Addition of 100 ng/ mL aTc to induce pIG10 expression is indicated by + or - symbols. 169 An alternate approach was taken in which translational fusions were built to determine if the native promoter and RBS were sufficient to generate a protein product. This construct consisted of the region upstream of lgmE used for reporter assays (Fig. 3.4), the coding region of lgmE in frame with green fluorescent protein (GFP), and a C-terminal 6×His tag (Fig. 4.6A). The plasmid backbone was derived from pIG10 and thus incorporated into the chromosome at a defined location (BP0772) in single copy. Superfolder GFP (sfGFP) was used due to its ability to correctly fold and provide a strong fluorescent signal when expressed as part of a fusion protein (258). These translational fusion constructs were then transformed into wild type and the BP338ΔlgmE strain. Fig. 4.6 shows translational fusions grown in liquid media and imaged by fluorescence microscopy at early and late log phases. Weak fluorescence is observed in both strains at each time point suggesting low-level expression of the LgmEsfGFP fusion construct (Fig. 4.6C and Fig. 4.6D). This weak activity was further confirmed by immunoblotting for the C-terminal 6×His tag on sfGFP. Long exposure of the blotted membranes revealed faint bands corresponding to the expected size of the LgmEsfGFP fusion protein, yet this was only observed in the wild type strain (Fig. 4.6B). The lack of a linker between LgmE and sfGFP may have inhibited LgmE function or insertion into the inner membrane, contributing to the minor growth defect observed when growing the BP338ΔlgmE + LgmEsfGFP strain in liquid media. Furthermore, the absence of detectable protein expression by immunoblot may also be associated with this reduction in growth. 170 171 Figure 4.6 Assessment of native LgmE expression using translational fusions. A) Schematic of LgmEsfGFP translational fusion construct introduced into the pIG10 vector for detection of native expression. B) Immunoblot of C-terminal 6×His tag on sfGFP translational fusions with LgmE. The time point at which samples were taken and strain are indicated above each lane, and the band of fusion construct is indicated by the green arrow. C) Fluorescence microscopy of wild type and BP338ΔlgmE transformed with LgmEsfGFP fusion construct at 24 hrs and D) 48 hrs. Bright-field and fluorescence were captured using the “auto-exposure” feature. Identification of each strain is indicated on the left of each set of images. 172 4.4.4 Deletion and overexpression of lgmE suppress B. pertussis activation of hTLR4 Alongside the native expression of lgmE, other characteristic phenotypes were assessed. It has previously been shown that deletion of lgmE resulted in 2 phenotypes: A significant reduction in TLR4 activation (Fig. 3.7A) without the loss of the glucosamine modification (Fig. 3.7D), partnered with a 2-fold reduction in lgmB transcript abundance (Fig. 3.7C). Additionally, survival assays with antimicrobial peptides also showed that the deletion of lgmE does not impair polymyxin B resistance (data not shown). To investigate this further, several constructs were transformed into both wild type and ΔlgmE strains to determine conditions for complementation and to see if LgmE overexpression has an impact on the outer membrane properties of B. pertussis. Here, the pIG10-xr1LgmE plasmid was utilized again. Given that translational fusions indicated lgmE expression, a C-terminally tagged construct of lgmE was also cloned into pIG10 (pIG10-LgmEChis; Fig. 4.5) for induction with aTc. Additionally, as lgmE may also be acting as a regulatory RNA, a third plasmid construct was built, pBBR2-LgmE, which would provide constitutive ectopic expression of the lgmE transcript. Once transformed into B. pertussis, these constructs were grown to mid-log phase and heat killed for TLR4 activation assays. Fig. 4.6A shows that neither of the three constructs used was sufficient to restore TLR4 activation by BP338ΔlgmE strain to wild type levels. Additionally, overexpression of lgmE in wild type B. pertussis also resulted in a significant reduction of TLR4 activation. Given that lgmE may be functioning as a regulatory RNA, the impact on other lgm locus transcripts was assessed. RT-qPCR analysis of the lgmE overexpression strains showed an up to a 14-fold increase in lgmE 173 transcript abundance in these strains, yet gene expression of the other lgm locus genes appears to be unaffected (Fig. 4.6B-E). As shown here, both deletion and overexpression of lgmE resulted in a significant reduction in TLR4 activation, implying that lgmE may be a negative regulator of lgm locus function. Furthermore, ectopic expression of lgmE appeared to have no impact on the stability of lgmA-D transcripts. These results indicate that the LgmE protein is the determining factor of negative regulation, the position of lgmE within the lgm locus is required for any post-transcriptional regulation, or lgmE may be involved in the regulation of an alternate gene locus. 174 175 Figure 4.7 Deletion and overexpression of lgmE impact B. pertussis TLR4 activation without altering the transcript stability of other lgm locus genes. A) hTLR4 activation by wild type and BP388ΔlgmA-D compared to BP338ΔlgmE, and wild type and BP338ΔlgmE strains expressing lgmE. Data are representative of three experiments with four technical replicates per experiment. Strains were compared to the wild type strain using a one-way ANOVA with Bonferroni post-hoc test, p-value < 0.0001 (****). RT-qPCR analysis of lgm locus transcripts in B) ΔlgmE, C) wild type BP338+pBBR2-lgmE, D) wild type BP338+pIG10-LgmE, and E) wild type BP338+pIG10-xr1-LgmE, compared to wild type. Error bars represent the standard deviation of two technical replicates. Plotted qPCR data are representative of three experiments. 176 4.4.5 Structural modelling of LgmE As its function is still currently unknown, a series of in silico approaches were used to elucidate a potential role of LgmE. LgmE is predicted to be a GtrA-like family protein consisting of four transmembrane helices. Several family members have been suggested to be involved in flippase activity (151, 259, 260), yet, many appear to be closely linked to glycosyltransferase activity in bacterial systems (152). Through the Pfam database, GtrA-like proteins are found in 4800 species with a large number forming domains in other proteins. Given the large diversity of GtrA-like proteins, structural features of LgmE were modelled to determine a potential function. Fig. 4.8, shows a model of LgmE generated using the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) (254). This model was produced with 99.1% confidence (where confidence is calculated on true homology with the template) based on the membrane anchor of Dolichol-phosphate Mannose Synthase (DPMS) from Pyrococcus furiosus (261). The predicted structural model LgmE shows the arrangement of the four transmembrane helices with TM1 and TM2 more perpendicular to the membrane, with TM3 and TM4 rotated 60° where they are likely embedded within the membrane. Hydrophobic residues line the surface in regions likely to be in contact with the hydrophobic region of the inner membrane (Fig. 4.7C). Surface details of the LgmE model show that this arrangement forms a cavity between the two pairs of TM helices. DPMS is involved in protein glycosylation, catalyzing the transfer of mannose from GDP-mannose to dolichol-phosphate to form Dol-P-Mannose. Interestingly, the catalytic domain of DPMS is a homologue of LgmA and a member of the same glycosyltransferase (GT)-fold superfamily, GT-A (146). DPMS proteins are found in prokaryotes and higher organisms of which there are two main classes. In the first class, the C-terminal transmembrane domain is 177 covalently linked to the catalytic domain. In the second class, the transmembrane domain is encoded and translated separately but acts as an anchor bringing the GT domain to the membrane (262). Indeed, the transmembrane domain of DPMS from P. furiosus was shown to not be required for glycosyltransferase function (261), suggesting GtrA-like proteins, such as LgmE, may act as membrane anchors for glycosyltransferases in prokaryotes. 178 Figure 4.8 Structural modelling of LgmE. A) Phyre2 predicted model of LgmE based on homology to DPMS from P. furiosus. B) Rotated view of LgmE model showing arrangement and distributions of transmembrane helices. C) surface model showing hydrophobic residues in orange. Arrow indicates the cavity formed between pairs of transmembrane helices. Nt = N-terminus Ct= C-terminus, TM = Transmembrane helix. 179 Recently, a GtrA family protein from Mycobacterium tuberculosis was shown to be involved in the biosynthesis of the cell wall components (263). The protein, Rv3789, was shown to be required for arabinan biosynthesis. Protein interaction studies showed that Rv3789 was able to interact with itself and likely served as a membrane anchor for the arabinosyltransferase, AftA. AftA is a GT-C fold superfamily glycosyltransferase with structural homology to ArnT. As LgmB has structural and functional homology with ArnT and given the known interaction between GtrA and AftA in M. tuberculosis, it was hypothesized that protein-protein interactions may occur between LgmE and LgmB. The structure of LgmB was initially predicted using Phyre2. This prediction (Fig. 4.9) was based on the solved structure for ArnT from C. metalidurans (148) with 100% confidence (Fig. 1.5C). Potential interactions between LgmB and LgmE were then modelled using PyDOCK a webserver for the prediction of protein-protein interactions (255). This resolves for the lowest binding energies of 10, 000 interaction conformations. The top 100 (lowest energy) structures are then generated as .pdb files. The terminal ends of LgmE are predicted to be cytoplasmic, thus interaction models were manually screened using PyMol for conformations in which the orientation of LgmE terminal ends corresponded correctly with LgmB. This identified three potential models with 2 docking positions for LgmE. Fig. 4.9A-C shows the two interaction models. The first is the lowest energy solution of the two (ranked 11th), in which LgmE is aligned with juxta-membrane (JM) helix 2 of LgmB. As JM2 of ArnT is part of the first periplasmic loop this predicted interaction with LgmE may suggest how the protein is positioned into the membrane. The second model (Fig. 4.9D-F) shows LgmE interacting with JM3 of 180 LgmB. This JM helix is involved in positioning substrates within the active site of ArnT (148). The groove present in LgmE also aligns with this active site cavity suggesting this interaction with LgmE may be involved in substrate binding. Finally, both models show that helices TM2 and TM3 are most likely involved with the interaction with LgmB. 181 182 Figure 4.9 pyDock modelled interactions between predicted protein models of LgmB and LgmE. A) lowest energy model (rank 11) of LgmB (yellow) and LgmE (cyan) interaction in which LgmE terminal ends (labelled) are cytoplasmic facing relative to LgmB predicted model. The model shows transparent surface interaction with LgmB JM2 from the front and B) side views. C) surface of LgmBE interactions indicating the location of the LgmB active site cavity (purple arrow) and groove in LgmE (black arrow). D) Second (higher) energy model (rank 85) of LgmB (gold) and LgmE (blue) interactions with correct LgmE orientation. Models show interactions with LgmB JM3 from the side and E) rear views. F) surface model showing alignment of LgmB active site cavity (purple arrow) and LgmE groove (black arrow). 183 4.5 Discussion Lipid A modifications in the Gram-negative pathogen B. pertussis appear to involve several overlapping regulatory networks that fine-tune responses to the host environment. The lgm locus is required for the glucosamine modification of lipid A and consists of lgmA-D and the antisense ORF lgmE. lgmE was originally proposed to influence the regulation of the lgm locus at the post-transcriptional level. The work presented here provides evidence that lgmE may act as a dual function asRNA in which both the RNA and protein product of lgmE may be involved in the regulation of outer membrane remodeling. In E. coli and B. pertussis, overexpression of an N-terminally 6×His tagged LgmE construct resulted in a protein product detectable via immunoblotting (Fig. 4.2 and Fig. 4.3). Crude membrane preps showed that the majority of the LgmE protein product was found associated with the insoluble fraction, likely as inclusion bodies. However, LgmE expression was still abundantly associated with the membrane fraction. To conclusively determine membrane association, protein markers of the membrane fraction would also need to be detected. Nonetheless, the data here implies that the predicted ORF of lgmE is likely to encode a genuine, membrane-bound protein. Antisense proteins are defined as ORFs which are found overlapping at the 5’ or 3’ end in antisense to a gene on the opposite strand (243). The original identification of lgmE described an antisense overlap of the sense strand at both ends of lgmE, however, data shown here provided evidence that the 5’ ends of lgmC and lgmE may not be overlapping (Fig. 4.4) Given this, an N-184 terminal 6×His tag was inserted to the chromosomal sequence of lgmE to determine if native expression could be detected. Interestingly, this construct could not be identified via immunoblotting (Fig. 4.5). Here, the translation or correct folding of LgmE may have been interrupted due to the introduction of the tag sequence. Additionally, previously reported promoter activities for lgmE (Fig. 3.5) may also suggest that the chromosomally tagged constructs were below the limit of detection due to low transcription levels. As an alternate approach, translational fusions of LgmE and sfGFP were used to identify if the native promoter and RBS were sufficient to produce a detectable protein product. Indeed, at early and late log phases, fluorescence from LgmEsfGFP expressing strains could be observed. Also, immunoblots indicate that the LgmEsfGFP fusion protein was detectable at low levels in the wild type B. pertussis strain transformed with this construct (Fig. 4.6B). The BP338ΔlgmE strain did not show the presence of the translational fusion construct via immunoblotting, yet weak GFP fluorescence was observed from this strain suggesting a level of expression had been attained. The data here provides evidence that the lgmE promoter and RBS are sufficient to produce detectable protein, however, the degree of expression detected suggests that lgmE is both transcribed and translated at a low level. Cis-encoded asRNAs are typically transcribed at a low level (264), yet the ratio of transcription relative to the sense mRNA often changes across the growth phase or during stress (72, 231). As previously discussed (Section 3.5), the promoter activity of plgmE is increased earlier in the growth phase and diminishes towards late-log phase. This may also suggest that translation of LgmE may follow the same pattern and is enriched during early growth in order to modulate lgm locus activity. Alternatively, complementary RNA-RNA binding often is associated with changes to transcript stability of the sense mRNA while 185 mitigating translation of both ORFs (72, 264). Thus, this increased lgmE transcription early in the growth phase may provide a means to reduce translation of the sense strand through mRNA-asRNA interactions. The data here suggest that even though translation is likely scarce across the growth phase, the lgmE promoter, RBS, and ORF possess the capacity to produce a small membrane protein potentially associated with lgm locus function. To determine if lgmE plays a role in outer membrane modifications in B. pertussis, strains lacking, and overexpressing the lgmE ORF were assayed for their impact on TLR4 activation. Fig.4.6A shows that the loss of lgmE resulted in a significant reduction in TLR4 activation, yet this phenotype is intermediate to the deletion of the full lgm locus. Three constructs were designed to restore lgmE transcript or protein production to the ΔlgmE strain; however, the data indicate that neither of these constructs was sufficient to complement the lgmE deletion mutant. Furthermore, wild type B. pertussis strains expressing these lgmE constructs were also shown to have a significant reduction in TLR4 activation compared to the wild type strain without lacking an lgmE construct (Fig. 4.6A). Taken together, these data suggest that both lgmE overexpression and deletion have a significant negative impact on the glucosamine modification of B. pertussis lipid A. This reveals an interesting role for lgmE in the lipid A modification of B. pertussis, in which an intricate balance of lgmE transcript and protein abundance may be required for proper lgm function. Importantly, relative gene expression analysis showed that the wild type strains expressing the lgmE constructs presented up to a 14-fold increase in transcript abundance, yet no notable impact 186 on other lgm locus transcripts was observed (Fig. 4.6B-E). Ectopic expression of antisense transcripts is commonly used to determine the effects of a regulatory RNA (72). Interestingly, this appears to not be the case for the lgm locus, and the cis- orientation of the antisense transcript may be required for function. This has been observed in S. aureus within a locus structured as a non-contiguous operon, where ectopic overexpression of the antisense transcript did not replicate phenotypes seen when the promoter region of the asRNA was replaced with a strong constitutive promoter (227). This may suggest that RNA polymerase interference may be one of the mechanisms involved in the regulation of the lgm locus (76, 227). Evidently, the ectopic expression of this gene still had an impact on TLR4 activation suggesting that the protein product itself may be implicated in the regulation and function of the lipid A modification machinery. GtrA-like family proteins are widespread amongst bacterial species. Functionally speaking, not much is known about the role of these proteins within their various systems yet appear to be closely related to the function of glycosyltransferases involved in membrane biogenesis (152). Members of the GtrA-like protein family have been associated in interactions with both GT-A and GT-C fold superfamily glycosyltransferases of which LgmA and LgmB are members of respectively (146, 152). As seen in M. tuberculosis, a GtrA family protein was shown to interact with itself and the inner membrane protein AftA (263), a glycosyltransferase related to ArnT and LgmB. Using this information an interaction model was produced from the predicted structures of LgmE and LgmB. This model suggested that the TM2 and TM3 of LgmE would be involved in the interaction with JM-helices of LgmB, potentially involved in anchoring the protein to the 187 inner membrane. Given the limited knowledge of this protein family, this data fits the notion that GtrA-like proteins are closely linked to glycosyltransferase function. Indeed, this is an interaction model of predicted protein structures. The likelihood for uncertainty is high with such an approach, yet this process provides a basis for assigning a putative function for LgmE. As previously discussed, deletion and overexpression of LgmE significantly reduced TLR4 activation in B. pertussis. It’s an interesting notion that the absence and over-abundance of a gene product result in the same phenotype, and this has indeed been described with the chemotaxis protein CheW in E. coli (265). The overexpression of CheW prevents the proper assembly of the chemotaxis signaling complex by sequestering complex components, and the complex cannot form in the absence of CheW (266). A similar scenario may explain what was observed with an improper LgmE abundance, ultimately suggesting that LgmE could be involved with complex formation of lgm locus components. For example, in the absence of LgmE, lgm locus glycosyltransferases can indeed perform their functions, yet may require to be complexed with LgmE to efficiently glycosylate B. pertussis lipid A. This may in part explain the significant reduction in TLR4 activation while the glucosamine modification is still detectable via mass spectrometry analysis. Furthermore, overexpression promotes monomers of LgmE to interact while complexed with LgmB to form LgmBEn, where n is any number of LgmE monomers. In this circumstance LgmB is kept an improper distance from other lgm locus proteins, thus inhibiting efficient modification of lipid A. As mentioned previously with the co-translation of LgmA and LgmB, the correct molar quantities of lgm locus proteins appears to be important for function (Fig. 3.8) (233). Complex formation of glycosyltransferases is indeed seen 188 in LPS biosynthesis pathways, such as for lipid A translocation to the outer membrane (122, 267), and hypothesized to be required for O-antigen biosynthesis (268, 269). However, as far as I know, no such interactions to date have been proposed for lipid A modification systems such as lgm or arn. The interactions involved may only be transient and only be required at certain points during bacterial growth (270). Within B. pertussis, it is intuitive to infer that LgmABC function proximal to each other. As N-acetylglucosamine is an important molecule to many bacterial cell functions, such as cell wall and LPS biosynthesis, the lgm locus genes acting near each other may be required to ensure the glucosamine sugar is committed to modify lipid A before it is utilized in other pathways. Indeed, further work and biochemical characterization is required to determine the function and potential interactions of LgmE within B. pertussis. In any event, the model suggested here postulates a highly regulatory role of an antisense ORF involved in modulating the activity of neighboring genes potentially at both the post-transcriptional and post-translation levels. 189 Chapter 5: Conclusion 5.1 Overview The work in this dissertation highlights and expands upon the complex regulation of virulence factor expression in B. pertussis. The regulation of B. pertussis pathogenesis is already regarded as a complex and exquisitely controlled system integrating signals from several overlapping regulatory networks to establish infection within a host (13, 56). As regulatory RNAs play an important role in fine-tuning gene expression in many Gram-negative pathogens, mutations to ribonucleases involved in processing regulatory non-coding RNAs were generated to explore the post-transcriptional landscape in B. pertussis. RNA-Seq analysis of mutant strains of RNase III or RNase E in B. pertussis demonstrated differential expression in about ¼ of the B. pertussis transcriptome. Alongside defined impacts on ABC transporters associated with amino acid uptake, these endonuclease mutations were shown to drive the stability of several key virulence factor transcripts in opposite directions. As shown here, autotransporters vag8, tcfA, and brkA are prime examples of this where each gene was more abundant in the RNase III mutant, yet substantially reduced in the RNase E mutant. This suggests that virulence factor production is intricately modulated by RNase III and RNase E through mediating transcript stability or other post-transcriptional regulatory mechanisms which are yet to be elucidated. This work provides the first analysis of altered RNA degradation in B. pertussis, expanding the current understanding of complex regulatory networks in B. pertussis, and providing further insight into gene loci potentially influenced by regulatory RNAs. 190 In the RNase E mutant background, one of the most upregulated gene loci was identified as BP0399-BP0397, three genes found as part of lgm locus (117). The lgm locus is a five gene locus required to modify B. pertussis lipid A with glucosamine sugar providing increased resistance to cationic antimicrobial peptides and moderate increases to TLR4 activation (117, 145, 154). The locus contains an antisense ORF known as lgmE, which was recently identified from analyses of Bordetella genomes (145). As this one modification provides both activation of and protection from the human innate immune system the modification machinery is likely to be tightly controlled during infection to correctly modulate host responses. Complex regulation is often seen in LPS remodeling systems in Gram-negative pathogens. In this context, it was hypothesized that several overlapping regulatory networks are involved in lipid A modifications in B. pertussis, and lgmE may function as an asRNA that fine-tunes lgm locus expression. In terms of post-transcriptional regulation at this locus, the mutations to both RNase III and RNase E in B. pertussis significantly reduced and increased TLR4 activation respectively. Even with these changes in activation, analysis of lipid A structure by MALDI-ToF showed that the glucosamine sugar was still present suggesting these endonucleases may be involved in regulating the quantity of the glucosamine modification presented on the cell surface. As sense and antisense transcripts are produced from the lgm locus at the same time, the activity of these endonucleases may be dependent on the presence of lgmE transcripts. Mutations to RNase E substantially increased lgm locus transcript abundance where nuclease activity appears to be targeted in a C-terminal domain dependent manner. This suggests that the lgm locus is inherently susceptible to RNase E degradation, potentially involving sRNAs in directing this activity. However, given a substantial peak in mapped RNA-Seq reads centered around where lgmE is 191 present in the genome, lgmE transcripts may provide a means of antisense regulation by protecting the sense strand from degradation from RNase E. Additionally, since complementary RNAs are being produced, RNase III may be involved in processing RNA-RNA duplexes involving lgmABC and lgmE transcripts. As mutations to RNase III lead to a decrease in lgmB and lgmC transcript stability, RNase III activity may be involved in stabilizing the lgmB-C region. However, given the modest effect of these mutations, this may be a minor role in the regulation of the lgm locus. At the transcriptional level, several environmental conditions appeared to influence lgm locus activation. The lgm locus had previously been reported to be activated by BvgA~P (14, 143). This was confirmed by a reduction of both luciferase and β-galactosidase reporter strains grown under modulating conditions (50 mM MgSO4). Furthermore, iron limitation, divalent cation availability, pH, and CO2 % had a direct impact on the activity of the lgm locus. Given this, the lgm locus appears to show differential activation under varying environmental stressors. A motif with weak similarity to the E. coli Fur binding site upstream of lgmA does imply iron responsive regulation and a predicted heat-shock sigma factor (RpoH) binding site may indicate additional stress-responsive promoter elements. These reporter assays also suggest that a long 5’ untranslated region (UTR) may be present upstream of lgmA. Given that both ribonuclease mutations and deletion of hfq impact the lgm locus (44), an extended 5’UTR provides further indication that sRNA mediated regulation or even RNA secondary structure may act in this region to modulate lgm locus activation (63). Additionally, a reciprocal relationship of lgmA and lgmE promoter activation was observed when cells were grown at low pH or high CO2 %. That 192 is, conditions that increased lgmA promoter activity were paired with a reduction of lgmE promoter activity. This reciprocal activity has been identified as a mechanism of antisense regulation (73) and a feature of noncontiguous operons (227). Importantly this relationship was only observed during early-log phase (24 hr time point). Promoter activity of lgmE was substantially decreased by late log phase (48 hr time point). This suggests a growth phase dependency on the expression of lgmE, implying a role or potential requirement on lgmE expression during early B. pertussis growth. The lgm locus serves as a prime example of the complex gene regulation in B. pertussis. The work presented here indicates that the lgm locus is influenced by BvgAS mode, ribonuclease cleavage, nutrient availability, pH, atmospheric CO2 concentrations, and likely several other regulatory mechanisms. The lgm locus genes form a non-contiguous operon and provide a distinct example of a gene locus that integrates regulatory signals from transcriptional and post-transcriptional inputs. This multifaceted regulation is likely involved in fine-tuning remodeling of the B. pertussis outer membrane, ensuring the coordinated expression of the modification machinery with other virulence factors to best promote host colonization. Finally, as the lgmE ORF has all the hallmark characteristics of a translated protein, it was investigated if the lgmE ORF encodes a functional protein and acts as a dual-purpose asRNA involved in lgm locus function. A gene expression system was developed to overexpress the lgmE ORF in B. pertussis and showed that a protein of approximately the predicted size of the putative proteins encoded by lgmE associated with the membrane fraction. Furthermore, it was 193 shown that both deletion and overexpression of lgmE lead to a significant reduction in TLR4 activation when compared to wild type. It was speculated that an inappropriate amount of LgmE interferes with lgm locus function. A GtrA-like family protein from M. tuberculosis was recently identified to interact with itself, and the GT-C family glycosyltransferase AftA (263). Taking this into account, protein-protein interactions were modelled between the predicted structures of LgmE and LgmB. It was then proposed LgmE may serve as a membrane anchor for LgmB, in which stable or transient interactions modulate the activity of the glycosyltransferase. Furthermore, in the context of studies of the deletion and overexpression of lgmE, both the absence and overabundance of LgmE results in the improper assembly of a potential lipid A modification complex. Taken together, these data provide evidence that lgmE may be acting as a dual-function RNA. The development of genetic tools here allowed for the overexpression of lgmE indicating a potential function for the protein. Keeping in mind that co-translation of lgmA and lgmB is likely required for lipid A glycosylation, this suggests that production of the lgm locus proteins must be balanced, ensuring correct molar quantities of each gene product to promote lipid A modification. Overall, investigation of the transcriptional, post-transcriptional, and translational properties, the following model of lgm locus regulation is proposed in Fig. 5.1 where the model can be characterized by two main features: elements either promoting or inhibiting lipid A modification. 194 195 Figure 5.1 Proposed model of lgm locus regulation involving dual functions of lgmE. A schematic of the lgm locus is shown below each model. LgmABC transcripts are represented as orange to green lines, lgmE transcripts are represented as light blue lines. Red boxes indicate transcriptional regulation, purple boxes indicate post-transcriptional regulation, and cyan boxes indicated post-translational regulation. A) Features promoting GlcN modification of B. pertussis lipid A. The lgmA promoter is strongly activated during mid-log growth, under stress or other yet to be identified conditions (large orange-green arrow), and an abundance of lgmABC transcripts are produced and translated. At the same time, but to a much lesser degree, lgmE is transcribed and translated (small blue arrow). The LgmE protein product in this small quantity is sufficient as a membrane-bound anchor where it interacts with primarily LgmB, to bring the modification machinery within close proximity, allowing for efficient lipid A glycosylation. A subset of lgmABC and lgmE mRNAs interact to form a stable population of lgmABC-lgmE duplexes. This is readily cleaved by RNase III, allowing for rapid changes to outer membrane properties in response to stress. B) Features inhibiting GlcN modification of B. pertussis lipid A. During early growth, or yet to be identified conditions, the activity of the lgmE promoter (large blue arrow) is greater than the activity of the lgmA promoter (small orange-green arrow). To modulate lgm locus activity, lgmABC transcripts are readily cleaved by RNase E. This is dependent on the RNase E C-terminal domain and potentially involves the role of Hfq and a putative sRNA. Under these conditions, lgmE transcripts are translated, leading to an excess relative to lgmABC. Any aberrant expression resulting in dysregulated production of LgmABC results in LgmE binding, primarily to LgmB, where it prevents appropriate complex formation through subsequent interactions with itself. 196 This model is inferred from data collected in this thesis, what is currently known about non-contiguous operon function, and knowledge based on characterization of GtrA-like family proteins. The role of lgmE in this model is presented as having a substantial impact on the lgm locus; however, the importance of this dual function RNA during infection and how this regulation impacts B. pertussis pathogenesis is unknown. Regardless, the data presented in this dissertation provide an intriguing line of evidence that lgmE is a protein-encoding asRNA involved with fine-tuning outer membrane remodeling in B. pertussis through transcriptional, post-transcriptional, and post-translational mechanisms. 197 5.2 Limitations and future work The model proposed in this thesis was derived from promoter activation and relative gene expression studies of the lgm locus genes in wild type and endonuclease mutants of B. pertussis. To better determine the role of lgmE as an asRNA, RNA-RNA interaction studies should be performed in vitro and in vivo to confirm interaction and determine patterns (e.g. timing, relative amount) of nuclease cleavage. Furthermore, as transcriptional bounds of lgm locus transcripts were not identified in this body of work, assessment of the B. pertussis transcriptome by differential RNA-seq would most efficiently determine these transcriptional bounds. The putative role of LgmE as a membrane anchor to LgmB could be probed through pull-downs with epitope-tagged fusions of either of these proteins or using bacterial two-hybrid systems. Additionally, RNA-Seq analysis of the endonuclease mutants identified a remarkable impact on virulence factor transcript stability. Vag8 and tcfA are two autotransporters that are drastically differentially expressed in both RNase III and RNase E mutants (216) as well as in studies of Δhfq strains (44). Analysis of the B. pertussis primary transcriptome showed that the transcript for Vag8 has an extended 3’ UTR. This fits many paradigms of post-transcriptional regulation, in which an extended 3’ UTR is often a determinant of transcript stability and processed to give rise to sRNAs. With TcfA, even though this experienced a similar impact on transcript stability in the endonuclease mutant backgrounds, the primary transcriptome indicates a very concise mRNA structure, predominantly just featuring the TcfA ORF. As very little is known about post-transcriptional regulation, and the targets of sRNAs in B. pertussis, it would be opportune to use 198 the strains developed here to derive the mechanism by which these virulence factors are regulated. However, it should be noted that studies of the B. pertussis transcriptome and potential regulatory RNA processing by RNase III and RNase E performed here looked at only one time point. The role of regulatory RNAs is often growth phase or stress dependent, thus an expansion on these growth conditions and time points before transcriptomic analysis may shed further light on post-transcriptional regulation in B. pertussis. Furthermore, exoribonucleases have also been shown to be important factors in the post-transcriptional regulation of virulence factors in several bacterial pathogens. Targeting specific exonucleases, such as PNPase, would further expand the knowledge of ribonuclease-dependent regulation and how this also influences the B. pertussis transcriptome. The pIG10 plasmid built here shows promise as a gene expression system for B. pertussis as strong activation is seen at tetracycline concentrations 20-fold below the MIC. Tetracycline responsive elements are present within the B. pertussis genome (271), thus this should be considered when using the system. 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Schubert M, Edge RE, Lario P, Cook MA, Strynadka NCJ, Mackie GA, McIntosh LP. 2004. Structural Characterization of the RNase E S1 Domain and Identification of its Oligonucleotide-binding and Dimerization Interfaces. Journal of Molecular Biology 341:37-54. 228 Appendices Appendix A A.1 Validation of RNase E dependent phenotypes through the generation of the temperature sensitive mutant strain BPrneG66S To verify the RNA turnover phenotype observed in the rneΔCT strain, a second, viable RNase E mutant was generated. RNase E temperature-sensitive mutants are viable below 30°C, yet at temperatures greater than 35°C the S1 domain destabilizes and leads to a non-functional enzyme (272). To achieve this, a point mutation in the S1 domain of RNase E was introduced by allelic exchange. A 1.5 kb fragment centered around the rneGly66 codon was amplified with upstream and downstream fragments (~750 bp each) joined at a PvuI site. This involved generating two silent mutations at Arg75 and Ser76 codons to form the restriction site motif CGATCG. The reverse primer of the upstream fragment contained a single point mutation at Gly66 (G-A) to generate the temperature-sensitive strain BPrneG66S. This mutation was confirmed by PCR and sequencing. Preliminary experiments with BPrneG66S show a similar growth rate to wild type at 30°C but a substantial growth defect at 37°C (Fig. A.1A), indicative of the temperature-sensitive phenotype. Additionally, the expression of BrkA and TcfA deteriorates after a shift to 37°C in the BPrneG66S where it is increased in the wild type strain (Figure A.1B). This is a similar impact on protein expression to what is seen in the BPrneΔCT strain, suggesting the observed instability of these transcripts is dependent on fully functional RNase E. 229 Appendix figure A.1 Preliminary characterization of temperature sensitive RNase E mutant, BPrneG66S A) Comparison of growth rates of the wild type B. pertussis and BPrneG66S mutant at 30°C and 37°C. B) Immunoblots of BrkA (left) and TcfA (right) at 37°C in wild type and the BPrneG66S strain. Cultures were grown at 30°C for 48hrs before being shifted to 37°C for 4hrs with samples taken at the indicated time point. 230 A.2 Refined tables and lists associated to RNA-Seq analysis of endonuclease mutants BPrncD45A and BPrneΔCT. Full tables available from Ifill et al. (2021) (216) Appendix Table A.1 Well characterized virulence factors found to be differentially expressed in the endonuclease mutants Well characterized VAGs found to be DE in BPrncD45A Well characterized VAGs found to be DE in BPrneΔCT Locus tag Gene pvalue FC Locus tag Gene pvalue FC BP3795 ptlG 5.94E-18 -13.21 BP2315 vag8 1.85E-134 -48.22 BP3794 ptlF 2.25E-12 -13.15 BP1201 tcfA 9.26E-178 -27.69 BP3796 ptlH 5.10E-18 -13.15 BP2236 bscW 6.96E-14 -27.15 BP3793 ptlE 2.69E-11 -13.02 BP2239 bscS 5.02E-18 -15.35 BP3792 ptlI 8.75E-09 -9.56 BP2237 bscU 4.38E-19 -11.81 BP3791 ptlD 5.86E-16 -8.73 BP2258 BP2258 8.70E-11 -11.49 BP3790 ptlC 5.84E-09 -7.38 BP2246 bscL 5.62E-14 -11.26 BP3789 ptlB 1.13E-15 -4.98 BP2238 bscT 1.31E-17 -11.21 BP3788 ptlA 3.13E-11 -4.65 BP2250 bcr4 7.28E-15 -10.68 BP2236 bscW 3.54E-05 -4.49 BP2240 bscR 1.21E-18 -10.27 BP2235 bscC 7.04E-08 -4.22 BP2235 bscC 1.28E-17 -9.96 BP3787 ptxC 9.39E-09 -3.77 BP2249 bscI 3.62E-17 -9.35 BP2237 bscU 6.89E-06 -3.35 BP2253 bopD 1.08E-22 -8.96 BP2238 bscT 0.00019 -2.71 BP2256 bsp22 1.06E-20 -8.75 BP2245 bscN 0.00137 -2.61 BP2254 bcrH1 1.44E-17 -8.44 BP2239 bscS 0.00033 -2.54 BP2263 bscE 7.76E-22 -8.40 BP2263 bscE 3.46E-05 -2.49 BP2257 bopN 4.92E-13 -8.28 BP3786 ptxE 7.81E-07 -2.47 BP2245 bscN 6.07E-12 -8.15 BP1112 bipA 2.63E-10 -2.44 BP2255 BP2255 6.05E-21 -7.68 BP2240 bscR 0.00051 -2.42 BP2241 bscQ 8.40E-26 -7.42 BP2264 bscF 0.0002 -2.38 BP2251 bcrH2 7.10E-14 -7.32 BP2246 bscL 0.01556 -2.10 BP3792 ptlI 3.34E-07 -7.09 BP2667 fhaS 0.00125 -2.04 BP2247 bscK 1.33E-10 -6.88 BP3785 ptxD 0.00063 -1.96 BP2264 bscF 1.26E-15 -6.50 BP3784 ptxB 0.00367 -1.73 BP3793 ptlE 1.11E-06 -6.44 BP2247 bscK 0.05369 -1.72 BP3796 ptlH 3.58E-10 -6.24 BP1119 fim2 0.00281 -1.60 BP2244 bscO 1.13E-39 -6.14 BP2255 BP2255 0.02002 1.61 BP3794 ptlF 8.20E-07 -6.02 BP1882 fimC 1.57E-08 1.62 BP2259 BP2259 1.05E-08 -5.59 BP2738 bapC 0.00042 1.62 BP2248 bscJ 2.80E-11 -5.26 BP2256 bsp22 0.01504 1.75 BP2738 bapC 1.87E-33 -5.24 BP1881 fimB 8.43E-07 1.83 BP3795 ptlG 2.12E-08 -5.13 231 BP1878 bvgA 1.66E-07 1.88 BP2252 bopB 1.44E-12 -5.03 BP2674 fimX 1.24E-08 1.89 BP1119 fim2 1.33E-23 -4.84 BP1201 tcfA 1.23E-10 2.11 BP2243 bscP 2.45E-18 -4.75 BP3494 brkA 8.26E-16 2.39 BP2674 fimX 3.63E-35 -4.72 BP3494 brkA 8.26E-16 2.39 BP3791 ptlD 5.25E-09 -4.69 BP2315 vag8 1.65E-19 4.00 BP0856 bfrD 6.11E-121 -4.22 BP2261 bcrD 1.70E-18 -4.12 Not Significant BP3790 ptlC 3.77E-05 -4.11 BP2241 bscQ 0.10528 -1.34 BP3783 ptxA 7.07E-29 -3.95 BP2251 bcrH2 0.33167 -1.28 BP3787 ptxC 8.92E-09 -3.78 BP2259 BP2259 0.41175 -1.25 BP3494 brkA 2.83E-34 -3.74 BP0763 cyaE 0.11327 -1.25 BP3494 brkA 2.83E-34 -3.74 BP2248 bscJ 0.39489 -1.23 BP3789 ptlB 1.11E-10 -3.55 BP2257 bopN 0.57563 -1.17 BP3786 ptxE 1.03E-11 -3.46 BP0762 cyaD 0.08565 -1.16 BP2667 fhaS 4.36E-08 -3.36 BP2258 BP2258 0.66947 -1.13 BP3785 ptxD 1.07E-09 -3.31 BP2262 bscD 0.38043 -1.13 BP3788 ptlA 6.76E-07 -3.14 BP2250 bcr4 0.71764 -1.11 BP2907 fhaL 9.68E-34 -3.08 BP2243 bscP 0.60928 -1.09 BP2262 bscD 9.99E-16 -3.05 BP2249 bscI 0.72048 -1.09 BP0216 sphB1 1.53E-35 -2.74 BP0761 cyaB 0.56506 -1.06 BP1878 bvgA 2.31E-13 -2.42 BP1877 bvgS 0.90947 -1.01 BP0760 cyaA 6.66E-26 -2.40 BP2907 fhaL 0.9149 -1.01 BP1881 fimB 4.59E-12 -2.35 BP1884 fhaC 0.20221 1.09 BP0763 cyaE 1.92E-09 -2.33 BP2261 bcrD 0.59635 1.09 BP1883 fimD 4.20E-35 -2.29 BP2252 bopB 0.49891 1.16 BP0758 cyaC 7.62E-06 -2.26 BP1879 fhaB 0.07817 1.16 BP1877 bvgS 3.20E-14 -2.25 BP0856 bfrD 0.00024 1.25 BP1884 fhaC 5.65E-31 -2.10 BP0758 cyaC 0.16792 1.27 BP3784 ptxB 7.71E-05 -2.10 BP1054 prn 0.00183 1.29 BP0761 cyaB 4.15E-11 -2.06 BP1876 bvgR 0.014 1.29 BP1882 fimC 1.16E-13 -1.89 BP1883 fimD 4.84E-05 1.31 BP0762 cyaD 6.37E-12 -1.83 BP2253 bopD 0.16378 1.34 BP1054 prn 2.44E-09 -1.63 BP0216 sphB1 0.0003 1.34 BP1112 bipA 2.42E-18 3.42 BP2254 bcrH1 0.21187 1.34 BP2244 bscO 0.02113 1.35 Not significant BP3783 ptxA 0.01008 1.37 BP1879 fhaB 0.0923973 -1.16 BP1055 cysG 0.00396 1.45 BP1876 bvgR 0.8406184 -1.02 BP0760 cyaA 1.60E-06 1.49 BP1055 cysG 0.2227201 1.17 232 Appendix Table A.2 Genes differentially expressed in both BPrncD45A and BPrneΔCT. Table shows the top 10 genes (of 444) sorted by lowest calculated p-values in BPrneΔCT. Table this shows examples of where negative differential expression in one strain is commonly matched by positive differential expression in the other endonuclease mutant. The full list of genes can be accessed from Ifill et al. (2021) (216) gene_name P-value BPrncD45A FC BPrncD45A vs wild type P-value BPrneΔCT FC BPrneΔCT vs wild type tcfA 1.23E-10 2.11 9.26E-178 -27.69 BP2925 3.12E-09 1.77 7.53E-151 -12.81 vag8 1.65E-19 4.00 1.85E-134 -48.22 pstS 2.13E-09 -1.82 2.41E-119 -11.23 BP2924 5.47E-06 1.79 8.32E-94 -14.04 BP2486 9.29E-07 1.52 1.92E-89 -6.31 BP0499 1.22E-40 3.67 8.03E-89 -8.99 BP2232 3.59E-48 3.53 4.32E-88 -8.05 brpL 9.59E-07 1.68 2.21E-83 -8.22 ompQ 7.69E-24 2.25 1.12E-73 -4.40 BP0500 1.62E-29 4.09 2.47E-69 -9.45 … Appendix Table A.3 sRNAs identified in RNA-Seq analysis of endonuclease mutants Strain Predicted sRNA p-value FC vs wild type Approx. length (predicted; bp) Genome coordinates BPrncD45A bprB 1.18E-06 1.99 80 (77) 1494130-1494207 bprJ 5.92E-05 2.24 70 (80) 3605317-3605397 bprM 0.016494 1.65 190 (86) 3896371-3896457 bprM' 1.45E-06 1.72 190 (86) 3896457-3896371 BPrneΔCT bprK 0.000133 -1.73 200 (135) 3619230-3619365 bprM' 1.95E-10 -2.10 190 (86) 3896457-3896371 233 Appendix Table A.4 Gene loci found near IS elements influenced by antisense RNA found to be differentially expressed in endonuclease mutants. BPrncD45A BPrneΔCT gene_name FC gene_name FC aroE -1.68 BP0119 -2.08 BP0356 2.21 BP0356 2.22 BP0515 -2.08 BP0364 2.57 BP0566 2.53 BP0538 1.87 BP0675 -1.90 BP0732 1.97 BP0689 -2.12 BP0744 -1.91 BP0732 2.85 BP0870 -1.71 BP0810 1.63 BP0872 2.24 BP0956 -1.51 BP1079 1.81 BP0998 1.81 BP1131 -1.64 BP1131 -2.74 BP1558 2.40 BP1362 -1.70 BP1646 -2.44 BP1718 1.83 BP1718 -2.17 BP2019 -2.18 BP1946 -1.67 BP2103 -1.51 BP2019 -3.92 BP2220 -1.84 BP2354 -2.20 BP2354 -2.81 BP2486 -6.31 BP2569 -1.88 BP2569 -1.94 BP2689 -1.52 BP2722 6.29 BP2813 1.67 BP3115 -1.75 BP2975 2.40 BP3518 -1.68 BP3066 -2.24 BP3606 -1.61 BP3090 2.05 BP3689 -2.13 BP3163 -2.47 BPt05 -2.84 BP3689 -2.69 BPt43 -1.69 BP3809 -1.51 fabI 1.83 BP3838 1.66 fim2 -4.84 fim2 -1.60 metH 1.85 metX 1.86 nadC -2.00 nadC -1.54 phaG 2.34 paaG -3.61 recR -1.54 BP0119 -4.80 BP0364 1.52 BP0744 1.90 BP0872 2.05 BP1757A 1.77 BP2486 1.52 BP3391 -1.52 BPt05 2.22 fadD -2.27 234 Appendix Table A.5 Operons differentially expressed in endonuclease mutants. Operon locus tags Direction FC vs wild type OperonID First gene last gene BPrncD45A BPrneΔCT 25927 BP0119 BP0121 - - 25948 BP0246 BP0247 + + 25968 BP0359 BP0362 + + 25971 BP0379 BP0380 + - 26034 BP0731 BP0732 + + 26125 BP1203 BP1205 + - 26147 BP1314 BP1315 - + 26148 BP1320 BP1321 + + 26156 BP1358 BP1360 - - 26201 BP1573 BP1575 - - 26211 BP1623 BP1626 - - 26228 BP1708 BP1709 - + 26232 BP1736 BP1738 - - 26258 BP1886 BP1887 - - 26282 BP2019 BP2020 - - 26323 BP2230 BP2231 - - 26337 BP2325 BP2326 + - 26396 BP2632 BP2633 -/ + + 26404 BP2669 BP2670 - - 26416 BP2717 BP2718 - - 26451 BP2903 BP2905 - - 26455 BP2924 BP2927 + - 26456 BP2930 BP2933 + + 26478 BP3050 BP3051 - - 26486 BP3086 BP3087 + - 26561 BP3497 BP3501 - - 26575 BP3569 BP3570 - - 26577 BP3573 BP3577 - - 26599 BP3695 BP3696 - - 26600 BP3701 BP3702 - - 26601 BP3703 BP3705 - - 26610 BP3753 BP3754 + - 26615 BP3788 BP3796 - - 26624 BP3828 BP3831 - - 235 Appendix B Appendix Table B.1 Sequences identified from lgmA and lgmE upstream regions most likely to contain sigma factor binding sites as predicted by BacPP. The score is representative of the percent change of a given sigma factor binding a given region (221). Sequences shown are 80 bp regions with the highest score for the indicated sigma factor. Regions were mapped to the lgm locus to define the region of sigma factor binding and identify potential promoter elements (Fig. 3.3). Promoter region pLgmA Score Sequence 5’-3’ RpoE 35% ATTTGATGAAAATTTGACGGGGCTTCACGAATACTGGCGGCCGTTCCCGTCAATTTCGCCGTGGTGTGTTCATGTATACC FliA 61% AACGCTCCCAACGGCAGCGGATTTGATGAAAATTTGACGGGGCTTCACGAATACTGGCGGCCGTTCCCGTCAATTTCGCC RpoH 57% ATTTGATGAAAATTTGACGGGGCTTCACGAATACTGGCGGCCGTTCCCGTCAATTTCGCCGTGGTGTGTTCATGTATACC RpoS 70% GATTTTACCGAAGCCCGGCGAACGCTCCCAACGGCAGCGGATTTGATGAAAATTTGACGGGGCTTCACGAATACTGGCGG RpoD 60% TTGATGAAAATTTGACGGGGCTTCACGAATACTGGCGGCCGTTCCCGTCAATTTCGCCGTGGTGTGTTCATGTATACCGA pLgmE FliA 52% CCGCGCCGGCCAATTCGATCAGCGCGCCATCGATGGCCTCGTTCATGCCAAAATCATCGCCGCACACCGCGATGCGCCGG RpoH 66% CGCCGGCCAATTCGATCAGCGCGCCATCGATGGCCTCGTTCATGCCAAAATCATCGCCGCACACCGCGATGCGCCGGCAA
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Fernandez, Rachel C.
Lipid A modifications in Bordetella pertussis : regulation and function of the lgm locus
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