Science, Faculty of
Microbiology and Immunology, Department of
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UBCV
Santamaría Amorocho, Valentina
Master of Science - MSc
University of British Columbia
In the context of infection, a challenge for pathogenic bacteria is to maintain iron homeostasis with varying iron availability due to changes in pH, oxygen concentration, and host nutritional immunity. Organisms such as Campylobacter jejuni and uropathogenic Escherichia coli (UPEC) strains use the Fet system to acquire iron and overcome these changes. In C. jejuni, the Fet system is composed of eight genes (fetMP-fetABCDEH). In both species, FetM and FetP interact and are proposed to uptake ferric and ferrous iron. The role of fetABCDEH in iron uptake or as a mechanism to overcome stress has not been characterized in UPEC and is still not well understood. The iron/manganese-deficient strain ECA458 (ΔentC ΔfecABCDE ΔfeoABC ΔmntH) was engineered to characterize the Fet system in a gain-of-function model. Using the chassis-independent recombinase-assisted genome engineering (CRAGE) system, fetMP, fetMP-fetABCDEH and fetAH were integrated into the genome of ECA458. The resulting strains (ECA-fetMP, ECA-fetMH, ECA-fetAH) were used for phenotypic characterization of the Fet system with E. coli K-12 W3110 (WT), ECA458 and ECA-LP (ECA458 with a landing pad in fliK) as controls. Growth on LB and iron-limited LB, response to oxidative and nitrosative stress, and tolerance to streptomycin were assessed for these strains. Strains ECA458 and ECA-LP had similar phenotypes such as limited growth, increased oxidative stress sensitivity and streptomycin tolerance compared to WT. The gain-of-function approach validated the role of FetMP in growth enhancement under iron restriction, increased sensitivity to oxidative stress and streptomycin compared to ECA458. The phenotypes for strains expected to express fetMP-fetABCDEH and fetABCDEH were similar to those seen in ECA458 and ECA-LP. In these strains, the expression of the fet genes in this model may be limited by unknown factors and further studies are required to define expression of fet genes by strains ECA-fetMH and ECA-fetAH. This work was a novel approach to assess the role of the Fet system in E. coli and support a role of FetMP in iron uptake and oxidative stress.
Characterization of an iron uptake system from uropathogenic Escherichia coli by Valentina Santamaría Amorocho B.Sc., Pontificia Universidad Javeriana, 2022 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2025 © Valentina Santamaría Amorocho, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Characterization of an iron uptake system from uropathogenic Escherichia coli submitted by Valentina Santamaría Amorocho in partial fulfilment of the requirements for the degree of Master of Science in Microbiology and Immunology Examining Committee: Dr. Michael Murphy, Professor, Department Head, Microbiology and Immunology, UBC Supervisor Dr. James Kronstad, Professor, Microbiology and Immunology, UBC Supervisory Committee Member Dr. Elitza Tocheva, Professor, Microbiology and Immunology, UBC Supervisory Committee Member Dr. Thomas Beatty, Professor, Microbiology and Immunology, UBC Additional Examiner iii Abstract In the context of infection, a challenge for pathogenic bacteria is to maintain iron homeostasis with varying iron availability due to changes in pH, oxygen concentration, and host nutritional immunity. Organisms such as Campylobacter jejuni and uropathogenic Escherichia coli (UPEC) strains use the Fet system to acquire iron and overcome these changes. In C. jejuni, the Fet system is composed of eight genes (fetMP-fetABCDEH). In both species, FetM and FetP interact and are proposed to uptake ferric and ferrous iron. The role of fetABCDEH in iron uptake or as a mechanism to overcome stress has not been characterized in UPEC and is still not well understood. The iron/manganese-deficient strain ECA458 (ΔentC ΔfecABCDE ΔfeoABC ΔmntH) was engineered to characterize the Fet system in a gain-of-function model. Using the chassis-independent recombinase-assisted genome engineering (CRAGE) system, fetMP, fetMP-fetABCDEH and fetAH were integrated into the genome of ECA458. The resulting strains (ECA-fetMP, ECA-fetMH, ECA-fetAH) were used for phenotypic characterization of the Fet system with E. coli K-12 W3110 (WT), ECA458 and ECA-LP (ECA458 with a landing pad in fliK) as controls. Growth on LB and iron-limited LB, response to oxidative and nitrosative stress, and tolerance to streptomycin were assessed for these strains. Strains ECA458 and ECA-LP had similar phenotypes such as limited growth, increased oxidative stress sensitivity and streptomycin tolerance compared to WT. The gain-of-function approach validated the role of FetMP in growth enhancement under iron restriction, increased sensitivity to oxidative stress and streptomycin compared to ECA458. The phenotypes for strains expected to express fetMP-fetABCDEH and fetABCDEH were similar to those seen in ECA458 and ECA-LP. In these strains, the expression of the fet genes in this model may be limited by unknown factors and further studies are required iv to define expression of fet genes by strains ECA-fetMH and ECA-fetAH. This work was a novel approach to assess the role of the Fet system in E. coli and support a role of FetMP in iron uptake and oxidative stress. v Lay Summary Iron is required by most forms of life, including bacteria that cause infection. Within the host, iron uptake can be a challenge for these bacteria due to a competition for this metal between both organisms. Some bacteria have developed systems to overcome this limitation, one of them being the Fet system. This system facilitates iron uptake when availability of this metal is restricted. However, this is possibly only one of the functions of this system. This work aimed to understand the role of the Fet system in response to stress conditions, in addition to iron uptake. Characterizing the Fet system contributes to further understanding microbial responses to environmental conditions that might be challenging, especially in the context of infection. vi Preface Bacterial strains for the microbial engineering work were kindly provided by Dr. Steven Hallam (University of British Columbia) and Dr. Dietrich Nies (Martin-Luther-University Halle-Wittenberg) from their collections. All other work described in this thesis is my original, unpublished work. This project required biohazard approval for the handling of Level I bacterial species. Approval was provided by the UBC Biosafety Committee, Certificate number: B17-0242. Generative artificial intelligence was not used for any aspects of this work. vii Table of contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of contents ......................................................................................................................... vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Chapter 1: Introduction ........................................................................................................... 1 1.1 Iron as an essential micronutrient ............................................................................... 1 1.2 Iron uptake in Escherichia coli ................................................................................... 1 1.3 Environment during urinary tract infections ............................................................... 3 1.4 The Fet system ............................................................................................................ 5 1.5 Oxidative stress in infection...................................................................................... 10 1.6 Nitrosative stress in infection.................................................................................... 11 1.7 Streptomycin resistance and iron .............................................................................. 11 1.8 Objectives ................................................................................................................. 13 Chapter 2: Methods ................................................................................................................ 14 2.1 Integration of fet genes into ECA458 ....................................................................... 14 2.1.1 Bacterial strains ..................................................................................................... 14 2.1.2 Primers .................................................................................................................. 14 2.1.3 Validation of ECA458 .......................................................................................... 16 viii 2.1.4 CRAGE system landing pad integration into ECA458......................................... 17 2.1.5 Validation of LP in ECA-LP................................................................................. 18 2.1.6 Integration of fet genes into ECA-LP ................................................................... 18 2.2 Phenotypical characterization ................................................................................... 20 2.2.1 Growth experiments .............................................................................................. 20 2.2.2 Response to oxidative and nitrosative stress ......................................................... 21 2.2.3 Sensitivity to streptomycin with or without iron supplementation ....................... 21 Chapter 3: Results................................................................................................................... 23 3.1 Engineering of ECA458 ............................................................................................ 23 3.1.1 The strain ECA458 harbors the zupT gene ........................................................... 23 3.1.2 Genes fetMP, fetMH and fetAH were integrated into ECA458 for further characterization as a gain-of-function model .................................................................... 23 3.2 Phenotypical characterization ................................................................................... 25 3.2.1 ECA-fetMP had improved growth compared to ECA458 in LB and iron-limited LB……………………………………………………………………………….……………………………………………..25 3.2.2 ECA-fetMP was more sensitive to oxidative stress than ECA458 and other ECA-derived strains ................................................................................................................... 27 3.2.3 The Fet system did not show a phenotype in response to nitrosative stress ......... 28 3.2.4 Iron deficiency increases resistance to subinhibitory concentrations of streptomycin ...................................................................................................................... 30 Chapter 4: Discussion ............................................................................................................. 33 4.1 Phenotypical differences between the iron deficient strains (ECA458 and ECA-LP) compared to WT ................................................................................................................... 33 ix 4.2 Phenotypes of ECA-fetMP validated the gain-of-function model and the role of FetMP in iron uptake ............................................................................................................ 37 4.3 Phenotypical characterization of ECA-fetMH and ECA-fetAH suggests limitations of the gain-of-function model ............................................................................................... 38 4.4 Conclusion ................................................................................................................ 40 Bibliography .................................................................................................................................41 x List of Tables Table 2.1. Primers used for engineering and validation of ECA458 and ECA-derived strains ... 14 xi List of Figures Figure 1.1. Proposed structure of the fet cluster in Campylobacter jejuni wild-type strain 81–176................................................................................................................................................... 5 Figure 1.2. Model of the Fet system in Campylobacter jejuni wild-type strain 81–176 based on predicted and known functions ....................................................................................................... 7 Figure 3.1. Strains for phenotypical characterization and relevant genetic traits ........................ 23 Figure 3.2. ECA-fetMP improved growth in LB and iron-limited LB ........................................ 25 Figure 3.3. ECA-fetMP had increased sensitivity to oxidative stress ......................................... 27 Figure 3.4. None of the strains showed a phenotype related to nitrosative stress ...................... 28 Figure 3.5. Iron-limited strains have increased resistance to streptomycin ................................ 30 xii List of Abbreviations ATCC American Type Culture Collection CDTA 1,2-Cyclohexanediaminetetraacetic acid CFU Colony Forming Unit CRAGE Chassis-Independent Recombinase-Assisted Genome Engineering DAP Diaminopimelic acid DETA/NONOate Diethylenetriamine NONOate DFO Desferrioxamine DIP 2,2'-Dipyridyl DNA Deoxyribonucleic Acid DTNB 5,5'-Dithiobis(2-nitrobenzoic acid) Fe-S Iron-Sulfur Clusters GTP Guanosine Triphosphate LB Luria-Bertani broth MH Mueller-Hinton agar MH-TV Mueller-Hinton with Trimethoprim and Vancomycin NO Nitric Oxide OD Optical Density RNA Ribonucleic Acid RFP Red Fluorescent Protein xiii RNS Reactive Nitrogen Species ROS Reactive Oxygen Species rpm Revolutions per minute TMM Tris-buffered minimal medium UPEC Uropathogenic Escherichia coli UT Urinary Tract UTI Urinary Tract Infection WT Wild Type xiv Acknowledgements Firstly, I want to thank Dr. Michael Murphy for his guidance and encouragement since I was an undergrad in the lab. Michael, thank you for your support and for helping me in the process of becoming a better scientist. I am also grateful to the members of my thesis committee, Dr. Elitza Tocheva and Dr. James Kronstad, for their guidance and support on my work. I was very lucky to be a member of the Murphy lab. I want to thank Angelé Arrieta for always being available for my science questions and debrief chats; Lauren Pugsley, thank you for our science talks and non-scientific tea talks that always made my morning; Ben, thank you for letting me become a better mentor and for your support with this project. To the past members of the Murphy lab, Dr. Anson Chan, Dr. Daniela Morales and Brianne Newman, I am grateful for your guidance in my early lab days, I learned valuable lessons from each one of you. Thank you to our cousins in the Eltis lab! Thank you Lianne and Elise for your friendship, company and support during these past years. You were a fundamental piece of this journey. I am grateful to the family I made in Vancouver for their encouragement and for listening to my science even when it was overwhelming. Most importantly, I want to thank my parents, Abel and Luz Dary, brothers, Johan and Roger, grandma, Marina, and nephews Juan Diego and Jerónimo for always being a call away when I needed them. Thank you for always listening, for believing in me and for making me feel like you were right by my side regardless of the distance. 1 Chapter 1: Introduction 1.1 Iron as an essential micronutrient Microbial physiology is highly dependent on metal homeostasis. Iron is especially relevant for biological systems due to its two biologically relevant possible oxidation states, ferrous (Fe2+) and ferric (Fe3+) iron, making this metal a common cofactor for enzymes involved in metabolic processes including DNA replication, respiration, among others (1). Iron can also be found as Fe4+ and Fe6+.The oxidation state of extracellular iron can be shifted by environmental conditions such as oxygen concentration and pH. Reduced ferrous iron is favored by low oxygen concentrations and low pH, while oxidized ferric iron is favored by high oxygen availability and high pH. Microorganisms have developed uptake mechanisms that are specific to one of the two iron forms and thus are inhibited by changes in the environment that shift the iron oxidation state (2,3). Maintaining iron homeostasis under changing environmental conditions is an advantage for microbial survival that is especially relevant under infection since pathogenic bacteria and their host compete frequently for this metal. 1.2 Iron uptake in Escherichia coli Expression of iron uptake systems in most prokaryotes is regulated by the ferric uptake regulator (Fur) regulator. Fur is responsive to intracellular ferrous iron availability. Upon binding of Fe2+, Fur undergoes dimerization. Iron-coordinated Fur dimers bind to target sequences in promoter regions, or Fur boxes, upstream of iron acquisition genes and inhibit gene transcription by preventing binding of RNA polymerase (4–6). This regulator is crucial for maintaining iron homeostasis. Under low intracellular iron concentrations, genes involved in siderophore synthesis, as well as their receptors, are expressed (7,8). Siderophores are chemically diverse compounds of low 2 molecular weight (500-1500 Da) that bind ferric iron with high affinity (9,10). These molecules are produced under iron-limited conditions, secreted into the extracellular environment, where they form complexes with Fe3+. Fe-siderophores are imported into the bacterial cells through specialized systems (10). Inside the cell, ferric iron is reduced into ferrous iron, aiding release of the metal in the cytosol (11). E. coli produces siderophores such as enterobactin (10,11). This iron chelator is synthesized by EntABCDEFGH, transferred into the periplasm by EntS, and finally exported by TolC. Uptake of iron-bound enterobactin is mediated by the outer membrane receptor FepA, the periplasmic binding protein FepB and the cytoplasmic membrane permease FepCDG (11). Other siderophores have been identified in UPEC. Salmochelin is the product of modifications of enterobactin, including coupling to glucose, among others. This siderophore is larger and more hydrophilic than enterobactin, minimizing membrane partitioning and binding to hydrophobic surfaces, therefore enhancing UPEC iron uptake (10). Enzymes for salmochelin synthesis and transport are encoded in the iro cluster (11). Other siderophores such as aerobactin and yersiniabactin have also been associated with isolates from the urinary tract rather than fecal isolates (10,11). Ferrous uptake systems are also present in pathogenic and non-pathogenic E. coli. The Feo system is a ferrous iron uptake system regulated by Fur and Fnr, and expression of this system is repressed under high iron and high oxygen availability (2). This system is encoded in the feoABC operon. FeoB is a transmembrane protein that enables ferrous iron transport into the cytoplasm. FeoA and FeoC are cytoplasmic with poorly characterized functions (2,12). Iron uptake by the Feo system is GTP-dependent and FeoA might act as a GTPase activating protein, while FeoC might have a protective role towards FeoB. Other ferrous uptake systems include MntH, an H+-dependent importer for multiple divalent metals, including Fe2+, but with strong preference for 3 Mn2+ (13). Regulation of MntH expression involves Fur, the hydrogen peroxide-sensing protein OxyR, which suggests MntH is involved in response to oxidative stress; and MntS, which represses mntH expression under high Mn2+ conditions (14–16). Another metal uptake system is ZupT, a constitutively expressed broad-range transporter capable of Fe2+, Mn2+, Co2+ and, preferably, Zn2+ uptake. The uptake mechanism of this transporter is not clearly understood yet (2,17). 1.3 Environment during urinary tract infections Urinary tract infections (UTIs) are classified as one of the most prevalent human bacterial infections worldwide, affecting primarily women. It is estimated that 50-60% of women will be affected by a UTI at least once in their lifetime (18). UTIs include conditions in which pathogenic bacteria invade and colonize the urethra, bladder, ureters or kidneys, thus, being classified in lower or upper tract infections (19). Uropathogenic Escherichia coli (UPEC) is the main causative agent of UTIs (20,21). The gut is the main reservoir for UPEC, therefore, successful infection of the urinary tract (UT) involves overcoming changes in oxygen and nutrient availability, microbiota, and mucosal surface composition between these two niches (10). Effective colonization of the UT involves virulence factors related to motility and adhesion, such as FimH, a bacterial adhesin that facilitates attachment to host cells found at the tip of type 1 pili. Biofilm formation, antibiotic resistance genes and a broad range of iron uptake systems are additional virulence factors that favor UPEC colonization and infection (10,20,21). Usually, colonization begins in the urethra and ascends through the UT until the bladder is reached. In some cases, the infection extends to the kidneys, causing a more severe infection called pyelonephritis. Untreated infection can spread to the bloodstream leading to sepsis (21). UPEC not only colonizes the tissue surfaces, but can also form intracellular bacterial communities that can lead to recurrent infection (10,20). 4 A remarkable characteristic of UPEC is the capacity to grow rapidly in urine. Urea is a major component with a typical concentration of ~300 mM in normal urine. The preferred carbon source for UPEC in urine is unknown. Amino acids, free and in peptides, are proposed as the main carbon source, but carbohydrates and tricarboxylic acid intermediates are also present. Nitrogen is present in urea, ammonia and amino acids, suggesting that urine is not nitrogen-limited. However, gene expression analyses of UPEC grown on human or mice urine have shown upregulation of genes such as the glutamine synthetase, suggesting nitrogen limitation (21). Sulfur is present in sulfate at around 20 mM, as well as in cysteine and taurine, indicating that urine is not sulfur-limited. Urine in healthy patients is challenging for microorganisms due to its slightly acidic pH around 6.0 (with normal ranges between 5 and 8), high urea concentrations and presence of nitrates and proteins with antimicrobial properties (21,22). During infection there is a competition for iron between the host and pathogenic bacteria (10,23). Nutritional immunity is a host strategy to restrict nutrient availability to pathogens during infection. Specifically, in the context of UTIs, multiple proteins are implicated in iron restriction. Lactoferrin, calprotectin and siderocalin, for example, are found in increased amounts in urine from patients with UTI symptoms compared to asymptomatic patients (10). These proteins bind iron and contribute to metal starvation as part of the host immune response by limiting availability of this metal. Iron concentration in urine ranges between 0.89 and 5 μM. This iron-limited condition induces expression of iron uptake genes that are usually Fur regulated (21). Since iron is such a limiting factor in the context of UTIs, iron uptake systems contribute to UPEC virulence. The expression of siderophores, such as aerobactin, and diverse metal acquisition systems with redundant functions are associated with UPEC isolates as adaptation mechanisms to overcome iron scarcity in the host and are a possible advantage for successful 5 infection (10,21). UPEC can also utilize iron from host compounds such as heme and hemoglobin (24). 1.4 The Fet system Iron uptake systems are generally inhibited by iron oxidation changes due to their specificity for either ferrous or ferric iron. Notably, the Fet system is a transport system with the capacity to uptake ferrous and ferric iron from media (23). This system is found in bacteria occupying diverse ecological niches, including many human-associated species. Campylobacter jejuni 81-176 and uropathogenic strains of E. coli such as VR50 and ST131-668 are some examples of organisms harbouring this system. In fact, the Fet system is upregulated in Campylobacter jejuni cultured in MH-TV medium supplemented with 30% human fecal extracts as compared to supplemented with 30% chicken cecal extracts, suggesting that the Fet system is involved in infection of the human host (25). In C. jejuni, the fet gene cluster is formed by eight genes located downstream of a Fur box and a putative transcription start site. The proposed genomic organization of the fet cluster in C. jejuni is found in Figure 1.1. This gene cluster encodes proteins FetM, FetP, FetA, FetB, FetC, FetD, FetE and FetF. Figure 1.1. Proposed structure of the fet cluster in Campylobacter jejuni strain 81–176. TSS: primary transcription start site. Retrieved from Richardson-Sanchez et al. (26). 6 FetP from C. jejuni, formerly named P19, was the first gene in the cluster shown to be iron regulated and required for growth under iron-limitation (27). Crystal structures show that FetP is a homodimer and each protomer has a copper-binding site with an adjacent iron/manganese binding site (27). The copper site of FetP is required for growth under iron-restriction using the chelator DFO (27). Compared to WT, the ΔfetP mutant is more sensitive to streptomycin and more tolerant to hydrogen peroxide-derived oxidative stress, but these phenotypes are not clearly understood (25). To further investigate the role of the fet cluster in C. jejuni a strain with deletion of downstream genes (ΔfetABCDEF) was constructed (25). Similar to the ΔfetP mutant, the growth of ΔfetABCDEF is decreased compared to WT in MH medium under iron restriction. MH is a complex medium composed by beef infusion, casein hydrolysate and starch. Iron is not supplemented into this medium but the concentration of this metal in standard MH is estimated around 9 μm. Growth in MH is restored upon supplementation with ferric citrate suggesting a role in iron uptake for fetABCDEF (25). This deletion also phenocopied the fetP mutant with regards to oxidative stress tolerance and increased sensitivity to streptomycin. Subsequently, deletion strains were constructed for each gene in the cluster (26). Bioinformatic analyses indicate that FetM is part of the iron/lead transporter superfamily of permeases and the presence of a FTR1 domain suggests function as a ferrous iron permease (26,28). Unlike other Ftr1-like proteins such as EfeU, FtrC and Ftr1p, that contain membrane spanning domains, FetM has a non-membrane spanning periplasmic domain that interacts with FetP (28). Bioinformatic approaches suggest FetA is a membrane protein with a periplasmic region that contains a YHS domain and a DUF2318 domain (26). Regarding FetB, FetC and FetD, these proteins are predicted to form an heteromeric ABC transporter, with FetB and FetC as permeases 7 with MacB_PCD and FtsX domains, and FetD with the role of ATPase. This putative transporter is expected to have the role of exporter of unknown substrates. FetB and FetC are homologues with 28% of amino acid sequence identity. Finally, FetE is structurally similar to thioredoxins and has a conserved catalytic motif CXXC. A model for iron uptake by the Fet system in C. jejuni based on predicted and characterized functions is shown on Figure 1.2 (26). Figure 1.2. Model of the Fet system in Campylobacter jejuni wild-type strain 81–176 based on predicted and known functions. Retrieved from Richardson-Sanchez et al. (26). A series of individual gene deletions were constructed to validate the model (26). Strains with deletions of fetA, fetB, fetC, or fetD C. jejuni grew poorly under iron-limitation (26). Supplementing the medium with ferric chloride partially restored growth (26), in contrast to the supplementation with ferric citrate for the ΔfetP and ΔfetABCDEF in earlier work (25). An insulin disulfide reduction assay demonstrated that FetE is capable of reducing disulfide bonds. Deletion of fetE did not have an effect on C. jejuni growth under iron limitation. FetF has 22% amino acid 8 sequence identity compared to FetE and deletion of fetF also had no effects on growth. A double deletion mutant ΔfetEF has decreased growth compared to WT C. jejuni, indicating overlapping functions between FetE and FetF. Additionally, ΔfetEF has increased DTNB reduction activity compared to WT, ΔfetE and ΔfetF all incubated in iron-limited MH. These results suggest the role of FetE and FetF as disulphide oxidases (26). In C. jejuni, deletion of fetM (ΔfetM) reduces growth on iron-limited, standard, and iron-replete MH (supplemented with 100 µM FeCl3) compared to WT (26). Interestingly, iron supplementation is not significantly correlated with growth enhancement for this mutant strain unlike mutants of other genes in the fet cluster. Under the conditions tested, ΔfetM appears to be essential for full growth of C. jejuni. The Fet system is also present in uropathogenic strains of E. coli such as F11 and ST131(23,29) The Fet cluster in E. coli is formed by fetMP-fetABCDEH. This gene cluster is homologous to the one present in C. jejuni, with a promoter containing a Fur box followed by fetMP, an intergenic region, and fetABCDEH. In E. coli, fetF is not present, but the hypothetical protein FetH is the last component of the system. The fetMP promoter is Fur regulated and expression is iron regulated (23). FetM and FetP from UPEC F11 were characterized using a gain-of-function model with E. coli strain ECA458 (23). This strain is an E. coli K-12 W3110 derivative with markerless deletions of ferrous iron uptake system (feoABC), ferric citrate uptake system (fecABCDE), manganese / ferrous uptake system (mntH), isochorismate synthase (entC), required for enterobactin biosynthesis, and a divalent metal transporter (zupT) (17,23,30,31). With these deletions of multiple metal uptake systems, ECA458 remains capable of manganese and iron uptake, but growth compared to the parental strain is reduced on Tris-buffered minimal medium (TMM) (31). 9 Compared to WT, this mutant strain is also more sensitive to oxidative stress when exposed to paraquat. Manganese supplementation increases the tolerance of ECA458 to oxidative stress (31). To characterize the function of FetM, the gene fetM from UPEC F11 was integrated into the genome of ECA458. When cultured in TMM supplemented with the iron chelator CDTA, the fetM integration significantly increases the growth. Interestingly, this integration improves growth anaerobically in TMM with 0.2% glucose upon supplementation with either FeSO4 or FeCl3, suggesting that E. coli expressing FetM is capable of uptake of iron in its reduced and oxidized form (23). Using a 55Fe uptake assay, FetM is shown to be an iron transporter. When fetMP was integrated in ECA458 growth of the strain was enhanced over expression of FetM only when grown on iron-restricted TMM (23). Additionally, expression of FetMP enhances growth upon supplementation with FeSO4 and FeCl3 under anaerobic conditions to higher levels than expression of FetM only. Interestingly, this growth enhancement is greater with ferric iron compared to ferrous iron. FetP, is suggested to have ferrireductase activity that facilitates uptake of ferric iron by reducing it into ferrous iron for uptake by FetM (23). Integration of fetP alone did not enhance iron uptake or enhance growth aerobically or anaerobically; however the strain has enhanced iron reduction activity compared to ECA458 (23). Similar to its C. jejuni homolog, FetP from E. coli has a copper-binding site in each monomer, and a putative iron-binding site located adjacent to the copper site (28). Through Far-western dot blot analysis, E. coli FetP was shown to interact specifically with the periplasmic domain of E. coli FetM, but not with C. jejuni FetM (28). Note, the function of FetABCDEH has yet to be characterized. In addition, this system might be an adaptation mechanism to other forms of stress that are not well-characterized yet but 10 are relevant in the context of host-pathogen interactions such as oxidative stress and nitrosative stress. 1.5 Oxidative stress in infection Oxidative stress is one of the major challenges faced by pathogenic bacteria. Reactive oxygen species (ROS) are responsible for the damage of macromolecules and can lead to cell death. Some ROS, such as hydrogen peroxide, are generated in cells grown aerobically, and bacteria have enzymes to cope with this stress such as catalases, as well as non-enzymatic strategies such as glutathione-dependent reduction systems (32). Due to its high osmolarity, moderate oxygenation and limited iron and other nutrients, human urine is considered a stressful environment for UPEC. In addition to endogenous ROS, uropathogenic bacteria are also exposed to ROS derived from interactions with epithelial cells and the host immune response, mainly by neutrophils (21,33). One of the most relevant risks from hydrogen peroxide is reaction with ferrous iron to form toxic hydroxyl radicals through the Fenton reaction and therefore, maintaining iron homeostasis is crucial to reduce oxidative stress in bacteria (24,34). The role of the Fet system in response to oxidative stress was characterized in C. jejuni 81-176 by Liu et al. (25). Wild-type C. jejuni, ΔfetABCDEF, ΔfetP and their complemented strains (fetABCDEFC, fetPC) were challenged with 1 mM hydrogen peroxide in MH medium. After 3 hours of exposure, the cell viability of all strains decreases, but WT and both complemented strains are significantly more sensitive to oxidative stress compared to the deletion strains. At 6 hours, the deletions strains recover to normal growth levels, but WT and the complemented strains do not. This test was also performed under iron supplementation (100 µM iron (III) citrate). C. jejuni WT 11 and the complemented strains are significantly more sensitive to oxidative stress compared to the deletion strains. Iron supplementation increased the relative tolerance of ΔfetP to hydrogen peroxide; however, the ΔfetABCDEF strain is more sensitive than in the absence of added iron. Further characterization of oxidative stress responses by the Fet system from UPEC would contribute to better understand the link between this system, oxidative stress and iron uptake. 1.6 Nitrosative stress in infection Nitrosative stress is another challenge faced by pathogenic bacteria within the host. This form of stress is derived from exposure to nitric oxide (NO) and other nitrogen oxides (35). Production of NO is part of the innate immune response to control infections, but it can also be endogenously produced as a product of anaerobic nitrate respiration due to nitrite accumulation (36,37). This form of respiration is suggested to be an advantage for UPEC during UTIs associated with biofilm formation and pathogenicity (38). Additionally, in the context of UTIs, NO levels are increased in the bladder of infected patients compared to healthy individuals as a possible antimicrobial response from the host (35,37). NO can react with metal ions (mainly iron) and other radicals to generate reactive nitrogen species (RNS) that can react with proteins and inhibit their function as well as cause damage to DNA and lipids (35,36). An advantage for pathogenic organisms is adaptation to nitrosative stress to succeed in host colonizing and establishing infection. The Fet system has not been previously characterized regarding this form of stress. 1.7 Streptomycin resistance and iron Streptomycin is an aminoglycoside antibiotic that has recently regained interest due to the emergence of antibiotic resistance to other groups of antibiotics. Aminoglycosides are bactericidal molecules that typically bind to the 30S ribosomal subunit, disrupting protein translation and 12 leading to widespread cell damage (39). Additionally, streptomycin binds to the 16S rRNA at the A-site, inhibits protein synthesis, and induces misreading during translation. Uptake of aminoglycosides is dependent on bacterial respiration and the proton motive force (PMF) is suggested to contribute to internalization of these molecules (39). The electromotive component of the PMF is reduced under pH ~6 and, therefore, the bactericidal activity of aminoglycosides is diminished under acidic conditions. Exposure to NO can reduce the PMF reducing aminoglycoside efficacy. Regarding iron and the PMF, E. coli complex I has iron-sulfur clusters that work as cofactors for electron transfer in most respiratory chains. Electron transfer and proton translocation increases the PMF and may affect antibiotic susceptibility, including for aminoglycosides (40). In fact, E. coli with a defective Complex I has increased resistance towards streptomycin and kanamycin (41). Mycobacterium avium, an opportunistic pathogen that produces high-affinity iron chelators within macrophages, showed increased resistance against streptomycin under iron-limited conditions. In Mycobacterium tuberculosis, increasing intracellular iron concentration led to sensitivity to multiple aminoglycosides, including gentamicin, kanamycin and spectinomycin (40). Response to streptomycin was assessed in C. jejuni (25). Streptomycin tolerance is significantly decreased compared to WT upon deletion of fetM, fetP, fetA, fetB, fetC, fetD or fetABCDEF, reaching a lower final OD600 than WT when exposed to 1 to 4 µg/mL of streptomycin. Deletion of fetE, fetF or fetEF also increased sensitivity, but OD600 is intermediate between the level reached for WT and the previously described mutant strains. For fetP, addition of ferric citrate recovers the tolerant phenotype, while for fetABCDEF this phenotype is only partially recovered. Overall, these results suggest that the Fet system is implicated in streptomycin resistance and that this phenotype is not limited to a consequence of iron uptake (26). 13 1.8 Objectives Iron uptake is relevant in the context of infection since pathogens and their host compete for this metal (10,23). Pathogenic bacteria have developed mechanisms such as the Fet system to aid iron uptake under restricted conditions. In E. coli, FetM and FetP are shown to function in iron uptake; however, the role of FetABCDEH is not well understood in this organism. The aim of this work is to further explore the function of FetMP and the downstream cluster FetA-H in iron uptake and as a stress response mechanism. This work will contribute to understanding the whole Fet system in E. coli through a novel gain-of-function model. 14 Chapter 2: Methods 2.1 Integration of fet genes into ECA458 2.1.1 Bacterial strains E. coli K-12 substr. W3110 was obtained from the ATCC. The iron uptake system-depleted strain ECA458 (derived from E. coli W3110) was provided by the Nies laboratory (23). E. coli WM3064 and a strain expressing RFP (E. coli EPI300-J23100-RFP) were provided by Leah Fulton (42). E. coli WM3064 is a diaminopimelic acid (DAP) auxotroph and was cultured under supplementation with 0.3 mM DAP unless stated otherwise (43). E. coli pir+ strain ECR6K was obtained from the Nies laboratory harbouring pUC18R6K-mini-Tn7T-Gm (23). The plasmid was cured out from this strain by sequential passages on LB medium without antibiotic supplementation. Clinical isolate UPEC ST131-668 was provided by the Pitout lab (29). ECA458-derived strains ECA-LP, ECA-RFP, ECA-fetMP, ECA-fetMH, ECA-fetAH were engineered as described below. 2.1.2 Primers Primers used in this thesis for engineering and verification of ECA-derived strains are summarized in Table 2.1. Table 2.1. Primers used for engineering and validation of ECA458 and ECA-derived strains. Primer Sequence (5’-3’) fecAE_fwd cagtcgtggtttggttc fecAE_rev cgattgtcctttttaccc feoABC_fwd gcgagttttcatccc 15 Primer Sequence (5’-3’) feoABC_rev ctctcctttgttggc mntH_fwd ggcggataaggcgtttac mntH_rev ggctatgttttagaggcac entC_fwd gcgaccgaaaatataaatg entC_rev gaatggaatgctcatcc zupT_fwd gcactcgccatccggtaatt zupT_rev tcatcattgttatccgggttcttac| zupT_mid_fwd cgcagcgctgccagtttgcg zupT_mid_rev ggggcgtgctgattaagatgta pW17_LP_fwd tattgtttccgacgcgcaacaagctaatttactgatccctgtggatgaaacaccgcctgtcatcaacatcgtgcaaagcctcgcaatc pW17_LP_rev gtgaaaccatctggatttgcgcctggttatcatccactttgagggagatttgcacttcacctaaatgtaatgctctgccagtgtcgcg LP_Kan_fwd gacagaagcattttccag Rec_Apr_fwd aggctcttctccttgagc fetE_int_fwd gtggtggttgtggcg fliK_rev gtttgctgtgcgttgg pW34_Lux(-)_F ctgtagcggcgcattaaggc pW34_Lux(-)_R actagttctagagcggccgc 16 Primer Sequence (5’-3’) mRFP1_fwd caccgcggtggcggccgctctagaa mRFP1_rev aattcctgcagccttaatgcgccgctacagat fetM_fwd caccgcggtggcggccgctctagaactagtttacagcgtcttgccagc fetP_rev aattcctgcagccttaatgcgccgctacagggcgcgttggttagtcgg RE_fetPA_fwd caataaaaacggctttggcgcaggggaatggatcccgtacctgaccatcag RE_fetPA_rev aggtcgacggtatcgataagcttgatatcggaattcctccctcatgtcgg RE_fetBC_fwd agcagtttgttcctgccgacatgagggaggaataatcatgctgtggcg RE_fetBC_rev ggtcgacggtatcgataagcttgatatcgggaattcttacgctgattgtg RE_fetCH_rev cgctggtggatcacaatcagcgtaagaatttgcagattaaaggcattgtg RE_fetCH_rev ggtcgacggtatcgataagcttgatatcggtcgcaaaacgggagattc pW34-prom-fetMP(-)_fwd ctgccggttatcttctggaatctcccgttttgcgactgtagcggcgcattaaggc pW34-prom-fetMP(-)_rev aagacctgtagcgtcgtgacgaaaaagtaactcatgcttataaacaagccaaagagagta fetAH_fwd atgagttactttttcgtcacgacg fetAH_rev tcgcaaaacgggagattccag fetE_int_fwd gtggtggttgtggcg 2.1.3 Validation of ECA458 Primers flanking fecABCDE (fecAE_fwd and fecAE_rev), feoABC (feoABC_fwd and feoABC_rev), entC (entC_fwd and entC_rev), mntH (mntH_fwd and mntH_rev) and zupT 17 (zupT_fwd and zupT_rev) were used for PCR amplification of these genes in E. coli K-12 W3110 as control and their expected truncated version in ECA458. Further validation for zupT was performed by amplifying this gene from WT and ECA458. Primers zupT_fwd and zupT_mid_rev were used to amplify the first half of the gene. Primers zupT_mid_fwd with zupT_rev were used for amplification of the second half of the gene. The fragments obtained from both strains were purified, sequenced and the resulting sequences aligned and compared. 2.1.4 CRAGE system landing pad integration into ECA458 Plasmid pW17 was provided by Leah Fulton (42–44). A modified version of the landing pad (LP) encoded in this plasmid was integrated into the fliK gene of the iron uptake-deficient strain ECA458 using lambda-red recombination (45). LP containing lox sites, Cre recombinase gene and a kanamycin resistance cassette was amplified by PCR with overhangs containing sequences homologous to two regions within the fliK gene. The primers for amplification of this fragment are pW17_LP_fwd and pW17_LP_rev. The LP amplicon was integrated into ECA458 as described previously (45). Briefly, ECA458 harboring an inducible lambda-red pathway encoded in plasmid pKM208 was subcultured on LB at 30°C until OD600 of ~0.2 was reached. Expression of the pathway was induced with 1 mM IPTG and cells were incubated until OD600 was between 0.4 and 0.6. Induced cells were electroporated with the landing pad fragment and plated on LB agar plates supplemented with 50 µg/mL of kanamycin. Resistant colonies were tested with colony PCR using primers LP_Kan_fwd and fliK_rev to verify integration of the landing pad into fliK. The resulting ECA-derived strain is referred to as ECA-LP further on. 18 2.1.5 Validation of LP in ECA-LP Plasmid pW34 was provided by Leah Fulton (42–44). Using primers pW34_Lux(-)_F and pW34_Lux(-)_R, the backbone of pW34 was PCR-amplified. This fragment will be referred to as pW34_BB. Colony PCR was performed on strain EPI300-J23100-RFP using primers mRFP1_fwd and mRFP1_rev to amplify mRFP1 and its promoter J23100 from genomic DNA to yield fragment J23100-mRFP1. Fragments pW34_BB and J23100-mRFP1 were assembled using Gibson Assembly Master Mix (NEB: #E2611S) and transformed into strain the E. coli pir+ strain ECR6K, since the construct would have an R6K origin of replication. Apramycin-resistant colonies were tested with colony PCR. After analysis of the PCR products, a positive ECR6K colony harbouring construct pW34_J23100_RFP was cultured overnight on LB supplemented with 50 µg/mL of apramycin. The plasmid was purified and transformed into E. coli WM3064. The transformed strain is used as donor for delivery of the pW34-derived construct. Conjugation was set up following (43), with ECA-LP as recipient strain. The gene mRFP1 was integrated into the landing pad of ECA-LP by Cre-lox recombination to generate the apramycin-resistant and fluorescent strain ECA-RFP. Fluorescence (λex = 575 nm, λem = 610 nm) was measured on overnight cultures from isolated colonies. Colony PCR was also performed to verify the integration with primers Rec_Apr_fwd and fliK_rev. 2.1.6 Integration of fet genes into ECA-LP Plasmids pW34_fetMP, pW34_fetMH and pW34_fetAH were built to integrate fetMP, fetMH, and fetAH into strain ECA-LP. Plasmid pB0018 from clinical isolate UPEC ST131-668 was the source of fet genes. All three plasmids were built using Gibson Assembly Master Mix (NEB: #E2611S). 19 To build pW34-fetMP, the backbone of pW34 (pW34_BB) was amplified using primers pW34_Lux(-)_F and pW34_Lux(-)_R. A fragment containing fetMP with overlapping regions to pW34_BB was amplified using primers fetM_fwd and fetP_rev. These fragments were assembled by Gibson Assembly. To build pW34_fetMH, a sequential cloning approach was used. Purified pW34_fetMP was digested with restriction enzymes BamHI-HF and EcoRI-HF following NEB protocols. This digestion yielded a linear product that contained the sequences of pW34_BB, fetM and a fragment of fetP. This was the first fragment used for this assembly. The second fragment was fetPA, which contained an artificial EcoRI restriction site. Fragment fetPA harbors the fetP sequence that was removed during the restriction enzyme digestion and part of fetA. This fragment was amplified with primers RE_fetPA_fwd and RE_fetPA_rev. The plasmid resulting from this assembly is pW34_fetMA. Plasmid pW34_MA was digested with EcoRI-HF and assembled with fragment fetBC. This fragment was amplified using primers RE_fetBC_fwd and RE_fetBC_rev and harbors fetB and part of fetC. A new EcoRI_HF site was added to the fragment. Plasmid pW34_fetMC was the product of this assembly. Plasmid pW34_fetMC was digested with EcoRI-HF and assembled with fragment fetCH. Fragment fetCH was amplified with primers RE_fetCH_fwd and RE_fetCH_rev. This PCR product harboured the remaining fetC sequence until fetH. Assembly between linearized pW34_fetMC and fetCH produced plasmid pW34_fetMH. For construct pW34_fetAH, primers pW34-prom-fetMP(-)_fwd and pW34-prom-fetMP(-)_rev were used with pW34_fetMP to amplify a single fragment that included pW34_BB and the upstream region (356 nucleotides) of fetM containing the natural promoter. Fragment fetAH was 20 amplified with primers fetAH_fwd and fetAH_rev. The pW34-derived constructs used for genomic integration were sequenced by Plasmidsaurus. The constructs pW34-fetMP, pW34-fetMH and pW34-fetAH were transformed into the strain E. coli WM3064, used as donor. Conjugation between ECA-LP and the transformed donor strain was set up following (43). Briefly, overnight cultures of ECA-LP and E. coli WM3064 harboring the selected pW34-construct were normalized to OD600 = 2 and combined in a 4:1 ratio. Cells were washed three times with LB supplemented with DAP and plated on plates of LB supplemented with DAP as a single spot. After overnight incubation, cells were collected with a loop, resuspended in LB, washed and diluted for plating in LB supplemented with 50 µg/mL of apramycin. The resulting strains from this recombination are ECA-fetMP, ECA-fetMH and ECA-fetAH. Colony PCR to verify the gene integration into the landing pad was performed with primers Apr_fwd and fliK_rev for ECA-fetMP and primers fetH_fwd and fliK_rev for ECA-fetMH and ECA-fetAH. 2.2 Phenotypical characterization All of the following experiments were performed on WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH strains. 2.2.1 Growth experiments Overnight cultures of WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH were grown in LB medium while shaking at 200 rpm. All the strains were subcultured in 5 mL LB broth until OD600 was in the range 0.4 - 0.6. Cells were harvested and washed with 0.9% saline solution once. Cells were resuspended and normalized to OD600 of 0.4 and inoculated into 100 µL of LB or LB supplemented with 40 µM of DFO to a final OD600 of 0.02 in 96-well plates. Cultures 21 were incubated at 37 °C for 30 h and every two hours, the cultures were shaken and OD600 was measured. 2.2.2 Response to oxidative and nitrosative stress Overnight cultures were inoculated into 5 mL of LB to an initial OD600 of 0.005 as subcultures. These subcultures were incubated for 3 h and challenged with 1 mM of hydrogen peroxide to test oxidative stress or 0.5 mM of diethylenetriamine NONOate (DETA/NONOate) for nitrosative stress. Dilutions of each subculture were plated on LB after inoculation, three hours of incubation (before challenge), two hours and six hours after addition of hydrogen peroxide or DETA/NONOate into the cultures. CFUs were determined after overnight incubation of the LB plates without antibiotic added. 2.2.3 Sensitivity to streptomycin with or without iron supplementation Strains were grown overnight and subcultured into LB without antibiotics added until OD600 was between 0.4 and 0.6. Cells were harvested and resuspended in 0.9% saline solution to an OD600 of 0.4. In a 96-well plate, 100 µL of LB broth with or without 100 µM FeCl3, was aliquoted into each well. One hundred microliters of LB or LB supplemented with 100 µM of FeCl3 with 128 µg/mL streptomycin was pipetted into each well of the first row of the plate to a final concentration of 64 µg/mL. Two-fold serial dilutions of the antibiotic were performed until the concentration was 0.5 µg/mL. Inoculated medium without streptomycin and uninoculated medium were used as controls. Cultures were incubated at 37 °C while shaking at 200 rpm for 18 h for OD600 to be measured. 22 23 Chapter 3: Results 3.1 Engineering of ECA458 3.1.1 The strain ECA458 harbors the zupT gene ECA458 is described as ΔentC ΔfecABCDE ΔfeoABC ΔmntH ΔzupT (23,30). The size of the PCR amplicons from fecABCDE, feoABC, entC, mntH and zupT from WT and ECA458 were compared. The amplicons for the first four genes were smaller from ECA458 than amplicons obtained from WT, indicating that these were successfully truncated as described previously (30). However, the PCR amplicon for zupT was the same size for both stains (data not shown). This indicated that this gene was not truncated in ECA458 as described (23). The zupT gene was amplified as two fragments and sequenced. The DNA sequences obtained from WT and ECA458 determined that zupT in both strains had the same sequence. Our validation of the gene deletions indicates that the strain as received is a ΔentC ΔfecABCDE ΔfeoABC ΔmntH mutant and encodes zupT. For the following sections, ECA458 will correspond to the validated strain lacking entC, fecABCDE, feoABC, mntH and encoding zupT. 3.1.2 Genes fetMP, fetMH and fetAH were integrated into ECA458 for further characterization as a gain-of-function model The function of FetM and FetP was characterized in ECA458 strain (ΔentC ΔfecABCDE ΔfeoABC ΔmntH ΔzupT) as a gain-of-function model (23). However, the role of FetAH in E. coli is poorly understood. Multiple strains with genomic integrations of different fet genes (fetMP, fetMP-ABCDEH and fetABCDEH) with the native fet promotor were engineered for phenotypic characterization following the gain-of-function approach. 24 The CRAGE system was used for integrating the selected genes. First, a landing pad was integrated into the fliK gene of ECA458 (ΔentC ΔfecABCDE ΔfeoABC ΔmntH, zupT+) building the kanamycin-resistant strain ECA-LP. This integration was validated by PCR with primers LP_Kan_fwd and fliK_rev. Plasmids pW34_fetMP, pW34_fetMH and pW34_fetAH were constructed and delivered to ECA-LP. Through Cre-lox recombination, fetMP, fetMP-ABCDEH and fetABCDEH were integrated into the landing pad of ECA-LP and the resulting apramycin-resistant strains were ECA-fetMP, ECA-fetMH and ECA-fetAH, respectively. The strains used for phenotypical characterization and their gene integrations are shown in Fig. 3.1. Integration of the fragments into fliK of ECA-LP was validated through PCR amplification. The resulting strains build on a previous gain-of-function approach with strain ECA458 (ΔentC ΔfecABCDE ΔfeoABC ΔmntH, zupT+); using a different integration system and site and a background strain with the zupT gene. Figure 3.1. Strains for phenotypical characterization and relevant genetic traits. From top to bottom: WT (green), ECA458 (blue), ECA-LP (black and white), ECA-fetMP (orange), ECA-25 fetMH (purple) and ECA-fetAH (yellow). For ECA-LP, lilac represents the disrupted fliK gene; red lines represent regions of homologous recombination for LP integration; blue triangles represent lox sites; integrated genes are labeled in the figure. For ECA-fetMP, ECA-fetMH and ECA-fetAH lox are represented as blue triangles; integrated fet genes are labeled in the figure. 3.2 Phenotypical characterization 3.2.1 ECA-fetMP had improved growth compared to ECA458 in LB and iron-limited LB To examine the role of fetMP and characterize the role of fetAH in E. coli growth, the strains WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH were cultured in LB with or without addition of 40 µM of the iron chelator DFO. Growth in 96-well plates for 30 hours was monitored by measuring OD600. Regarding growth in LB without addition of DFO, WT reached stationary phase faster and reached the highest OD600 of all strains (Fig 3.1A). The growth rate of ECA458 that lacks several metal uptake systems was slower than WT as expected. Similarly, growth of ECA-LP was also slower than WT. ECA-fetMP had improved growth rate and reached a higher OD600 compared to ECA458 and ECA-LP. Growth of this strain exceed all other ECA-derived strains in the first 18 hours of incubation. The strains ECA-fetMH and ECA-fetAH had similar growth to the iron uptake system-depleted strains ECA458 and ECA-LP (Fig. 3.2A). Unexpectedly, ECA-fetMH did not have improved growth, even though the fetMP genes are present in this strain as well. Growth was also characterized in iron-limited LB. Addition of 40 µM DFO was detrimental for the growth of all the strains (Fig. 3.2B). In this case, the WT strain had the fastest growth rate and it reached the highest OD600. Additionally, the growth of ECA-fetMP was improved compared to ECA458, ECA-LP, ECA-fetMH and ECA-fetAH. Similar to culture under 26 standard LB, growth of ECA-fetMH was similar to ECA458 even though fetMP were integrated in this strain. When the growth of ECA458 and the derived strains were compared, only ECA-fetMP showed improved growth under standard LB and iron-limited LB conditions. Figure 3.2. ECA-fetMP improved growth in LB and iron-limited LB. Growth of WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH on (A) LB and (B) LB supplemented with 40 27 µM DFO shaken every two hours. Error bars indicate standard deviation for the mean at each timepoint, n = 3 for all strains in all conditions. 3.2.2 ECA-fetMP was more sensitive to oxidative stress than ECA458 and other ECA-derived strains Oxidative stress is related to iron homeostasis. Ferrous iron can react with hydrogen peroxide to form toxic hydroxyl radicals that are harmful for cells (34). In C. jejuni 81-176, deletion of fetABCDEF increased tolerance to oxidative stress (25). The role of the Fet system in E. coli in response to oxidative stress is not described in the literature. To assess the role of the Fet system in mediating oxidative stress, the engineered strains cultured on LB were challenged with 1 mM H2O2 and cell survival was assessed by CFU counts. The concentration of viable cells was compared for all strains two and six hours after exposure to hydrogen peroxide (Fig. 3.3). The E. coli WT strain was insensitive to the oxidative stress condition tested. After 2 hours of exposure, the concentration of viable cells of this strain increased in comparison to the concentration when the cultures were challenged. ECA458 and ECA-LP, the iron uptake-impaired strains, were more sensitive to oxidative stress. The concentration of viable cells for both strains decreased after two hours of exposure to hydrogen peroxide. However, the growth of both strains recovered six hours after exposure. ECA-fetMH and ECA-fetAH followed a similar growth pattern (Fig. 3.3). In contrast, ECA-fetMP was more sensitive to oxidative stress than ECA458 and ECA-LP. The concentration of viable cells of ECA-fetMP strain two hours after exposure to hydrogen peroxide was significantly reduced compared to ECA458, ECA-LP, ECA-fetMH and ECA-fetAH. Six hours after challenge, the viable cell concentration of ECA-fetMP increased, recovering to concentrations similar to that of ECA458 28 and the other derived strains (Fig. 3.3). Under the conditions tested, only ECA-fetMP showed increased sensitivity to oxidative stress compared to the iron uptake-deprived strains ECA458 and ECA-LP. Figure 3.3. ECA-fetMP had increased sensitivity to oxidative stress. Cell viability of WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH before and after exposure to 1 mM H2O2 in 5 mL tubes while shaking at 200 rpm. Two-way ANOVA was performed for statistical analysis of WT and all ECA-derived strains. Significant differences are indicated as follows: (****) p-value < 0.0001. Green asterisks indicate significance for WT against ECA458, ECA-LP, ECA-fetMH and ECA-fetAH; orange asterisks indicate significance for ECA-fetMP against ECA458, ECA-LP, ECA-fetMH and ECA-fetAH. Error bars indicate standard deviation, n = 4. 3.2.3 The Fet system did not show a phenotype in response to nitrosative stress Pathogenic bacteria are exposed to nitric oxide as part of the host immune response. Nitric oxide can react with iron to generate RNS that can compromise cell homeostasis (35,36). Mechanisms to overcome nitrosative stress are an advantage for survival for bacteria within the 29 host. To our knowledge, the Fet system has not been characterized in either E. coli or C. jejuni against nitrosative stress. To assess the possible role of the system against this form of stress, all strains cultured on LB were challenged with 0.5 mM of the nitric oxide donor DETA/NONOate and cell viability was assessed by CFUs. Under the conditions tested, none of the strains showed a sensitive phenotype against nitrosative stress (Fig 3.3). Since ECA-fetMP had increased sensitivity to oxidative stress, I hypothesized increased sensitivity to nitrosative stress as well. For all the strains, the concentration of viable cells after two and six hours of exposure to 0.5 mM DETA/NONOate increased (Fig. 3.4). Thus, the Fet system did not show a role in response to nitrosative stress. Figure 3.4. None of the strains showed a phenotype related to nitrosative stress. Cell viability of WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH before and after exposure to 0.5 mM DETA/NONOate in 5 mL tubes while shaking at 200 rpm. Two-way ANOVA was performed for statistical analysis of WT and all ECA-derived strains. Error bars indicate standard deviation, n = 4. 30 3.2.4 Iron deficiency increases resistance to subinhibitory concentrations of streptomycin The role of the Fet system in streptomycin resistance was previously characterized in C. jejuni 81-176. The deletion mutant ΔfetABCDEF was more susceptible to streptomycin than WT C. jejuni on standard MH medium. Upon supplementation with 100 µM iron (III) citrate, streptomycin tolerance was partially restored in the ΔfetABCDEF deletion mutant (25). To assess if the Fet system has a role in tolerance to streptomycin in E. coli, all strains were exposed to concentrations of streptomycin going from 0 up to 64 µg/mL in LB medium with or without 100 µM FeCl3 added and incubate at 37 °C while shaking at 200 rpm for 18 h. For tests performed on LB, growth of the strains decreased with the addition of 4 to 8 µg/mL or greater of streptomycin (Fig 3.4A). Interestingly, the growth of ECA458 and derived strains increased when streptomycin was present in concentrations from 0.5 to 2 to 4 µg/mL. Notably, for ECA-fetMP, the phenotype was intermediate between the WT and the other ECA458 derived strains. The growth of ECA-fetMP increased when streptomycin was present in concentrations from 0.5 to 2 µg/mL, but reached the same final growth as with no streptomycin when 4 µg/mL of this antibiotic was present in the medium (Fig. 3.5). Since only the ECA458-derived strains showed an increase in growth at subinhibitory concentrations of streptomycin, I hypothesize that this phenotype is related to iron limitation. To test this hypothesis, tolerance to streptomycin was characterized under iron supplementation. The expected outcome was that the ECA458-derived strains would demonstrate growth more similar to that of WT. Interestingly, under these conditions, WT experienced a growth increase when the medium was supplemented with 0.5 to 2 µg/mL of antibiotic. Also, a growth increase was observed for the ECA458, ECA-fetMH and ECA-fetAH strains with 0.5 to 4 µg/mL 31 antibiotic. ECA-LP also had a growth increase, when the concentration of streptomycin was 2 µg/mL. ECA-fetMP had decreased tolerance to streptomycin upon iron supplementation (Fig. 3.5). Figure 3.5. Iron-limited strains have increased resistance to streptomycin. OD600 of WT, ECA458, ECA-LP, ECA-fetMP, ECA-fetMH and ECA-fetAH exposed to 0 - 64 µg/mL of streptomycin on (A) LB or (B) LB supplemented with 100 µM FeCl3 was measured after 18 hours 32 of incubation with constant shaking at 200 rpm at 37°C. The OD600 was normalized for each strain in both media conditions to the OD600 obtained with no streptomycin added. For (A), n = 3. For (B), n = 3 for ECA-fetMH, n = 4 for the remaining strains. Error bars indicate standard deviation. Since the ECA458-derived strains lack multiple iron uptake systems, iron supplementation might not be sufficient to characterize the effect of intracellular iron in streptomycin resistance, but the hypothesis of increased streptomycin resistance under iron limitation could be characterized with WT or ECA-fetMP under supplementation with iron chelators. Manganese limitation is not expected to have a role in streptomycin resistance, but it could also be characterized since ECA458-derived strains lack mntH. 33 Chapter 4: Discussion 4.1 Phenotypical differences between the iron deficient strains (ECA458 and ECA-LP) compared to WT To establish a gain-of-function model for characterization of the Fet system, the growth of ECA458 (ΔentC ΔfecABCDE ΔfeoABC ΔmntH) and the derived strain ECA-LP was compared to wild-type E. coli K-12 (W3110). Strains ECA458 and ECA-LP encode zupT, a broad-spectrum metal transporter that can uptake Fe2+ and Mn2+, among other metals, but is not sufficient to maintain growth to WT levels (17). The growth defects of these strains are likely derived from defects in iron uptake since E. coli metabolism is iron-centric since Mn2+ is required only under oxidative stress conditions (14). However, the growth of iron-limited strains can be partially restored by manganese supplementation rather than iron supplementation (17,31). The growth differences between WT and the iron/manganese depleted strains ECA458 and ECA-LP suggest a potential use for a gain-of-function approach to characterize the Fet system, similar to previous work characterizing FetM and FetP (23). ECA458 and ECA-LP have growth differences. Overall, when grown on standard or iron-limited LB, ECA-LP reached higher OD600 than ECA458. The genetic differences between these strains are disruption of the fliK gene due to integration of the landing pad in ECA-LP as well as the presence of the Cre gene, lox sites and the kanamycin resistance cassette in the landing pad. Juhas et al. disrupted the fliK gene of E. coli K-12 MG1655 and integrated genes related to sucrose catabolism and lactose metabolism. Since FliK is involved in flagellar functions, its disruption inhibited motility but did not have effects on growth of the MG1655 strain on LB in 96-well plates (45). Based on this work, growth differences between ECA458 and ECA-LP on LB were not 34 expected. Characterizing the effects of disrupting fliK in motility, and therefore, cell interactions or possible formation of aggregates, as well as assessing growth with fluorescence in addition to OD600 or culturing in bigger volumes could provide additional information regarding the growth differences seen under the tested conditions. In addition to the growth differences, compared to WT the iron- and manganese-limited strains ECA458 and ECA-LP were more sensitive to oxidative stress caused by addition of hydrogen peroxide. Oxidative damage by H2O2 is amplified by the Fenton reaction that occurs with ferrous iron to produce hydroxyl radicals within the cell. In E. coli, the positive regulator OxyR responds specifically to hydrogen peroxide stress (14). OxyR induces transcription of genes that encode proteins involved in iron scavenging, such as Dps, and removal of H2O2, such as the catalase KatG. The overall effect of these processes is preventing Fenton chemistry (46). Interestingly, the Fur- and OxyR-regulated manganese-uptake system MntH also plays an important role in E. coli to cope with oxidative stress due to its capacity to outcompete iron in mononuclear enzymes, acting as cofactor to restore metabolic functions without undergoing Fenton stress (14,47). Even though ECA458 and ECA-LP are expected to have less intracellular iron than WT due to the disruption of multiple iron uptake systems, sufficient ferrous iron may be present to react with hydrogen peroxide in the cells, causing a decrease in viable cells after two hours of exposure due to oxidative damage. Since mntH is not present in these strains, Mn levels in these cells may not be sufficient to compensate for exposure to H2O2, which would lead to the decrease in cell viability seen in the mutant strains but not in WT. The results presented here are consistent with the phenotype seen by Taudte et al. where an iron/manganese deficient strain (ECA458) was challenged with paraquat, which increases concentrations of ROS due to generation of superoxide 35 anions. On TMM, 6 µM paraquat completely restricts growth of ECA458, while for WT 60 µM was needed to see the same effect. Manganese supplementation had a protective effect against oxidative stress generated by paraquat. Additionally, expression of SodA (Mn-SOD) was improved by manganese addition and was required for aerobic growth of the iron/manganese-restricted strain (31). After six hours of exposure to hydrogen peroxide, ECA458 and ECA-LP showed growth recovery. Mn is not only involved in protection to oxidative stress, but also in restoring DNA replication after exposure to hydrogen peroxide (48). ECA458 and ECA-LP are able to uptake Mn through the broad-range metal uptake system ZupT or by an under characterized manganese uptake system (17,31). These results provided information regarding the background phenotypes of the gain-of-function model. To assess if the oxidative stress-sensitive phenotype seen on ECA458 and ECA-LP is related to manganese limitation, both strains could be exposed to hydrogen peroxide under manganese supplementation. Regarding nitrosative stress, ECA458 and ECA-LP were not affected by exposure to the nitric oxide donor DETA/NONOate. The response to nitrosative stress in E. coli is mediated by iron-dependent molecules, such as flavohemoglobin, and iron-independent molecules such as glutathione (49). Under the conditions tested for this form of stress, the protective mechanisms may be sufficient to maintain growth of the mutant strains regardless of iron availability. Furthermore, the slow release of nitric oxide by DETA/NONOate at neutral pH for up to 20 hours may have been an experimental limitation. A particularly interesting phenotype was observed upon exposure to subinhibitory concentrations of the aminoglycoside streptomycin. The final OD of ECA458 and ECA-LP was increased on LB supplemented with subinhibitory concentrations of antibiotic (0.5 to 4 µg/mL). 36 However, this increased growth with subinhibitory concentrations of streptomycin was not seen for WT. Iron limitation in E. coli can increase tolerance to aminoglycosides as described by Erzaty et al. (50). The efficacy of aminoglycosides is dependent of the cellular proton motive force (PMF) that facilitates the uptake of these antibiotics. Increased PMF leads to increased aminoglycoside uptake and their bactericidal activity is, therefore, increased as well. The PMF is increased by proton translocation activity of the respiratory complexes I and II. The respiratory complex I, NADH dehydrogenase protein Nuo, has nine iron-sulfur (Fe-S) clusters, while in complex II, the corresponding succinate dehydrogenase Sdh, contains three Fe-S clusters. The main Fe-S biogenesis systems are ISC and SUF, the latter being mainly relevant under stress conditions (51). Erzaty et al. characterized the role of the ISC and SUF systems in the formation of respiratory complexes I and II and their effect on gentamicin uptake and bactericidal activity. Disrupting the ISC system by deleting iscUA impaired the correct insertion on Fe-S into the PMF-producing respiratory complexes I and II, decreasing PMF and gentamicin uptake. However, mutations on the SUF system under iron replete conditions did not increase gentamicin resistance, due to preference for the ISC system under these conditions (50). In contrast, in iron-limited cultures generated by DIP addition, the activity of both respiratory systems is reduced and there is a switch from the ISC to SUF system. Under these conditions, gentamicin resistance is increased due to decreased PMF. Addition of iron into the cultures increased sensitivity to gentamicin and reduced expression of SufC, a protein involved in the SUF system. The reduced sensitivity to streptomycin and increased growth seen in the strains ECA458 and ECA-LP may be related to reduced PMF due to a switch from the ISC to SUF system in response to reduced iron uptake by these strains. Compared to WT, these mutant strains would have decreased levels of intracellular iron, leading to preference for the SUF system over ISC, decreasing the activity of 37 respiratory complexes I and II, and with this, the level of PMF and streptomycin uptake, leading to increased resistance compared to WT. 4.2 Phenotypes of ECA-fetMP validated the gain-of-function model and the role of FetMP in iron uptake Previously, expression of fetMP led to increased iron uptake and accumulation, supporting a role of FetMP in iron transport (23). These genes were integrated into the genome of the iron/manganese deficient strain ECA458 to yield strain ECA612. In addition to fetMP, a gentamicin resistance cassette is present in this strain. Strain ECA612 had improved growth in iron-limited TMM or supplemented with 10 µM of FeCl3 or FeSO4 at pH 7 compared to a control strain. In the present study, ECA-fetMP had improved growth on LB and iron-limited LB compared to the negative controls ECA458 and ECA-LP consistent with the results described by Koch et al. (23). The growth improvement of ECA-fetMP is presumably also due to successful expression and enhancement of iron transport into ECA-LP. These results indicated that the gain-of-function model is a feasible approach to assess the remaining proteins encoded in the fet cluster for an iron-uptake phenotype. Additionally, genomic integration of fet cluster genes alleviates the need of antibiotics to maintain plasmids, limiting stressing factors for the phenotypic assessment. Interestingly, ECA-fetMP had increased sensitivity to oxidative stress compared to ECA458 and ECA-LP. The expression of fetMP in ECA-fetMP increases iron uptake capacity, that upon exposure to hydrogen peroxide undergoes Fenton chemistry. Cell damage is amplified in this manganese-limited strain due to the deletion of mntH. Characterizing the growth of ECA-fetMP upon being challenged with hydrogen peroxide in presence of an iron chelator would further assess if the increase in sensitivity is related to an increase in iron uptake. Additionally, 38 supplementing with manganese would validate the protective role of this metal in iron-replete conditions, compared to the intracellular iron-limited conditions of ECA458 and ECA-LP. The increase in iron uptake of ECA-fetMP possibly led to reduced streptomycin tolerance compared to the iron-deficient strains ECA458 and ECA-LP. I hypothesize the mechanism is an increased PMF due to activity of the respiratory complex I and II, enhancing streptomycin uptake and inhibiting the phenotypical resistance seen in the iron-limited strains. To assess if this is the case, ECA-fetMP and control strains could be grown under iron-replete and iron-limited conditions and assayed for respiratory complex function. As well the expression of ISC and SUF proteins could be assessed through RT-qPCR or Western blotting. 4.3 Phenotypical characterization of ECA-fetMH and ECA-fetAH suggests limitations of the gain-of-function model The engineered strains ECA-fetMH and ECA-fetAH had phenotypes similar to ECA458 and ECA-LP. Under the tested conditions, all these strains had limited growth on LB and iron-limited LB, increased oxidative stress sensitivity and tolerance to streptomycin compared to WT and ECA-fetMP. Based on characterization of the Fet system in C. jejuni, integrating fetMP-fetAH in ECA-LP was expected to improve over integrating fetMP only. Even though fetMP and its promoter was present in ECA-fetMH, unexpectedly, this strain did not have similar phenotypes to ECA-fetMP. In C. jejuni 81-176, expressing only FetMP (ΔfetABCDEF) compared to the whole Fet system (WT) decreased growth in MH and under iron-limited conditions (MH + 5 µM DFO), and growth was restored with iron-supplemented MH (25). In the E. coli gain-of-function approach, integration of fetMP increased sensitivity to oxidative stress, while fetMP-fetAH or fetAH did not. In contrast, wild-type C. jejuni was more 39 sensitive to oxidative stress than the strain expressing FetMP only (ΔfetABCDEF). These results are consistent in linking iron-uptake with oxidative stress in that strains defective for growth under iron-limitation are less sensitive to oxidative stress. Finally, the phenotypic resistance to streptomycin seen in the iron-limited strains ECA458 and ECA-LP was also seen in the ECA-fetMH and ECA-fetAH strains compared to WT. In ECA-fetMP this phenotype is intermediate, probably due to the enhanced iron uptake of this strain that leads to increase streptomycin uptake. In C. jejuni 81-176, expressing the whole Fet system (FetMP-FetAH) increases tolerance to streptomycin compared to expressing FetMP only (ΔfetABCDEF). However, supplementation with iron partially restores the resistant phenotype of the ΔfetABCDEF mutant (25). In contrast, integrating fetMP reduced resistance of the ECA-LP strain, while integrating fetMH or fetAH did not show significant changes regarding streptomycin resistance. The gain-of-function model used here has advantages for phenotypic characterization since genomic integration is stable and the natural promoter was used to express the fet genes. The growth enhancement seen in ECA-fetMP indicated that the gain-of-function approach was feasible to characterize the Fet system. However, the lack of phenotypes observed for strains ECA-fetMH and ECA-fetAH has limitations that should be considered. For instance, integration of the fet genes does not ensure their expression. It is possible regulatory mechanisms that limit the expression of the integrated genes are present in the non-pathogenic ECA-LP, but are not present in the source UPEC strain. These expression changes may be related to the integration of only fetMP in contrast to fetMP-fetAH. Characterizing the engineered strains with RT-qPCR to assess if the integrated fet genes are expressed and if the phenotypes can be related to the activity of the Fet system in E. coli would be an initial approach to validate the gain-of-function model. 40 4.4 Conclusion Characterization of the Fet system from UPEC in a non-pathogenic E. coli through a gain-of-function model provided new insights into the iron-deficient strain ECA458 and its response to multiple forms of stress. The engineering of ECA-LP generated a new strain for phenotypic characterization of genomically integrated genes in an iron-limited background. This was demonstrated by the integration of fetMP, building strain ECA-fetMP, which validated the role of FetMP in iron uptake. 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Thesis/Dissertation
eng
Microbiology and Immunology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Murphy, Michael E. P.
Characterization of an iron uptake system from uropathogenic Escherichia coli
Text