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Salmonella Typhimurium genes induced upon bacterial invasion into mammalian cells Pfeifer, Cheryl Gurine 1999

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SALMONELLA TYPHIMURIUM GENES INDUCED UPON BACTERIAL INVASION INTO MAMMALIAN CELLS by CHERYL GURINE PFEIFER B . S c , U n i v e r s i t y o f Saskatchewan, 1989 M . S c , U n i v e r s i t y o f Saskatchewan, 1992 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department o f M i c r o b i o l o g y and Immunology and the Bio technology Laboratory W e accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A u g u s t 1999 © C h e r y l G u r i n e Pfeifer, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of MKEOfclDiafrK / W T MtAUtiO LO 6rV The University of British Columbia Vancouver, Canada DE-6 (2/88) ii Abstract In causing gastroenteritis, Salmonella typhimurium are able to invade and grow within non-phagocytic intestinal cells. They are also able to survive within phagocytic cells, and have the potential to cause systemic disease. The ability of these bacteria to be intracellular is important in the disease process. Salmonella express an unique set of genes inside macrophages (not expressed with other stresses, e.g. heat, low p H , starvation), however, only a few have been identified. Therefore, the goal of this study was to search for Salmonella genes only expressed inside cells, thereby identifying genes essential for intracellular survival and perhaps virulence. A light-based reporter system was developed to specifically detect gene expression from intra-cellular bacteria. Vibrio harveyi luciferase genes, luxAB, were fused to Salmonella plasmid virulence genes, spvRAB, and the construct was shown to be upregulated after Salmonella invasion into epithelial cells. Upregulation was also demonstrated using a similar B-galactosidase (lacZ) reporter gene fusion. The results indicated luciferase was a sensitive reporter, able to monitor bacterial gene expression from within host cells, and able to differentiate live from dead bacteria. Subsequently, a library of S. typhimurium mutants was made by randomly inserting promoterless luxAB genes into the bacterial chromosome. Individual mutants were screened for luciferase expression (i.e. light production) within phagocytic cells. Extracellular bacterial mutants expressing light during growth in rich media or in the presence of cell-secreted factors were eliminated. From this screen, four S. typhimurium genes were identified as upregulated within both phagocytic and non-phagocytic cells. Three genes were found located within "Salmonella pathogenicity islands" (SPI): sigD/sopB and pipBwithin SPI-5 and ssaR within SPI-2. The sigD/sopB gene encodes an inositol phosphate phosphatase affecting host cell signalling pathways and chloride secretion. The ssaR gene potentially encodes a type III secretion apparatus component. Bacterial type III secretion systems are highly regulated and specialized iii f o r s ecre t ion o f bacter ia l pro te ins d i rec t ly into hos t c e l l s . T h e f o u r t h g e n e (iicA f o r i n d u c e d m t r a c e l l u l a r l y ) w a s c o m p l e t e l y n o v e l . T h e f o u r bac ter ia l mutants re ta ined their abi l i ty to i n v a d e a n d g r o w w i t h i n c u l t u r e d ce l l s , a n d a l l b u t iicA w e r e r e q u i r e d f o r v i r u l e n c e i n a m o u s e m o d e l . T h i s w o r k c o n f i r m s Salmonella p a t h o g e n i c i t y i n c l u d e s genes e x p r e s s e d in the i n t r a c e l l u l a r e n v i r o n m e n t . T h e abi l i ty to ident i fy s u c h g e n e s , a n d their fur ther character izat ion has l e d to an e n h a n c e d u n d e r s t a n d i n g about h o w Salmonella f u n c t i o n s as an intracel lular p a t h o g e n , i n a d d i t i o n to i d e n t i f y i n g s p e c i f i c v i r u l e n c e factors . iv Table of Contents Abstract ii Table of Contents iv List of Tables viii List of Figures i x List of Abbreviations xi List of Bacterial Genetic Abbreviations xiii Acknowledgements xvi Dedication xvii Chapter 1: Introduction 1 1.1. Salmonella and salmonellosis 1 1.2. Model Systems for the Study of Bacterial Pathogens 2 1.2.1. Bacterial culture 3 1.2.2. Cell culture 4 1.2.3. Animal models 7 1.3. Bacterial physiology 9 1.3.1. Secretion systems (Gram-negative bacteria only) 9 1.3.2. Virulence genes 13 a. Pathogenicity islands 15 b. Other virulence genes 17 1.3.3. Regulation of virulence genes 21 a. Environmental regulation 22 b. Genetic regulation 22 1.4. Reporter systems for bacterial gene expression 26 1.4.1. Reporter genes 28 a. B-galactosidase 28 b. Bacterial luciferase 28 V c. Other reporter genes 29 1.4.2. Other detection methods 30 1.5. Summary of thesis 32 Chapter 2: Materials and Methods 35 2.1. Media and chemicals 35 2.1.1. Chemicals and Assay Reagents 35 2.1.2. Antibiotics 36 2.1.3. Molecular Biology Reagents 36 a. Reagents 36 b. Primers 37 2.1.4. Media 38 2.1.5. Buffers 38 2.2. Equipment 39 2.3. Strains and Plasmids 40 2.3.1. Cells lines 40 2.3.2. Bacteria 40 2.3.3. Bacteriophage 41 a. Preparation of phage P22 stock 42 b. Transduction of Salmonella with phage P22 42 2.3.4. Plasmid Preparation 43 2.4. Molecular Biology 44 2.4.1. D N A Isolation 44 a. Plasmid preparation 44 b. Chromosome preparation 44 2.4.2. Basic Method of D N A Precipitation with Ethanol 45 2.4.3. Isolation of D N A from Agarose 45 a. Sephaglas Bandprep Kit 45 vi b. Freeze Squeeze Method for Isolating D N A Fragments 45 c. Spin-column Method for Isolating D N A Fragments 46 2.4.4. Electroporation 46 a. Preparation of electrocompetent bacteria 46 b. Electroporation 47 2.4.5. Two plasmid competition system 47 2.4.6. Inverse P C R 48 2.4.7. Sequencing 48 2.5. Invasion and Survival Assays 49 2.6. Reporter gene assays 50 2.6.1. B-galactosidase assays 50 a. B-galactosidase assay using fluorescent substrate 50 b. B-galactosidase assay using chemiluminescent substrate 51 2.6.2. Luciferase assay 51 2.7. Screen for Transformed Salmonella Exhibiting Low Luciferase Activity Outside Host Cells 52 2.7.1. Extracellular Bacterial Screen 52 2.7.2. Intracellular versus Extracellular Bacterial Screen 52 2.11. Mouse Studies 54 2.11.1. Typhoid Mouse Model 54 Chapter 3: Development of an Intracellular Reporter System 55 3.1. Results 55 3.1.1. Invasion assay to determine intracellular bacterial numbers 55 3.1.2. B-galactosidase as a reporter of intracellular bacterial expression 58 3.1.3. Luciferase as a reporter of intracellular bacterial gene expression 62 a. Measurement of light production 62 b. Effects of aldehyde concentration on bacteria and luciferase activity 65 v i i 3 .1 .4 .Compar i son o f 6-galactosidase and luciferase as reporters o f intracellular bacterial gene expression 71 3.2. D i s c u s s i o n 72 Chapter 4: Development o f Screen for Bacterial Genes 76 4 .1 . Resul ts 76 4.1.1. Transformation o f Salmonella and Screen for Upregulated Bacter ia l Genes 76 4.1.2. Screening for Bacter ia l Gene Induction Inside Cul tured Macrophages . . 81 4 .1 .3 . Transfer o f Genes to Ensure Induction Phenotype is L i n k e d to Gene Insertions 85 4.2. D i s c u s s i o n 85 Chapter 5: Characterization of Genes Upregulated by Intracellular Salmonella 90 5.1. Results 90 5.1.1. Inverse P C R and Sequencing 90 5.1.2. Extent of Gene Express ion by the Mutants 95 5.1.3. Compar i son o f G r o w t h Rate of the Mutants 102 5.1.4. Compar i son o f Invasiveness o f the Mutants 104 5.1.5. V i ru lence o f Mutants in T y p h o i d M o u s e M o d e l 105 5.2. D i s c u s s i o n 108 5.2.1. D 1 1 H 5 (ssaR) 108 5.2.2. A I M (sigD/sopB) and E 1 2 A 2 (pipB ) 109 5.2.3. G 7 H 1 (iicA) I l l 5.3. Overa l l Conc lus ion 112 References 114 Append ix : D N A and Predicted Protein Sequences Surrounding Insertional Mutat ions 152 viii L i s t of T a b l e s Table 1: Primers for Inverse P C R 37 Table 2: Bacterial strains 41 Table 3: Bacterial plasmids 43 Table 4: Induction of expression of reporter enzyme fusions by intracellular S. dublin 71 Table 5: Growth of Bacteria Over Time in Various Conditions 103 Table 6: Relative Invasion of Bacterial Mutants Into Cultured Cells 104 Table 7: Bacterial dose affects kinetics of mortality of mice 107 ix List of Figures Figure 1: Schematic diagram of the type I, type II, type III, and type IV secretion systems of gram-negative bacteria 12 Figure 2: Schematic representation of Salmonella pathogenicity islands 18 Figure 3: Schematic representation of the Salmonella typhimurium chromosome, which is divided into 100 centisomes 27 Figure 4: The effect of the detergents Triton X-100 (1.0%) and SDS (0.1%) on the recovery of intracellular S. typhimurium from HeLa cells 57 Figure 5: Plasmid maps of pFF14 and pSPLUX 59 Figure 6: Expression of spvB::lacZ by bacteria inside non-phagocytic cells 60 Figure 7: Comparison of two different methods to detect bacterial light production: Luminograph LB980 versus X-ray film 64 Figure 8: Effect of aldehyde concentration on bacterial viability and light production 66 Figure 9: The action of different concentrations of gentamicin on the luciferase activity within bacteria 69 Figure 10: Detection of light production in Salmonella pSPLUX in an intracellular versus an extracellular environment 70 Figure 11: Schematic of the luciferase reporter gene cassette inserted within the bacterial chromosome, and the orientation of the primer pairs used for inverse PCR and subsequent D N A sequencing 77 Figure 12: Luminograph images of L B plates with S. typhimurium colonies transfected with both pTF421 and pFUSLUX plasmids 79 Figure 13: Luminograph images of bacteria expressing light from a single sample 96-well plate of mutants 83 Figure 14: Comparison of the luciferase activity from the P22-transductional mutants with the original S. typhimurium insertional mutants 86 Figure 15: D N A bands resulting from inverse PCR are visualized on a 1% agarose gel 91 X Figure 16: Position of luciferase gene insertions within known S. typhimurium genes 92 Figure 17: S. typhimurium mutants show increased light production inside mammalian cells 96 Figure 18: Comparison of light production of luciferase-expressing bacterial mutants exposed to different environmental conditions 97 Figure 19: Relative luciferase activity by extracellular and intracellular Salmonella mutants 100 Figure 20: Virulence of S. typhimurium mutants in typhoid mouse model 106 List of Abbreviations xi amp ampicillin B A L B . B M 1 cultured bone-marrow-derived macrophages from B A L B / c mice B A L B / c inbred Bagg albino mice; used for models of various bacterial infections c A M P cyclic-adenosine monophosphate CDC42 cell-division-cycle; small GTP-binding protein cfu colony-forming unit cm chloramphenicol D M E M Dulbecco's modified Eagle's medium D M S O dimethyl sulfoxide dNTP deoxy-nucleotide tri-phosphate E D T A ethylene-diamine tetra-acetic acid EEA1 early endosomal antigen 1 F B S fetal bovine serum F D G fluorescein-di-galactopyranoside HeLa human cervix epitheliod cell line ID50 bacterial dose at which 50% of cells or animals become infected IgA immunoglobulin A in vitro (Latin: in glass) here, to mean within labware or within cultured cells in vivo (Latin: in the living organism), here, to mean within the host animal IP intraperitoneal rv intravenous J774A. 1 cultured macrophage-like cells from B A L B / c mice J N K c-Jun N-terminal kinase L A M P iysosomal-associated membrane protein L B Luria-Bertani; rich bacterial media LD50 bacteria] dose at which 50% of animals die L P S bacterial lipopolysaccharide M D C K cultured Madin -Darby canine kidney cells M E M minimal Eagle 's medium M H C major histocompatibility complex M O I multiplici ty o f infection N A P S U n i t Nuc le i c A c i d and Protein Services U n i t at Univers i ty of Br i t i sh C o l u m b i a N P - 1 neutrophil peptide defensin 1 N r a m p natural resistance-associated macrophage rjrotein O R F open reading frame P B S phosphate-buffered saline P C R polymerase chain reaction P E T G phenyl-ethyl-thio-galactoside P M N polymorphonuclear cel l or neutrophil p o l y m y x i n - C A P cyclic-antibacterial peptides P V C polyvinylch lor ide S C V 5a/mone//a-containing vacuole S D S sodium dodecyl sulfate S O C r ich bacterial media containing tryptone, yeast extract and glucose str streptomycin T E buffered solution containing T r i s - H C l and E D T A tet tetracycline U B C Univers i ty o f Br i t i sh C o l u m b i a L i s t o f B a c t e r i a l G e n e t i c A b b r e v i a t i o n s xm A 1 A 1 S. typhimurium mutant wi th insertion in sopB/sigD gene agf thin aggregative f imbrial gene ahp a lky l hydroperoxide gene A I D A - l adhesin invo lved with diffuse adherence in E P E C atr acid tolerance response gene brk gene encoding Bprdetella resistance to complement cys gene encoding a protein component in the cysteine pathway D l 1H5 S. typhimurium S L 1 3 4 4 wi th insertion in ssaR gene E 1 2 A 2 S. typhimurium S L 1 3 4 4 wi th insertion in pipB gene emrR E-mul t idrug resistance gene; involved with low-energy shock adaptation envZ histidine kinase; sensor component of two component regulatory system O m p R / E n v Z E P E C enteropathogenic E. coli fhlA formate hydrogenlyase fim type 1 fimbrial gene fliA flagellar gene; specifically an alternative sigma factor flgM flagellar gene; specifically an anti-sigma factor fur ferric uptake regulator gene G + C total guanosine plus cytosine content of the D N A G 5 D 5 see: G 7 H 1 G 7 H 1 S. typhimurium S L 1 3 4 4 wi th insertion in iicA gene G 8 B 1 s e e : G 7 H l iic gene induced intra-cellularly inv invasion gene ipa invas ion p lasmid antigen gene XIV kat gene encoding catalase lacZ gene encoding B-galactosidase, from lactose operon Ipf l ong polar f imbrial gene luxAB genes encoding bacterial luciferase marR multiple antibiotic resistance gene mgt magnesium transport gene mutS mutator gene involved in methyl-directed mismatch repair ompR regulator component of two component regulatory system O m p R / E n v Z orf open reading frame P 2 2 Sa/mone/Za-specific bacteriophage pag PhoP-activated gene pef plasmid-encoded fimbrial gene phoP regulator component of two component regulatory system P h o P / Q phoQ histidine kinase; sensor component of two component regulatory system P h o P / Q pip pathogenicity island-encoded protein pmr p o l y m y x i n resistance gene prg PhoP-repressed gene rho gene encoding transcription terminator factor R h o rpoS gene encoding R N A polymerase alternative sigma factor 38 sap genes corresponding to sensitivity to antimicrobial peptides sec amino-terminal secretion signal of a protein selC selenocysteine t R N A gene; selenium metabolism sif Salmonella-induced filament gene sig Salmonella invasion gene sip Salmonella invasion protein gene sod superoxide dismutase gene XV sop Salmonella outer protein gene spa surface presentation of antigen gene SPI Salmonella pathogenicity island spi Salmonella pathogenicity island encoded gene spv Salmonella plasmid virulence gene ssa Salmonella secretion apparatus gene sse Salmonella secretion effector gene ssr Salmonella secretion regulator gene yop Yjersinia outer protein gene xvi Acknowledgements I would like to thank the many individuals who have helped me during my studies: : thank you to my supervisor: Dr. B. Brett Finlay - for his continued support, : thank you to the Finlay Lab - for help above and beyond the call of science... both present: Sharon Ruschkowski, Gwen Lowe, Elizabeth Frey, Olivia Steele-Mortimer, Rebekah DeVinney, Jean Celli, Sandra Marcus, Michelle Zaharik, Carrie Rosenberger, John Brumell, Leigh Knodler, Mitchell Uh, Ben Goh, Wanyin Deng, Myriam de Grado, Ursula Heczko, Annick Gauthier, Danika Goosney, Derek Knoechel, Yuling Li and past: Francisco Garcia del Portillo, Han Rosenshine, Ka Leung, Murry Stein, Scott Mills, Patrick Tang, Dan Wilson, Agneta Richter-Dalfours, Akio Abe, Jose Puente, Annette Siebers, Vida Foubister, Markus Stein, Dieter Reinsheid, Maryse St. Louis, Brendan Kenny, Graciella Pucciarelli, Oliva Ortiz-Alvarez, Carol Cordeiro : thank you to the departments of Microbiology and Immunology, and The Biotechnology Laboratory for the many learning opportunities, : thank you to the members of my supervisory committee: Dr. R. A. J. Warren, Dr. G. Spiegelman, and Dr. W. R. McMaster. My studies were made possible by operating grants to B. B. Finlay from the Medical Research Council of Canada. Dedication This thesis is dedicated to a number of special people: to my husband, Tom Pfeifer - for all his understanding and loving support, to my parents, Ken and Myrtle Morrison - who helped me believe in myself, to my grandma, Anne Borgeson - who always thought education was important. Chapter 1 1 C h a p t e r 1: I n t r o d u c t i o n 1.1. Salmonella a n d s a l m o n e l l o s i s Salmonella are Gram-negative bacteria wi th in the family Enterobacteriaceae, tribe Salmonellae, genus Salmonella, and (according to the E w i n g classification scheme) are divided into three species: typhi, choleraesuis, or enteritidis (168). S. typhi is solely a human pathogen, and is the causative agent o f typhoid fever (169, 252). Humans are the reservoir for this bacterium, and spread is most often through the consumption o f water contaminated with human feces. Symptoms o f typhoid fever may take from one week to one month after ingestion of the bacteria to manifest and are characterized by a sustained high fever, bacteremia fo l lowed by infection o f the bilary system and other tissues, and an initial constipation period fo l lowed by diarrhea, poss ibly bloody (169). The disease is often severe, wi th a death rate from 2-10% and a 2 0 % rate o f relapse. Even after the person has apparently recovered, bacteria may survive in the gall bladder and be shed for up to a year (even wi th antibiotics and surgery) or longer (168, 169, 274). S. choleraesuis is able to cause disease in both animals and humans, wi th the reservoir being farm animals, inc luding swine. In humans it causes a severe disease in the form of a prolonged bacteremia characterized by fever, ch i l l s , and anorexia. Gastroenteritis is not c o m m o n , however due to its systemic nature, focal lesions may develop in any tissue to cause osteomyelitis, pneumonia , pulmonary abscesses, meningitis or endocarditis. In patients wi th depressed immune systems (e.g. due to A I D S , organ transplantation, or cancer), S. choleraesuis is able to cause a severe typhoid- l ike disease (168, 274). S. enteritidis is the most common o f the three species, consist ing o f over 2000 serotypes according to the Kauffmann-White antigen classification scheme, and are almost ubiquitous in the environment (14, 20). They are found associated wi th many different types of animals and food products ranging from radish sprouts and eggs (227) to chicken and beef (14, 168, 274) . M o s t often the bacteria are referred to by their common serotype Chapter 1 2 nomenclature, i.e. S. typhimurium rather than S. enteritidis serotype typhimurium (20). Of the more than 2000 serotypes existing, only 40% of them account for over 95% of all the clinical isolates (168) and some clinically relevant serovars include: S. typhimurium, S. dublin, and S. paratyphi A. In humans, the ingestion of food or water contaminated with S. enteritidis results in gastroenteritis (274). Symptoms appear within 6 to 24 hr after ingestion and last up to a week, with severity varying from person to person, depending on serotype and dose ingested. The disease is characterized by initial nausea and vomiting followed by abdominal pain and diarrhea, with or without fever. After recovery, a person may continue to shed the bacteria in their feces for up to three months. Interestingly, antibiotic treatment of uncomplicated gastroenteritis has been reported to prolong the earner state (168). Most often the disease is self-limiting and localized to the intestinal tract, but in a small number of people, especially young children or immunocompromised patients, the infection may become systemic and thereby more severe (227). In rare cases, S. enteritidis serotypes have been implicated in systemic disorders, including arthritis (313), pancreatitis (8), endocarditis (168), pericarditis (168), and mediastinitis (234). S. enteritidis serotypes are also able to cause disease in a number of animals, resulting in symptoms which range from barely detectable to severe typhoid-like resulting in death. This has allowed for the development of animal models. For example, infection with either S. typhimurium or S. dublin causes gastroenteritis in humans, yet result in a severe typhoid-like disease in BALB/c mice (10, 156). The remainder of this introduction describes research concerning Salmonella enteritidis species, specifically S. typhimurium or S. dublin. Many of the aspects described below also apply to S. choleraesuis and S. typhi (218), although the differences will not be expounded on. 1.2. M o d e l S y s t e m s f o r the S t u d y o f B a c t e r i a l P a t h o g e n s The study of bacteria and their interaction with the environment and hosts is done through the use of different model systems, all of which have their advantages and their limitations. Chapter 1 3 1.2.1. Bacterial culture The growth of a pure bacterial culture in the lab can yield a wealth of information regarding the physiology of the organism. This is considered one of the most basic and essential aspects of bacteriology, and in the case of pathogenic bacteria, it is required to fulfill Koch's postulates to determine the cause of a disease (274). Specific biochemical and genetic information regarding the bacteria can only be obtained through the use of a pure culture. Furthermore, environmental cues can be controlled either individually or in combination, and thereby the specific bacterial responses to them determined. Some examples of parameters which can be tested include bacterial responses to changes in temperature, pH, osmolarity, nutritional requirements, resistance to complement, oxidative radicals, heavy metals, antibiotics, the production of toxins, and growth phase (115, 119, 295). However, the growth of a single culture in the lab does not reveal all aspects of how bacteria are able to survive within an environment or cause disease. Often, the synergism and competition with other living organisms in the environment cannot be duplicated in a test tube (in vitro). Furthermore, species or tissue tropisms associated with a particular bacterial disease cannot be deduced. It is the rule rather than the exception that clinical isolates of bacteria display very different phenotypes than those passaged many times over in the lab. It is clear that while bacteria are free-living entities, they are greatly influenced by their surroundings and spend a great deal of their time reacting to and altering their environment (115). Salmonella are able to survive in a wide range of temperatures and pHs. They are not only able to react to environmental cues (e.g. pH, temperature, organic and inorganic substrates, oxidative radicals, etc.) (295), but are able to interact with and actually communicate at the cellular level with animate entities such as other bacteria, and even plants and animals (97, 250). It is this interaction and communication (or 'argument') between bacteria and host that is the basis of disease in salmonellosis, and most other bacterial diseases. The chemical signaling between bacteria and other organisms is very complex and is extremely difficult to reproduce in the lab with a pure bacterial culture. For example, the bacteria are able to survive in contaminated Chapter 1 4 water and food products. Once ingested, they are subjected to the salivary enzymes of the mouth and then the bile salts and low pH of the stomach. After the shock of the stomach, the bacteria pass through into the small intestine where they encounter the peristaltic movement of the intestines and the mucus continually moving along the ciliated brush border lining the intestinal surface. Furthermore, the ingested bacteria encounter many other microorganisms that are already resident in the intestine. The Salmonella are able to bypass these barriers, to interact with the intestinal cell surfaces and ultimately engineer their own uptake into these cells. Within the host cells, Salmonella remain inside a vacuolar space, where they must adapt in order to survive. 1.2.2. Cell culture To avoid confusion throughout this discourse, bacterial cells will not be referred to as "cells" but as simply "bacteria"; while host cells will be referred to as "cells". The interactions of bacteria with host cells can be studied more closely with the use of cultured mammalian cells. There are a number of host cell types available for study, from various tissues, and from various types of animals. Examples of epithelial cells include HeLa cells which originated from a human cervical tumor and MDCK cells which originated from immortalized canine kidney cells. Examples of phagocytic cells include J774A.1 and BALB.BM1 cells, which are both mouse macrophage-like cells originating from BALB/c mice. The discovery of numerous Salmonella virulence factors has been made through the controlled infection of cultured host cells (22), specifically many factors required for bacterial invasion of cells and for survival' within cells. The discovery of a new bacterial protein secretion system, the type III secretion system (TTSS), is especially noteworthy since bacterial effectors are secreted into host cells predominantly upon contact of the cells with the bacteria (188, 208). Salmonella were long thought to produce disease from extracellular locations within the body for a number of reasons. The bacteria could be isolated from fecal and blood samples, and in EM studies, the destruction of S. typhimurium within host polymorphs and macrophages was seen (160). Moreover, the bacteria were able to replicate in extracellular Chapter 1 5 spaces, such as the peritoneal cavity (160). Over the past decade, there has been a shift, and many researchers now believe that the ability of Salmonella to persist intracellular^ greatly influences the extent and severity of the disease (51, 76, 81, 85, 195, 247, 263). The intracellular stage appears to be especially important as mutants which either cannot invade cells or survive within cells are attenuated (76, 195, 247). However, the bacteria are able to move via flagella (171) and adhere to cells via various fimbrial adhesins and pili (183, 324). They ultimately engineer their own uptake into the intestinal cells, targeting the M cells of Peyer's patches (165, 288). During invasion Salmonella induces selective aggregation and internalization of host cell surface proteins, such as M H C class I heavy chain, fibronectin-receptor, CD-44 and B2-microglobulin (109). The bacteria also trigger host cell signaling pathways in a CDC42-dependent manner (42, 172), (although uptake is Fc receptor independent (205)), that lead to cytoskeletal and nuclear responses resulting in the uptake of the bacteria (5, 90, 91, 267, 272). The mechanisms required for the invasion of cells by Salmonella and for their subsequent survival within cells are complex and require the coordination of a number of bacterial factors (see virulence factors section below) (79). In culture, Salmonella are able to enter every mammalian cell type tested (81, 328). However, in order to separate the bacteria-mediated invasion from phagocytic uptake, invasion studies have used non-phagocytic epithelial cells (79). There is evidence that similar events take place in phagocytes and that the bacteria mediate their own uptake into these cells as well rather than relying on Fc-receptor or complement-receptor mediated uptake (110). Salmonella do not bind intimately to the cell surface in order to enter into cells, rather the bacteria initiate a splash or membrane-ruffling event which causes the cell to macropinocytose everything within the immediate vicinity (90, 105, 169). The bacteria are thus taken up into a large spacious vacuole, where they can be seen to actively move around (4, 5, 84). Once the bacteria have been engulfed, the cell surface returns to normal (84). Inside the cell, the Sa/moneZ/a-containing vacuole (SCV) does not traffic in the same manner as would a regular vacuole and the intracellular environment of the SCV is actively Chapter 1 6 modif ied by the bacteria (31, 108). W h i l e the p H o f the vacuole is lowered, it only falls to around p H 5.0, rather than to p H 4.0 (3, 108). There is evidence that whi le the S C V s do accumulate some o f the markers o f the regular trafficking pathway ( E E A 1 indicating fusion wi th early endosomes; lysosomal glycoproteins and L A M P s indicating fusion with late endosomes (219, 240, 241) and N r a m p l (220, 284)), they prevent the accumulation of other markers in any great amount such as cathepsin D or the mannose-6-phosphate receptor. It was earlier reported that the mannose-6-phosphate receptor was not associated wi th the S C V at all (106, 256), but recently more sensitive evidence indicates that as many as 10% o f S C V s may contain this marker; however this is still significantly different from the normal trafficking pattern seen wi th a cell (219). Abou t 4-6 hr after invas ion, the cell is seen to produce filamentous structures wh ich extend out from the S C V , called Sifs (Salmonella-induced filaments), wh ich correspond to bacterial replication wi th in the cells (195, 221, 299). The Sifs can be labeled wi th lysosomal glycoprotein, and are connected to the S C V . They are not actually large enough to contain a bacterium although bacterial membrane blebs are found wi th in these structures (299). Other stimulated pathways include inositol phosphate signaling pathways (270) and those stimulated by rapid calc ium fluxes within cells (65, 243), although cycl ic adenosine 3':5'-monophosphate ( c A M P ) levels do not increase (37). The result is the secretion o f fluid into the intestine and the influx o f neutrophils to the site o f infection, resulting in diarrhea (78, 206). Furthermore, Salmonella are toxic to cel ls . Invasion o f the bacteria into macrophages induces apoptosis, w h i c h can be seen both in vitro (224) and in vivo (263). Al though ultimately more complex than the study o f single bacterial cultures, the limitations o f us ing cel l culture are very s imilar , and this model is still considered an in vitro model . Cul tured cells exist in an artificial environment where they cannot interact wi th other cell types for st imulation, cross-feeding, differentiation, or to get r id o f toxic wastes. Often they are transformed such that they w i l l not have a limited life-span but w i l l be able to be passaged many times, s imi lar to the passage of bacteria. However , as occurs wi th bacteria, the Chapter 1 7 more a cell is passaged in a lab setting, the more it is removed from specific contacts, hormones and other chemical gradients, the less likely it is to resemble the tissue from which it was taken. The use of primary cell cultures for study helps to lessen this 'de-differentiation effect', although they are more difficult to work with in the lab and often their availability is limited. Similarly, combined cell and bacterial cultures are difficult to work with and the results difficult to interpret because there are many variables, known and unknown. 1.2.3. Animal models Ultimately, the best way to determine bacterial virulence and tissue specificity is to infect the host. In place of experimenting on humans, animal models are used to reproduce a given disease and determine whether a specific bacterial factor, or conversely a host factor, may increase or decrease the extent of bacterial pathogenesis. A good animal model requires that the bacterial infection can be established reproducibly. However this trait may actually make them less sensitive when assaying for bacterial virulence, as the animals are often deficient in some way or immunologically immature in order to give the bacteria an edge. Furthermore they are fairly crude measures of virulence as they reflect the cumulative effects of many steps including bacterial colonization and the production of symptoms. This makes it possible to overlook important virulence determinants. There are a number of ways to determine virulence using an animal model. Traditionally researchers have used a whole animal model, looking at either the infectious dose (ID50) or the lethal dose (LD50) (274). The infectious dose (ID50) is the number of bacteria necessary to infect 50% of the exposed animals. This parameter only reflects the infectivity of the bacteria and not the morbidity of the disease resulting from infection. The lethal dose (LD50) is the number of bacteria necessary to kill 50% of the infected animals. A variation of this involves the determination of the day on which 50% of the animals died. Other parameters also used as indicators of virulence include the measurement of signs of disease such as weight loss, diarrhea, presence of bacteria in animal fluids (e.g. blood, urine, cerebrospinal fluid) or organs (e.g. spleen, liver), or the extent of the immunological response (e.g. P M N influx, Chapter 1 8 number and size of infection foci). Another model is the ileal loop model, where the intestine within an anesthetized animal is tied off and the bacteria injected directly into it. Only more localized effects of the bacterial infection can be determined with this model (i.e. fluid accumulation or P M N influx), however it has the advantage in that more than one bacterial strain at a time may be analyzed within the same animal. For salmonellosis, a number of animal models have been used. The most widely used model involves the infection of B A L B / c mice with S. typhimurium resulting in a disease which resembles typhoid fever in humans (233, 306). The B A L B / c mice are particularly susceptible to intracellular bacterial infections, which has been attributed to a defective host resistance locus nrampl (formerly known as the ity/bcg/lsh locus) (64, 116, 327). Other animals used include guinea pigs (160), rabbits (330), chickens (53, 142, 318), pigs (123, 314), sheep (27), and cows (94, 330). In the mouse, Salmonella are able to cause disease when given by various routes. The bacteria may be induced orally, which is the route thought to most mimic the natural course of infection (112, 156). A typical L D 5 0 is around 1 x 10 6 bacteria per mouse (195, 299, 306). The bacteria are forced to contend with not only the acid and gastric juices within the stomach, but also with various cellular barriers. Although Salmonella invade many cell types in vitro, the bacteria appear to be targeted to the membranous cells (M-cells) in vivo (94, 156). M-cells are specialized cells, found in Peyer's patches within the intestine, which are thought to internalize luminal materials for presentation to underlying antigen-presenting cells (e.g. monocytes, macrophages, and neutrophils). Salmonella are able to pass through the intestinal cell layer to the sub-layers (83, 85, 170), where they are able to colonize (204) and/or are picked up by waiting macrophages. The bacteria are able to survive within phagocytes (263), and possibly travel to other organs via this intracellular route (57). Using this route of infection, factors required for invasion are often detected as their loss results in a loss in virulence. When bacteria are injected intraperitoneally (IP) into mice (10), they no longer have to deal with these particular barriers, nor do they have to compete with the normal intestinal Chapter 1 9 flora. Consequent ly , the number o f Salmonella required to cause disease is reduced us ing IP inoculat ion, wi th an LD50 around 100 bacteria per mouse (299). A s w e l l , bacteria with mutations in invasion genes are not attenuated for infection resulting f rom IP inoculat ion, but those wi th mutations in genes needed for subsequent bacterial growth wi th in cells are attenuated. Intravenous ( IV) inoculation o f Salmonella into mice bypasses the most barriers, putting the bacteria into direct contact wi th circulating macrophage cells and prov id ing the most direct route to the organ o f choice in wh ich to establish an infection (e.g. l iver and spleen). A g a i n , the number o f Salmonella required to cause disease is drastically reduced, wi th an LD50 around 10 bacteria per mouse (131, 263). 1.3. Bacterial physiology 1.3.1. Secretion systems (Gram-negative bacteria only) The type I sec-independent secretion system involves the three components: an inner membrane transport A T P a s e (coined A B C protein for A T P - b i n d i n g cassette), a periplasmic-spanning protein w h i c h is anchored in the inner membrane, and an outer membrane protein which is secreted v ia a sec-dependent pathway (80, 161, 208). The genes encoding the secretion apparatus and the secreted protein are usually clustered. Proteins secreted via the type I pathway contain the signal for secretion in their carboxy terminus, however this signal appears to vary sl ightly between subfamilies o f the secretion system. In contrast to the sec-dependent secretion pathways, this carboxy signal sequence is not cleaved off and there is no periplasmic intermediate o f the secreted protein. Examples of proteins secreted v ia this pathway include the subfamily o f hemolysins Escherichia coli a lpha-hemolysin, Bordetella pertussis adenylate cyclase, and Pasteurella haemolytica l eukotoxin , as we l l as the subfamily o f proteases from Pseudomonas aeruginosa and Erwinia chrysanthemi (80, 161). There are no reported Salmonella proteins secreted by this pathway so far, however the outer membrane component o f the secretion apparatus has been described and is functionally interchangeable wi th the E. coli homolog ( T o l C ) (305). Chapter 1 10 Both the type II and type IV secretion pathways are sec-dependent and involve two separate steps for transport across the inner and outer membranes. The first step (the sec-dependent step) is energized by ATP hydrolysis and requires the protein to have an amino-terminal signal sequence which is cleaved off during export to the periplasm. Components of this first step include a number of inner membrane proteins, an inner membrane associated ATPase, a chaperone to bind presecretory target proteins, a periplasmic signal peptidase, as well as a number of accessory proteins (161, 208). Type II secretion is thought to be the main export pathway for gram-negative bacteria and is known as the generalized secretory pathway (80, 208, 273). The second step for translocation through the outer membrane includes a periplasmic chaperone and a number of outer membrane associated proteins which multimerize to form a channel (273). It is thought that the secretion signal for this step may be encoded by multiple segments on the protein (273), and no signal sequence cleavage occurs. Secretion components usually are encoded by about 13-15 clustered genes. The type II secretion pathway is primarily used for the secretion of degradative enzymes. Some examples of proteins secreted via this pathway are Klebsiella oxytoca pullulanase, P. aeruginosa phospholipase C, exotoxin A , and elastase, and Xanthomonas campestris polygalacturonase (161). Proteins which use the type IV secretion pathway are able to induce their own transport across the outer membrane, and are therefore also known as auto transporters (80, 149). Once through the inner membrane, the proteins are able to associate with the outer membrane to form a pore and mediate their own passage out of the cell, in the absence of any energy coupling or accessory factors. Once outside, the protein may remain associated with the outer membrane or be cleaved off. Examples of proteins secreted via this pathway include gonococcal IgA protease, Haemophilus influenzae IgA protease, Helicobacter pylori vacuolating cytotoxin, enteropathogenic E. coli EspC, and Bordetella pertussis pertactin and BrkA (80, 149, 161) The type III secretion pathway is especially important for the specific secretion of a number of virulence factors. This secretion system has been found to be conserved amongst a Chapter 1 11 number of gram-negative pathogens including Salmonella (80, 98, 268), Shigella (98, 268), Yersinia (268), E. coli (80), Erwinia (325), and Pseudomonas (311, 325) species. Proteins of the type III secretion system, as well as many of the proteins to be exported, are contained within large regions of D N A dubbed pathogenicity islands (111, 208). These regions may be chromosomally encoded, as in Salmonella, or contained on the virulence plasmid, as in Yersinia. Type III secretion involves more than twenty components, many of which are homologous to those involved with flagellar assembly, including a multimeric ATPase and various proteins involved in the synthesis of a specialized surface organelle (113, 182). Amino terminal signal sequences target proteins to the secretion machinery, although the secretion sequences are not cleaved (188). There is some evidence to indicate that some secretion information is contained within the structural configuration of the mRNA (7), and that at least for some proteins (but not all (96)), secretion and translation may be coupled (188). Each protein to be secreted also associates with a corresponding specific cytosolic chaperone. The whole system is highly regulated and proteins are only secreted upon the bacteria receiving specific environmental cues. The method is sec-independent and proteins are secreted across both the inner and outer membranes in one step. Furthermore, proteins may be secreted from the bacteria directly into host cells, where they can influence host cell signaling. In contrast to other secretion systems, many of the structural components of the type III system are considered to be virulence genes rather than housekeeping genes because their only function appears to be the selective secretion of proteins required for pathogenicity in the host (188). Moreover, mutations within these structural genes often leads to reduced bacterial virulence without greatly affecting bacterial growth in broth culture. Chapter 1 12 s CD 0> a ra •z. g ro O - i 0) X = s o c ra sL 5 E 2 E Cl o s. 0 2 CO 0) E 2 a ro 0 = = 0 o = = o r ii CV •o c i s s 8 01 01 9 j IX ~ o> o 11 ex £ e g c i a i i 3 £ & a 3 !_ C Z CJ £ $ I a •>-. — 5 = Ol 0> 2 s a 01 . .5 cs * = — 0> 0) — a >. * • — o — 01 Ol 0> "S & E | 11 «I E | u = d5 < Is • s t. 3 E 5 2 a * 1 ^ i_ > ** CX •a § • 3 — "O S £ C 9 E 5 01 ~ I 2 u c 11 i t t 3 IX LZ Chapter 1 13 1.3.2. V i r u l e n c e genes a . P a t h o g e n i c i t y i s l a n d s The definition of a 'virulence' gene is a gene that is required by the bacterium to specifically cause disease in the host or to subvert the host's immune system. This differs from a 'housekeeping' gene which is required for maintenance of regular bacterial functions for growth, D N A division, protein production, nutrient acquisition, etc. Generally, if a mutation is made within a virulence gene, the bacteria wil l show increased susceptibility within the host, but wi l l not be greatly affected during growth in regular rich media (122). While virulence genes can be found scattered throughout the Salmonella chromosome, the latest research has shown the existence of large regions of D N A encoding groups of genes which appear to coordinate various aspects of pathogenesis known as pathogenicity islands (122). These islands have also been described in other gram-negative pathogens and frequently include the genes needed for the type III secretion system, including the L E E (locus of enterocyte effacement) region of pathogenic E. coli (67, 249), the two P A I (pathogenicity island) regions of uropathogenic E. coli (264), the Mxi /Spa region of Shigella (161, 268), the Exs/Psc region of P. aeruginosa (161), the Hrc/Hrp region of Ralstonia solanacearum (161, 325), and the virulence plasmid-encoded Lcr /Vi r region of Yersinia (161, 325). Pathogenicity islands are clusters of horizontally acquired virulence genes which are generally found inserted at t R N A loci (24) and have a G+C content that is lower than that of the bacterial chromosomal D N A (around 52%). Within Salmonella there have been five Salmonella pathogenicity islands described so far (SPI-1, -2, -3, -4, and -5), although only two encode the machinery for type III secretion. The genes within these islands are important for coordinating and secreting other virulence factors encoded outside of these specialized areas on the bacterial chromosome. In S. typhimurium, SPI-1 is a 40 kb region located at 63 minutes on the chromosome between mutS and flilA (49, 217). SPI-1 appears to be present in all S. enterica subspecific groups (238). The overall G+C content of the gene cluster was found to range from 42-47%. SPI-1 has been shown to be especially important for bacterial invasion of epithelial cells and Chapter 1 14 M-cells (6, 47, 49, 98, 99, 331). This active invasion of cells by the Salmonella is in contrast to the antiphagocytosis activity of Yersinia species (207). Many of the SPI-1-encoded genes are also required for cytotoxicity in macrophages (43) and for virulence in vivo. This region contains genes encoding components of a type III secretion system which encode structural proteins (invA, invC, invG, invJ, spaO, spaP, spaQ, spaR, spaS, prgH, prgl, prgj, prgK, and orgA) (50, 99, 174, 277, 304), secreted proteins (avrA, sptP, spaO, invJ, sipA, sip/sspB, sip/sspC, and sip/sspD) (48, 50, 162, 175, 176, 277) and their corresponding chaperones (sicA, sicP , invl, and prgP)(95, 98), and proteins for regulation of secretion (sip/sspD, and invE) (162). Proteins required for regulation of gene expression are also encoded within the SPI-1 region (Hi lA and I n v F ) ( l l , 174). Additional proteins secreted by the SPI-l-encoded type III secretion system (e.g. SopB/SigD, SopE, SopD), as well as additional regulatory factors of this region (e.g. PhoP/PhoQ), are located in unlinked chromosomal locations (60, 101, 173). Many of the SPI-1 secreted proteins are translocated directly into the host cell and elicit direct effects on the host cell signal transduction pathways. SptP has tyrosine phosphatase activity within host cells which disrupts the actin cytoskeleton, likely freeing up G-actin monomers necessary for the formation of host membrane ruffles during Salmonella invasion (96, 177). SptP also has sequence similarity to the cytotoxins YopE from Yersinia spp. (268) and ribosyltransferase exoenzyme S from P. aeruginosa (177). A v r A has significant sequence similarity to A v r X , an avirulence protein from the plant pathogen Xanthamonas campestris pv. vesicatoria, and to YopJ/P from Yersinia spp. (311, 325). SipB and SipC are also secreted and are thought to affect the host actin cytoskeleton, as deduced by their ability to block Salmonella invasion when they exist as membrane-associated receptor chimeras in the host cell (36). They are further required for secretion of other proteins into host cells via the SPI-1 type III system (96). Without SipB and SipC, bacteria are able to secrete proteins but are not able to translocate them into host cells (339). InvH also appears to be important for secretion of proteins via the SPI-1 type III system, however its mechanism is unclear. It may Chapter 1 15 be acting to post-translationally modify proteins to be secreted via the type III system, but it has not been shown to be secreted itself (55). SPI-2 is located at 31 minutes and also encodes components of a type III secretion system (150, 286). SPI-2 is present in most subspecific groups of S. enterica, but not all (238). It is interesting to note that while cross complementation between type III systems of different species can occur, e.g. Salmonella sipB can complement a Shigella ipaB mutant (98, 154) and a Shigella spa24 could complement a Salmonella spaP mutant (121), many of the type III components of SPI-2 do not compensate for mutations in the components of SPI-1 within the same bacterium. However, this may reflect other factors, such as regulation of each SPI, since certain mutations in SPI-2 affect the transcription of SPI-1 genes and the ability of S. typhimurium to secrete SPI-1 effector proteins and to invade cultured cells as well (60). The function of the genes within SPI-2 appear to be divergent from those of SPI-1, and are necessary for intra-macrophage survival and dissemination throughout the host rather than invasion. Mutations within various SPI-2 genes resulted in reduced systemic spread of the infection within the mouse, although colonization of the intestinal Peyer's patches was still seen (46). A recent report has further implicated the SPI-2 region as necessary for bacterial replication in the host rather than bacterial survival per se (285). The secretion apparatus (ssa) is encoded within two regions: a 10 kb operon containing the genes ssaK, L, M, V, N, O, P, Q, R, S, T, and U (152) and a second smaller operon containing the genes spiC, A, B (239) (ssaED, C, B) (153). These two regions are separated by a region containing the potential effector (^e)/chaperone genes sseA, B, C, D, E, F, and G (153). The regulatory genes, ssrA and ssrB, are transcribed in the opposite direction to all the other genes within SPI-2. While many of the potential effector proteins have not been shown conclusively to be translocated into host cells, their expression is upregulated by intra-macrophage Salmonella (46, 321, 322). Mutations within various structural genes have been observed to have pleiotropic effects resulting in the increased sensitivity of the bacteria to complement, gentamicin, and polymyxin (60). Chapter 1 16 SPI-3 (24) is a 17 kb region located at 82 minutes which is inserted within the selC t R N A locus. It contains at least, two genes which contribute to bacterial survival within macrophages, mgtCB (24). Although the G+C content of mgtCB is similar to the overall G+C content of 52-54%, they are immediately surrounded by regions with lower G+C content, (39.8 and 49.3%). These genes are transcribed in an operon and enable Salmonella to transport magnesium in low M g 2 + conditions (225). Although Salmonella contain other magnesium uptake systems, mgtCB are tightly regulated by the PhoP/PhoQ two-component regulatory system (see below) and required for full virulence in mice (312). It has been suggested that MgtB has another function besides magnesium transport that is required by the bacteria inside host cells (290, 291). SPI-4 is a 27 kb region located at 92 minutes on the S. typhimurium chromosome which is inserted between ssb (encoding the single-stranded D N A binding protein) and SoxSR loci (encoding a superoxide regulatory gene) at sites encoding a tRNA-like gene (337). There are two 9 kb regions which have lowered G+C contents (37% and 44%), and one 7 kb region with a G+C content of 54%. SPI-4 encodes for 18 putative proteins, including seven with homology to type I secreted toxin proteins including Pasturella haemolytica leukotoxin and Bordetella pertussis adenylate cyclase-hemolysin. This region is also important for intramacrophage survival. A n insertional mutation in this region renders the bacteria unable to grow within macrophages (13) SPI-5 is an 8 kb region located at 25 minutes in S. typhimurium and in S. dublin (159, 338). It is also present in other Salmonella serovars but not in Shigella sonnei, E. coli, or Yersinia pseudotuberculosis. This island appears to consist of six genes: pip D, orfX) sigD/sopB, sigE /pipC, pip B, and pip A, which are flanked by the t R N A serT on one side and copS on the other side. Of these genes, sigD/sopB is secreted by the SPI-1 type III secretion apparatus and sigE /pipC is thought to be its specific chaperone (101, 159). In S. dublin, SopB has been shown to be an inositol phosphate phosphatase which acts to increase chloride secretion by the host cell. Activities of SopB include the hydrolysis of phosphatidylinositol Chapter 1 17 3,4,5-trisphosphate, an inhibitor o f calcium-dependent chloride secretion and o f inositol 1,3,4,5,6-pentakisphosphate, an indirect inhibi tor of phosphatidylinositol 3,4,5-trisphosphate-dependent chloride secretion (235). Mutations wi th in the S. typhimurium homologs , sigDE, are reported to reduce bacterial invas ion by ten-fold (159), however this is not observed wi th the S. clublin genes, sopB and pipC. Nonetheless , the enteropathogenicity o f S. dublin is greatly reduced in calf ileal loop models wi th mutations the sopB and all the pip genes, although systemic disease appears unaffected in a mouse model (338). b . O t h e r v i r u l e n c e genes S o p E is secreted by the type III secretion system of the SPI-1 (140, 339), yet it is encoded outside o f centisome 63 wi thin a cluster o f genes from a cryptic P2- l ike prophage (141). The sopE gene was not found to be present in all S. enterica serovars: S. dublin, a number o f S. typhimurium isolates, as we l l as S. typhi contained the gene, whereas S. choleraesuis d id not. The S o p E protein is secreted into the host cell cytoplasm in a ^ - d e p e n d e n t manner (339) where its actions include the activation of cytoskeletal rearrangements along a CDC42-dependent pathway (140) and the activation of J N K . Furthermore, S o p E was able to b ind to several (but not all) members o f the R h o subfamily o f G T P a s e s to promote nucleotide exchange in vitro (140). Altogether, S o p E is capable of stimulating signal ing pathways that can lead to both nuclear and morphological responses by the host ce l l (140). S o p D (in S. dublin) is translocated into host cells and is secreted without amino-terminal processing, however direct secretion v i a the type III pathway has not been shown. It is located downstream of the cysJIH operon, outside o f the k n o w n pathogenicity islands. S o p D acts in concert wi th S. dublin S o p B ( S i g D in S. typhimurium) to cause enteropathogenicity in the host, although the nature of its activity remains obscure (173). A number o f flagellar genes have also been implicated in Salmonella virulence (82). W h i l e flagellar mutants are still able to invade cells wi th high efficiency in vitro, the ability to Chapter 1 Chapter 1 19 smooth s w i m has been shown to be advantageous during the initial interactions wi th the cells (171). Smooth s w i m m i n g may cause the formation of propulsive bundles and free the majority of the bacterial surface from steric constraints, wh ich may occur when the flagellar filaments are randomly distributed during tumbly s w i m m i n g . Furthermore, the s w i m m i n g motion may help the bacteria propel through mucus layers towards the cells. Other flagellar genes involved wi th regulation, such as the alternative s igma factor F l i A and a corresponding anti-sigma factor F l g M , may act to coordinately regulate diverse virulence-associated genes (283). M u l t i p l e f imbr ia l adhesins, i nvo lved wi th the ini t ial adherence of S. typhimurium to the intestinal mucosa, are required for full virulence of the bacterium in mice (324). There are at least four f imbrial operons k n o w n in S. typhimurium: Ipf ( long polar finbriae) (324), pef (plasmid-encoded fimbriae) (324), fim (type 1 fimbriae) (70, 82, 248, 324), and agf (thin aggregative fimbriae) (265, 307, 324). Mutat ions in any one of these operons only moderately affect virulence, but a combination o f mutations within all four operons results in more than 20-fold attenuation o f the bacteria when given orally to mice. H o w e v e r , there is evidence o f other f imbrial structures and/or adhesive organelles wh ich may a l low for colonization of the intestinal cells in the absence o f the fimbriae encoded by the k n o w n operons (324). A cell-contact-stimulated formation of filamentous appendages has been implicated in the triggering of bacterial entry into host cells (258) and these appendages have been termed "invasomes" (113, 340). The formation o f these contact-stimulated filaments is thought to be secreted v ia a type III secretion system, however they are formed in the absence o f the SPI-1 type III secretion system, and their presence is not itself sufficient to trigger bacterial invasion (258). Resistance to cationic antimicrobial peptides is mediated either directly or indirectly through P h o P / P h o Q regulation of a number o f genes including pagB-pmrAB, sapE, G, H, J, L, M, and various L P S biosynthetic genes (117, 120, 245). The expression o f these genes increases Salmonella resistance to the p o l y m y x i n - C A P family of peptides (216), and may have effects on other systems as w e l l , e.g. expression o f pmrE also results in modifications to the l ip id A portion o f L P S (134, 136, 148). A gene not regulated by P h o P , sapM, is involved Chapter 1 20 with resistance against the neutrophil peptide (NP-1) defensin (135). Resistance to antimicrobial peptides may be effected via an energized efflux pump system (117), biochemical alteration/inactivation of the peptide (117), or the prevention of peptide binding to the bacterial surface by L P S (319). Salmonella resistance to complement includes a number of other factors such as many of the SPI-2 genes encoding type III secretion apparatus, the virulence plasmid-encoded Rck protein (45, 145), as well as L P S structure. L P S oligosaccaride side chains act as a physical barrier to complement such that the components are deposited away from the membrane and the pore-forming complex cannot penetrate the bacteria (82, 223). Reactive oxygen intermediate-mediated damage, such as lipid peroxidation and amino acid carboxylation, is countered by Salmonella in a number of ways. A bacterial copper- and zinc-cofactored superoxide dismutase SodC (mapping between centisome 23.8 and 29.6) prolongs bacterial survival in the presence of superoxide in vitro and within a mouse model (75). SodA, a manganese-cofactored superoxide dismutase, may confer resistance to the early oxygen-dependent microbiocidal mechanisms of phagocytes, especially in the absence of iron (316). Resistance to alkyl peroxides is conveyed via an alkyl hydroperoxide (ahp ) (93), while resistance to hydrogen peroxide is conferred by catalase (katG and katE), as well as by a 59 kD protein (303). Reactive nitrogen intermediates are combated by homocysteine production by Salmonella which may directly interact with S-nitrosothiols such as S-nitrosoglutathione (59). S-nitrosothiols act as nitric oxide donors and have also been shown to have broad-spectrum microbiostatic activities themselves (59). Glucose 6-phosphate dehydrogenase has been shown to protect S. typhimurium against both oxygen and nitrogen intermediates in vitro, and is important during the early stages of salmonellosis (200). L P S and various LPS-biosynthetic genes play an important role in the virulence of Salmonella. Not only do they act to provide resistance to cationic peptides and complement, but the lipid A portion of L P S is toxic to host cells and causes an endotoxic shock in the host. Chapter 1 21 LPS further is able to act as a chemokine and a macrophage activation factor (16), and may also be involved with the ability of Salmonella to transcytose cell layers (85). Other factors known to be important for the survival of S. enterica species within the host are extra-chromosomal and found to be encoded within a large virulence plasmid (50-90 kb) (21, 128, 242). The Salmonella-plasmid virulence genes, encoded by the operon spvRABCD (129, 181), are upregulated within cultured cells (77) although they have not been found to be important for intracellular growth in vitro (128). These genes are important for extracellular growth under nutrient-limited conditions however (253). They are not required for bacterial invasion into cultured cells (44, 232, 330), but promote survival and growth of the bacteria within animals thereby increasing bacterial virulence (127, 130, 131, 327). The operon is regulated by a number of factors which include nutrient limitation (66, 72), short-chain fatty acids (66), RpoS (66), and SpvR (54, 296). SpvR is the operon regulator and is able to upregulate its own expression, while SpvA downregulates the operon (296). SpvB, C, D are thought to be membrane-associated proteins, although their activities remain obscure. The virulence plasmid may be involved with the lysis of resident and activated peritoneal macrophages in vivo which could influence the net growth of Salmonella during infection (124). 1.3.3. R e g u l a t i o n o f v i r u l e n c e genes It is known that bacterial pathogens, such as Salmonella, tightly regulate the expression of many of their genes required for virulence. Many of these genes are coordinated in response to very specific environmental conditions, e.g. in response to heat or osmolarity, and often there are multiple regulators for a particular virulence gene such that its expression, and subsequent translation, are in response to a variety of environmental parameters. The expression of Salmonella invasion genes are in response to specific environmental conditions resulting in the production of new proteins immediately prior to bacterial invasion (201). Chapter 1 22 a . E n v i r o n m e n t a l r e g u l a t i o n The environment provides many clues to pathogenic bacteria. Temperature, specifically the normal body temperature of the host, often serves to activate virulence genes, e.g. Yersinia only make Y o p s at 3 7 ° C , not at 2 6 ° C (211). Interestingly, the Salmonella virulence genes do not seem regulated to any great extent by temperature. p H serves to activate numerous Salmonella v i rulence genes (3, 211, 257), inc luding Fur-regulated genes wh ich are activated at l o w p H (317) and many o f the type III secretion genes in SPI-1 wh ich are expressed under slightly alkaline conditions (161). Starvation for different nutrients {e.g. carbon, nitrogen) results i n the activation o f R p o S regulated genes such as spvRABCD (66, 72). Environmental cues k n o w n to affect the expression level o f a number o f virulence genes, including those found in S P I - 1 , are osmolar i ty (12, 211), oxygen tension (12, 75, 91), growth phase (189), as wel l as the level o f D N A superhelicity (100, 178). The effects of supercoil ing on gene regulation is k n o w n to affect local gene expression, and can even affect the transcription of regions w h i c h are kilobases away {e.g. promoter relay) (74): b . G e n e t i c r e g u l a t i o n Salmonella encodes two-component systems for the global regulation o f genes, inc luding both virulence and housekeeping genes. Examples o f these two-component systems are encoded by the genes phoP/phoQ , pmrA/pmrB, and ompR/envZ. These systems consist of a sensor protein and a regulator protein. The sensor protein (PhoQ, P m r B or E n v Z ) is a histidine kinase wh ich spans the bacterial membrane with its extracellular domain acting to sense external signals and the intracellular domain acting as a kinase (329). U p o n stimulation, the sensor protein undergoes autophosphorylation and is then able to phosphorylate the regulator protein (PhoP , P m r A or O m p R ) . This phosphorylation changes the ability of the regulator protein to b ind to specific D N A sequences, a l lowing it to act as a transcriptional activator and/or repressor (12, 80, 102, 103). In addition, these genes are often positively autoregulated, and include regulation v ia two separate promoters, one of wh ich is environmentally-sensitive and one wh ich is not (135, 292). Moreove r , the two-component Chapter 1 23 regulatory systems can interact to process multiple environmental signals in a complex hierarchical system (118, 135, 294). The P h o P / P h o Q regulatory system (located at 27.4 centisomes (167) has been shown to modulate the expression o f over 40 genes within S. typhimurium including the phoPQ operon itself, resulting in the activation or repression of these genes in response to external magnesium and calc ium concentrations (103, 118, 211 , 293, 312), p H (12, 15, 118, 211) , osmolari ty (12), oxygen tension (18), and starvation for both phosphate and carbon. There is evidence for the direct involvement o f P h o P to all the conditions (329), however , P h o Q appears to respond specifically to magnesium and calcium (102, 118). The pathogenic properties w h i c h are regulated by the P h o P / P h o Q system include intra-macrophage survival (118, 215), resistance to ant imicrobial peptides, such as p o l y m y x i n and N P - 1 defensins (104, 135, 137, 212) , the formation o f spacious vacuoles (5), adaptive mutagenesis to growth-dependent mutations (139), and the down-regulation of the ability o f phagocytic cells to present bacterial antigens to T-cel ls (332). Genes activated by this system are referred to as PhoP-activated genes (pag) and many virulence pag's promote intracellular survival . Examples o f virulence genes activated by P h o P include mgtCB (293), pmrAB (135), pagP (137) and possibly pagC (214, 304), pagD, pagj, pagK, and pagM (19, 132-134, 136). Genes which are repressed are k n o w n as PhoP-repressed genes (prg), and many are invo lved wi th bacterial invasion and early survival wi th in the phagolysosome (213, 332). Examples o f virulence genes repressed by P h o P include SPI-1-encoded genes hilA, prgHIJK, sip/sspA, sipC, invF and orgA (246). A balance between activation and repression is needed for full virulence in vivo, since constitutive expression o f P h o P attenuates Salmonella virulence and survival (215). M a n y genes responding to regulation by P h o P / P h o Q are co-regulated by other systems, such as activation by S i r A (167), or may be responding to other regulators wh ich also respond to P h o P , such as H i l A (12, 18, 167) or P m r A (103, 120, 294). The P m r A / P m r B regulatory system has been shown to modulate genes directly in response to magnesium concentrations and to p H (120, 294), as we l l as indirectly in response Chapter 1 24 to regulation by P h o P / P h o Q (135). Furthermore, many genes wh ich are regulated by p H are coordinately regulated by both P h o P / P h o Q and P m r A / P m r B (15, 103, 294). The P m r A / P m r B system confers bacterial resistance to a number o f cationic antimicrobial peptides, such as p o l y m y x i n (but not N P - 1 defensin) through regulation of the pmrE/pagA and pmrF loc i (120). The O m p R / E n v Z regulatory system (located at 75 to 80 centisomes) (230) has been shown to regulate the formation o f spacious vacuoles in macrophages, and affect the ability of the bacteria to k i l l these cells (199), resulting in bacterial attenuation (61). The O m p R / E n v Z system controls the expression of Salmonella outer membrane proteins, such as O m p F , O m p C , and the tripeptide permease T p p B (40, 198, 199), and also affects the formation o f Salmonella-induced filaments wi th in host cells (221). The acid tolerance response also is dependent on this two component regulatory system during bacterial growth in min ima l media (89). S i r A (Salmonella invasion regulator) is a transcriptional activator w h i c h similar to the two-component response-regulator F i x J / U v r Y subfamily (167). F i x J o f Rhizobium meliloti, has been shown to activate target gene expression after phosphorylation in response to changes in environmental oxygen (167). Al though it is located outside o f any identified pathogenicity is land at 42.4 centisomes, S i r A is k n o w n to activate a number o f pathogenicity island-encoded genes, inc luding SPI-1 encoded hilA and SPI-5 encoded sigDE. It is unclear whether its effects on these genes are direct or indirect (167). H i l A is encoded within SPI-1 (190) and is a transcriptional activator related to O m p R (11), although it contains neither a phosphoryl acceptor nor a membrane-spanning domain . It is required for the activation of many SPI-1 encoded invasion genes including the inv-spa—prg operon and invF (which further promotes sipBCDA expression). The hilA locus is coordinately regulated by both P h o P (12, 167) and S i r A (167), although its expression does not necessarily affect the transcription o f other P h o P or S i r A regulated genes, e.g. sigDE transcription is unaffected by H i l A but is dependent on S i r A (159). S l y A , or ig inal ly reported to be a cytolys in (197), is actually a transcriptional regulator o f S. typhimurium wh i ch is upregulated within macrophages and by entry into stationary Chapter 1 25 phase (29, 269). Other members o f this family o f proteins include M a r R and E m r R which provide resistance to a wide range of toxic compounds by regulating the expression o f membrane efflux systems. S l y A is required for bacterial resistance to oxidative products such as hydrogen peroxide, but not to nitric oxide products! W h e n this gene is put into E. coli, it activates the cryptic hemolys in clyA, although the presence o f a C l y A - l i k e toxin in Salmonella has not been shown (29). Mutations within Salmonella slyA result in attenuation in mice by oral , IP , and I V routes of infect ion, and this gene has been implicated for intracellular survival rather than for the init ial invasion or colonizat ion steps (56). F u r (ferric uptake regulator) is a gene repressor (located at 16.9 centisomes (230)) wh ich is inactivated when iron availabil i ty is l o w a l lowing for the activation o f genes involved in iron acquisi t ion (88, 317). It is required for intra-macrophage survival (317). It is further required for a transient Salmonella acid-tolerance response (89, 191, 333), although this effect may be both direct and indirect as p H has a varied effect on different iron-regulated genes (88, 317). The F u r repressor may also act on genes required for resistance to reactive oxygen intermediates, such as sodA (316). The role o f F u r regulation in bacterial virulence is unclear (107). R p o S (sigma-38) is an alternative sigma factor wh ich is required for Salmonella virulence. The avirulence o f L T 2 strains o f S. typhimurium results (at least in part) from a defective rpoS gene (308, 334). R p o S is used to transcribe genes required during times of carbon and nitrogen starvation (73), during the accumulation o f metabolic products (such as short-chain fatty acids) (66), and when the bacteria enter stationary phase (71, 335). W h i l e the expression o f rpoS has been shown to increase after invasion into cells (41) and rpoS is thought to be exclus ive ly required for systemic infection (125, 330), more recently it has been shown to play a role in the colonization of Peyer 's patches (231). A n example of virulence genes regulated by R p o S include the spv plasmid-encoded genes, wh ich are required for long term survival o f Salmonella in the host. H o w e v e r , not all genes upregulated in stationary phase use R p o S . F o r example, the increase in slyA expression during stationary phase is Chapter 1 26 independent o f R p o S (29), and induct ion o f S p v R by R p o S occurs during exponential growth but not dur ing stationary phase (66). R p o S is also required for a sustained, low-pH- induc ib le ac id tolerance response in S. typhimurium that occurs in stationary phase (88, 191, 192). It is not required for the acid tolerance response in logarithmic phase growth , although it may co-regulate the atrB locus w h i c h is required (191, 333). S p v R is a virulence plasmid-encoded transcriptional activator wh ich is upregulated during stationary phase (54, 296, 335), as wel l as during logarithmic phase in minimal salts media (but not r ich media) (54, 335), and upon entry into host cells (77, 261 , 262). S p v R posi t ively regulates an operon immediately downstream encoding genes spvABCD (287, 296 , 323). These genes are required for both gastroenteritis and systemic infection by the bacteria wi th in animal models (196), and more recently they have been shown to be essential for replication o f Salmonella wi th in macrophages in the host (131). S p v R is acted upon by a number o f different factors, inc luding the upregulation by R p o S and autoregulation (335). It is downregulated by high levels o f S p v A (296, 323), by iron (296), as we l l as c y c l i c - A M P ( c A M P ) and the c A M P receptor protein ( C R P ) (71, 236). S p v R has been shown to be regulated by a two-step model of transcription activation in a s imilar manner to L y s R of the Vibrio luciferase operon (287). 1 . 4 . R e p o r t e r s y s t e m s f o r b a c t e r i a l gene e x p r e s s i o n Reporter enzymes make it possible to visualize the expression o f bacterial genes whose products are not readily assayable. The expression of a specific gene can be monitored by first fusing that gene wi th a promoterless reporter gene and then measuring activity of the reporter gene product. H o w e v e r , the reporter enzyme must meet at least two criteria. Firs t , it must have an activity that is distinct f rom endogenous cellular or bacterial enzymes in the system used. Second, (relative to the studies here), the reporter enzyme assay must be very sensitive, as numbers o f invasive bacterial are often low and bacterial gene expression may be moderated. Chapter 1 o _ cs E o c/l O cu © U JS cu CU 'hie X ide. ? o C/3 <u CU T 3 e • —• E w C/1 CU x o H i H i C/l 3 O © c E cu o o JS a s- •w X c © cu © X! 5 c C/l •2 cu u °C o es a a sh u *». CU © ph ar gulat ds gulat an CU L . isl CU fl Salmo nicity rice gei CU oge cu Xi ^ oge "3 O X - * H cs JIA s a =H-_© c o S3 CU © X sen Kn um cu i-l s a cs CU u E J U _cu itiso xi '& cs itiso E E CU CU CU Xi o CU i-CU C € o cu CU u 3 Chapter 1 28 1.4.1. R e p o r t e r genes a . B z g a l a c t o s i d a s e 6-Galactosidase has traditionally been the most widely used bacterial reporter enzyme and is a sensitive reporter o f gene expression (38, 89, 163, 289, 293). It is encoded by a single gene, lacZ, w h i c h may be fused to a gene o f interest as either a translational fus ion, resulting in a hybr id protein, or a transcriptional fusion, in wh ich both reporter and target genes are expressed under one promoter but the reporter protein is separate from the coding region (38). B-galactosidase hydrolyzes a number of commercial ly-avai lable substrates wh ich may be assayed colorimetrically (28, 163), fluorescently (28, 77, 108, 251 , 289) or chemiluminescently (28). The activity o f the enzyme may be detected under a wide range of b iochemical conditions (on agar plates, in solut ion, or wi th in host cel ls) , and does not require any co-factors for its activity (e.g. oxygen or bacterial energy). H o w e v e r , enzymatic activity persisting in a k i l l e d bacterium can not be differentiated from the activity in a viable bacterium and may poss ib ly result in an apparent higher activity per bacterium than really exists. Furthermore, the bacterial and host cell membranes are not permeable to B-galactosidase substrates and must be lysed in order to quantify the enzymatic activity in solut ion. F i n a l l y , whi le Salmonella do not produce an intrinsic B-galactosidase, host cells do have a l ow level endogenous activity (28). b . B a c t e r i a l l u c i f e r a s e Bacterial luciferases may be found in many different bacteria and are generally encoded by two genes, luxAB, w h i c h result in a heterodimeric enzyme (210), and are transcribed as part of an operon (222), w h i c h also encodes gene products necessary for the production o f an aldehyde substrate (210). In the absence o f the. substrate synthesis genes (e.g. luxAB on ly) , the substrate must be added to the sample (138). In addition to an aldehyde substrate, the luciferase requires the presence of oxygen (143) and the bacterial energy source, F M N H 2 (17, 186), but does not require metals or cofactors (210). The product o f enzymatic reaction is light in the v is ib le range between 478-505 n m (17, 266). M o s t eukaryotic luciferases (luc firefly or Chapter 1 29 beetle luciferases (28)) are encoded by a single gene and require A T P as an energy source. They are more susceptible to changes in p H , ionic strength, and temperature than are the bacterial luciferases, and also require a metal cofactor (266). O f the hundreds o f different strains o f luminescent bacteria, the most widely used reporter genes come from Vibrio fischeri (69, 92, 210), V. harveyi (187, 193, 210), Photorhabdus (Xenorhabdus) luminescens (52, 86, 210), Photobacterium phosphorum (210), or P. leiognathi (210). The most dist inguishing feature between the different luciferases is their optimal temperature for activity. Luciferase from V. fischeri is stable and active between 20"C and 30"C but not at 37"C, whereas luciferases from V. harveyi and P. luminescens are stable at 37"C or higher (209, 251). The use o f bacterial luciferases offers several advantages over other reporters. The luciferase aldehyde substrate, n-decanal, is volatile, amphipathic, and readily crosses membranes (138, 155, 210, 244, 276), unl ike most B-galactosidase substrates [Garcia-del Por t i l lo , 1992 #22; (28, 77). Therefore, luciferase activity can be assayed without the need for bacterial or host cell lys i s , and potentially, expression could be assayed wi th in the same sample over time (186, 251). It is also possible to monitor bacterial gene expression from wi th in a whole animal (35, 52). A s w e l l , unl ike B-galactosidase, most bacteria and tissue culture cells have no endogenous luciferase activity (52). A n y light produced is the direct result o f luciferase activity. Furthermore, light production is also an indicator o f bacterial viabi l i ty (180, 301, 302). In the absence o f a sufficient supply of flavin mononucleotide (FMNH2) wi th in the bacteria, light is not produced even i f luciferase is present, resulting in measurement of activity from viable organisms only (52). c. O t h e r r e p o r t e r genes It has been possible to demonstrate the upregulation o f various genes by Salmonella inside tissue culture cells us ing various other reporter genes. Examples include papA ( type I fimbriae) (262), cat (chloramphenicol acetyl-transferase) (297), phoA (alkaline phosphatase) (28, 157), gusA (B-glucuronidase) (28), and galK (galactokinase) (300). Chapter 1 30 1.4.2. O t h e r d e t e c t i o n m e t h o d s In Vivo Expression Technology (IVET) is a technique which enables researchers to positively select for Salmonella genes which are expressed upon infection of a host animal (147, 202, 203, 289). The bacteria are engineered with a promoter trap in which bacterial promoters drive the expression of a gene cassette. This cassette contains a gene which is required for growth in the host but can be supplemented for in vitro (e.g. either pur A (auxotrophy) or car (antibiotic resistance)) and the reporter lacZ gene (146, 202, 203). The final gene fusions are generated on chromosomal DNA, rather than on a plasmid, using gene duplication in a manner that avoids gene disruption unlike regular transposon insertional fusions. Since the genes are contained on the chromosome and not on a multi-copy plasmid, it is likely that the genes are regulated under the natural promoter environment. IVET identifies genes which are expressed upon infection of mice rather than upon invasion of a particular cell type. Therefore, bacterial genes that are necessary at any time during infection will be identified using this technique, providing the specific bacterial clones survive within the host until they can be recovered. IVET does not indicate at which point the bacterial genes are required, i.e. are they transiently induced or expressed in vivo all the time, and a large number of the identified bacterial genes are not specific for virulence (i.e. housekeeping). As well, genes which may be inhibitory or refractory to recombination events in the bacteria may be under-represented. Furthermore, complementation in the animal may demand high levels of expression relative to growth on laboratory medium, indicating that small increases in gene expression may be overlooked. Signature-tagged mutagenesis (STM) is a negative selection technique that is also based on in vivo selection of bacterial mutants (147, 151). Transposons carrying gene tags, which consist of 40 bp of unique DNA flanked by 20 bp regions of DNA common to all the tags, are randomly integrated into the Salmonella chromosome. A pool of tagged insertion mutants is then used to infect a host animal and those bacterial mutants which cannot be recovered from the animal are deemed to carry insertions within genes necessary for infection. The presence or Chapter 1 31 absence o f a particular bacterial mutant is assessed by dot blot hybr idizat ion, wi th the hybridization signals f rom the recovered pool compared with those from the input poo l . The complexi ty or size o f the pools must be restricted such that each mutant is present in sufficient numbers so that avirulent mutants are not falsely identified and that the hybridizat ion signals do not become too weak to identify. Differential display also utilizes an in vivo mouse model , however it looks for gene expression in the absence o f bacterial mutagenesis, i.e. without the use o f reporter genes. It requires the subtractive hybridization o f two bacterial c D N A libraries, wi th the resulting c D N A s are used as probes to search for in v/vc-expressed bacterial genes (147). One c D N A library is made from bacteria grown on laboratory medium and represents the pool of 'housekeeping' genes. The other c D N A l ibrary is made from bacteria recovered from infected tissue, and represents those genes required for bacterial virulence. The 'housekeeping' genes are subtracted or removed from the pool of genes expressed upon infection, and the remaining genes in the ' infected' pool are then used to screen the bacterial genome in search o f genes required specifical ly for virulence. The crucial step of this technique lies in the ability to isolate and stabilize the bacterial m R N A s in order to make a representative c D N A library. U n l i k e most eukaryotic m R N A s , bacterial m R N A s do not have a p o l y - A tail . Differential fluorescence induction (DFI) (320) is another technique ut i l iz ing a D N A promoter trap, where bacterial genes are selected on the basis of their expression within cultured macrophages rather than a whole animal (147, 321, 322). The gene encoding the green fluorescent protein ( G F P ) is transcriptionally fused wi th Salmonella genes and then, with the use o f a fluorescence-activated cell sorter ( F A C S ) , host cells containing intracellular bacteria wi th genes expressing high levels o f G F P are detected and sorted. A n advantage of this technique is the ability to determine the bacterial gene expression within a single macrophage (321). A disadvantage of this technique is that gene fusions to G F P are contained on a mult i -copy plasmid, rather than in the chromosome. This was done to increase the range of fluorescence obtained from the various gfp fusions, imp ly ing that l o w levels of gfp Chapter 1 32 expression may be missed with this technique. Furthermore, regulation of the genes may not have reflected the true nature of the expression within the chromosomal environment, e.g. repressors and/or activator proteins may have been titrated out by the multiple copies of the gene promoters, and there would be no gene regulation in cis or by promoter relay. A second disadvantage arises from the use of pooled clones, where competition between bacteria during growth (either intracellular or extracellular expansion of the pools) may result in the loss of some fusions. Using two-dimensional protein gels, it has been shown that bacteria inside macrophage cells express different protein profiles than those grown outside cells (1, 30, 34, 254). With this technique, radiolabelled proteins in bacteria and/or cells can be separated into a discrete spots on a gel by separating them on both their isoelectric point and their molecular size. More recently, a technique involving the use of radiolabelled diaminopimelate (a lysine precursor specifically used by bacteria) has been used to identify the patterns of intracellular bacterial protein expression, without contamination by host proteins (33, 34, 147). These techniques are very powerful, however many of the proteins have yet to be identified. 1.5. S u m m a r y o f thesis In the past six years there has been a veritable explosion of data regarding the mechanisms of virulence for not only Salmonella but many other gram-negative bacteria as well. When this study was initiated, pathogenicity islands and type III secretion systems were unknown. At that time, it had been shown that Salmonella expressed an unique set of genes inside macrophages, that were not expressed in the presence of other stresses (heat, low pH, starvation, oxidative stress) either singly or in combination (1, 30). Moreover, this gene expression differed slightly depending on the isolate of Salmonella used as well as the type of cultured cells used. It was determined that over 100 bacterial genes were upregulated by intracellular S. typhimurium, with over 40 genes deemed unique to the intra-macrophage environment. However, only a handful of these genes could be attributed to those already known (30). It had furthermore been determined that the ability of Salmonella to invade cells Chapter 1 33 and to grow intracellularly were separate events and each were required for virulence in animal models (78). Therefore, the goal of this study was to develop a system to search for Salmonella genes wh ich were only expressed inside cel ls , and thereby uncover genes which may be essential for intracellular survival and perhaps virulence. The first section describes the development of a light-based reporter system by which to detect genes expressed from intracellular Salmonella. Ini t ial ly, reporter gene fusions were made to the Salmonella p lasmid virulence genes spvRAB, wh ich had previously been shown to be required for Salmonella virulence in a mouse model . The spv genes were also k n o w n to be induced by carbon and nitrogen starvation and during stationary phase growth. U s i n g the enzyme 6-galactosidase (lacZ), the spv genes were shown to be upregulated after Salmonella invasion into epithelial cells (77). S imi lar results o f induction were confirmed by two other studies, where the genes were shown to be upregulated within phagocytic cells as w e l l (262). Nevertheless, the use o f 6-galactosidase as a reporter was not ideal, and therefore bacterial luciferase was tested as alternate reporter o f intracellular bacterial gene expression. The luciferase genes were fused to the spv genes in order to be able to directly compare between the two reporter systems. The results indicated that whi le luciferase was also not a 'perfect' reporter, it was as sensitive as 6-galactosidase and offered a number o f advantages as a reporter, namely the ability to monitor gene expression whi le the bacteria were inside the cells and the ability to differentiate between the activity from l ive and dead bacteria (251). The second section describes the construction o f a library of Salmonella mutants to be used to search for genes upregulated by intracellular bacteria. A l s o described in this section is the initial screening procedure used to look for genes which were only upregulated once Salmonella had become intracellular. The promoterless luciferase reporter genes were inserted as single random insertions throughout the chromosome, us ing a modif ied two-plasmid competit ion system (138). Bacter ia were then tested for their ability to produce little to no light outside mammalian cel ls , whi le producing light from the intracellular environment. This Chapter 1 34 al lowed for the identification o f genes which were repressed during growth in rich media , but were activated or upregulated during Salmonella growth within cells. The final section describes the characterization o f the Salmonella mutants wh ich were found to contain upregulated gene fusions. T w o o f the genes identified in this screen were found to be part o f S P I - 5 , sigD/sopB and pipB one gene was found to be encoded within S P I - 2 , ssaR; and the fourth gene was completely novel , iicA. N o obvious difference in phenotypes could be identified whi le the bacteria grew in vitro when comparing the insertional mutants to the wildtype bacteria. The bacterial mutants retained their ability to invade and grow within cultured cells (at least during short time periods). Interestingly, all four gene fusions were not only upregulated wi th in phagocytic cells, but were upregulated wi th in non-phagocytic cells as w e l l . The insertions wi th in all o f these genes reduced the in vivo virulence o f S. typhimurium to vary ing degrees, except for iicA wh i ch retained its virulence in the mouse mode l . W i t h i n this study, a reporter system was described which was specif ical ly able to detect the gene expression from bacteria residing inside mammalian cel ls . Moreove r , the activity could be correlated to viable bacteria. B y incorporating this system into the bacterial genome, several Salmonella virulence factors were identified and characterized. That not all the genes upregulated by intracellular bacteria were found to be virulence factors indicates the increasing complexi ty o f animal models over ce l l culture models. Chapter 2 35 C h a p t e r 2: M a t e r i a l s a n d M e t h o d s 2.1. M e d i a a n d c h e m i c a l s 2.1.1. C h e m i c a l s a n d A s s a y R e a g e n t s Detergents Tr i ton X - 1 0 0 and sodium dodecyl sulfate ( S D S ) were purchased from S igma-Ald r i ch Canada, L t d . (Oakvi l le , O N ) and made up as 10% stock solutions in sterile water, wh ich were stored at room temperature. Tryps in (Gibco L i f e Technologies; Bur l ing ton , O N ) was stored at - 2 0 ° C and used undiluted during the passage o f epithelial cel ls . Dimethyl sulfoxide ( D M S O ) ( B D H Inc.; Toronto , O N ) was used as a cryoprotectant when freezing cultured cel ls , at a final concentration of 10%. Miscel laneous chemicals such as ethidium bromide, agarose, ethanol, methanol, phenol , ch loroform, sodium chloride, sodium hydroxide , hydrochlor ic acid , etc. were purchased from a number o f different companies including: S i g m a - A l d r i c h Canada, L t d , B D H I n c , V W R Scientific Canada, L t d . ( L o n d o n , O N ) , Amersham Canada, L t d . (Oakvi l le , O N ) , Baxter-Canlab ( V W R - C a n l a b ; Miss i s sauga , O N ) , D i f c o L a b s - F isher Scient if ic (Ottawa, O N ) , and G i b c o - B R L (Bur l ington, O N ) . n-Decanal (99%; Sigma) was used for luciferase assays and kept stored at 4 ° C in an airtight container. A range of aldehyde concentrations were made by first adding 1 p i rc-decanal into 1, 2, 4, or 10 m l o f a solution of 7 0 % (v:v) ethanol:8% (v:v) methanol. One mill i l i ter o f this solution was then added to 4.5 m l o f M E M + 10% F B S , resulting in concentrations o f 0 .02%, 0 .011%, 0 .0055%, and 0 .0022% (v:v) n-decanal, respectively. T w o further concentrations o f aldehyde were made by adding 1 pi n-decanal to 100 or 2 0 0 p i ethanol:methanol, and then adding 100 p i o f this solution to 1 m l o f tissue culture media, resulting in aldehyde concentrations o f 0 .099% and 0 .0495% (v:v) respectively. U s e of alcohol a l lowed the even dispersal o f the aldehyde in the solutions. The concentration of alcohol was 14.2% (v:v) for the four lowest di lut ions, and 7.8% (v:v) for the two higher di lut ions. Due to their long-term instability in suspension, aldehyde solutions were kept in airtight containers, at r oom temperature, for no longer than 4 h. (Note: the final concentration Chapter 2 36 of the aldehyde in the luciferase assay well is another 10-fold diluted: see "luciferase assay" below.) (251) For fluorescent B-galactosidase assays, the fluorescent substrate fluorescein-di-galactopyranoside (FDG) (25) and the B-galactosidase inhibitor phenyl-ethyl-thio-galactoside (PETG) (58) were used (Molecular Probes; Eugene, OR) (77, 108). FDG and PETG were initially dissolved in DMSO, and then diluted and stored at a concentration of 50 mM at -20°C for up to 1 month. The final concentration of DMSO in the stock solutions was 25% (v:v). For chemiluminescent B-galactosidase assays, the Galacto-iSTA/? ™ assay kit was used according to manufacturers instructions (Tropix - Perkin Elmer; Bedford, MA) . 2.1.2. A n t i b i o t i c s All antibiotics were purchased from Sigma. Ampicillin (amp; 100 pg/ml (w:v)), tetracycline (tet; 15 pg/ml (w:v)), streptomycin (str; 25 pg/ml (w:v)), and chloramphenicol (cm; 30 pg/ml (w:v)) were used as selection agents for plasmid maintenance and bacterial growth. Gentamicin was used to inhibit extracellular bacterial growth during invasion assays (310) at a concentration of 100 pg/ml (total weight per volume) for the first four hours and at 10 pg/ml for incubations longer than four hours. 2.1.3. M o l e c u l a r B i o l o g y R e a g e n t s a . R e a g e n t s Restriction enzymes Haelll, Rsal, and BamHI were purchased from both New England Biolabs (Mississauga, ON) and Boehringer Mannheim Canada (Laval, QC). T4 DNA ligase and deoxynucleotides (dNTPs) were purchased from Boehringer Mannheim Canada. The dNTPs were made as a 2.5 mM stock solution and stored at -20°C; all four deoxynucleotides were in equal proportions. The polymerase AmpliTaq (Perkin-Elmer Applied Biosystems; Norwalk, CT) was used for inverse polymerase chain reactions (inverse PCR). For sequencing reactions, ABI's AmpliTaq Dye Terminator Cycle Sequencing chemistry with FS Taq was used as recommended by the Nucleic Acid and Protein Services Unit (NAPS Unit) at the University of British Columbia. Centrisep spin columns (Princeton Chapter 2 37 Separations; Adelphia, NJ) were used to remove unused dyed dinucleotides from the sequencing reactions, b . P r i m e r s DNA primers were made by the NAPS Unit and are listed in Table 1. The primers LUX76 and LUX340 were made to regions within the luxA gene; all other primers were made to regions on the S. typhimurium chromosome. Primer set LUX76:LUX340 was used to amplify the regions upstream from the luxA gene in all six clones initially identified. Primer sets E-MINUS:E-PLUS and E3615:E3805 were used to amplify the region around the insertion site of E12A2; chromosomal DNA from both E12A2 and SL1344 was used. Primer set G-MINUS:G-Plus was used to amplify the region around the insertion site of G7H1; chromosomal DNA from both G7H1 and SL1344 was used. All primers except for LUX340 were used for sequencing. Table 1: Primers for Inverse PCR Primers Length Sequence (5' to 3') LUX76 18 nt CAA GCG ACG TTC ATT CAC LUX340 18 nt TGC CGC ACA TCT ATT AGG E-PLUS 20 nt CAG TTT TCC AAT TAC CTC CC E-MINUS 22 nt TTC TGG AGG ATG TCA ACG GGT G E3615 24 nt ACA GCG TGT AGA TTT GCA CAA CAC E3805 21 nt GAC AGG TAG TCA ACA TAC CCC G-MINUS 20 nt GGA GGA ATG CAC ACC TTT AG G-PLUS 19 nt TAG TCC CTA ACC CCC ATT G Chapter 2 38 2 .1 .4 . M e d i a Tissue culture cells were grown in either minimal essential media (MEM #410-1500; Gibco Life Technologies) or Dulbecco's modified Eagle medium (D-MEM #430-2800; Gibco Life Technologies), both of which were supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies). DMEM++ refers to basic D-MEM supplemented with both 10% serum and 20 mM HEPES (pH 7.4). Phosphate-buffered saline containing calcium and magnesium (PBS++) was used for washing and dilutions. Bacteria were grown in a number of different media. Luria-Bertani (LB) broth and agar plates, MacConkey plates, and SOC broth, were used for routine bacterial culture, and recipes can be found in Sambrook et al. (275) and Ausubel et al. (9). Special "green plates" were used to isolate phage-free bacteria after transductions and made as previously described (68, 300). The green plates contain the dyes Alizarin Yellow and Aniline Blue, and infected colonies appear dark green in color while uninfected colonies remain pale. Bacteria were also grown in DMEM++ where indicated. 2 . 1 . 5 . B u f f e r s A number of different buffers were used throughout this study. Phosphate-buffered saline (PBS— or PBS++) was used as a diluent for bacteria and cells where indicated. PBS consisted of 0.2 g/L KCI, 0.2 g/L K H 2 P 0 4 , 8 g/L NaCl, and 2.16 g/L N a 2 H P 0 4 . 7 H 2 0 and the final pH was adjusted to 7.4 with HC1. P B S - contained no calcium or magnesium, whereas PBS++ contained 130 mM calcium and 200 mM magnesium. HEPES buffer (pH 7.4) was used as an extra buffering agent in DMEM++. T A E buffer was used for making and running agarose gels (185). TE buffer was used for resuspending preparations of DNA (275), and 50 mM TE (i.e. 50 mM Tris pH 8.0 + 50 mM EDTA) was used with chromosomal DNA preparations, while a 5 m M TE concentration was used with plasmid DNA samples (i.e. 5 mM Tris pH 8.0 + 5 mM EDTA). Tris-HCl buffers (made in a range from pH 7.0 to 8.0) were used for molecular biological manipulations and made according to Sambrook et al. (275). P22 buffer was used during the dilution and storage of the bacteriophage P22 (300). Chapter 2 39 2.2. E q u i p m e n t H o r i z o n mini-gel apparatus ( B R L ; Bur l ing ton , O N ) were used in conjunction with an F B 1 0 5 power source (Fisher Scientific; Pit tsburgh, P A ) was used for resolving D N A on agarose gels as previously described (275). D N A gels were stained with ethidium bromide (275) and visual ized us ing a long-wavelength transilluminator (Fisher Scientif ic) . Pictures were taken us ing a Po la ro id camera and 667 f i lm (Polaroid , L t d . ; St. A l b a n s , Hertfordshire, England) . The Gene P u l s e r ™ from B i o R a d (R ichmond , C A ) was used to electroporate D N A into competent bacterial cel ls . The Er i comp T w i n b l o c k System thermocycler (Er icomp Inc.; San D iego , C A ) was used for P C R reactions (both inverse P C R and sequencing reactions). Sequencing gels were made and run by the N A P S U n i t and the D N A sequences were determined using an automated 373 D N A Sequencer. Sequences were further analyzed using N C B I ' s B L A S T program and the G e n B a n k database. Three different sets of apparatus were used for determining light production from bacterial samples. F i rs t , microtiter plate formats were exposed to X - r a y f i lm ( X - O M A T by Eastman K o d a k Company ; Rochester, N Y ) and the resulting spots were quantitated with a computing densitometer (Molecular Dynamics ; Sunnyvale , C A ) . Second, light from tubes of bacteria was measured us ing the 1250 Luminometer ( L K B - W a l l a c ; F in land) . T h i r d , the Luminograph L B 9 8 0 ( E G & G Berthold; Germany) (26) was used to measure light from a number of formats inc luding agar plates and microtiter plates. Fluorescence was monitored in 96-wel l microtiter F luo r i con assay plates ( I D E X X Corporat ion; Wesbrook , M E ) wh ich were read on a P A N D E X Fluorescence Concentration Ana lyze r ( I D E X X ; Portland, O R ) . Cul tured cells were routinely expanded in sterile, flat-bottomed, tissue culture flasks (Becton D i c k i n s o n Canada; Miss i s sauga , O N ) , wh ich were 25 c m 2 , 75 c m 2 , or 125 c m 2 in size. D u r i n g passage o f the cultured macrophages, the cells were scraped with 25 cm disposable ce l l scrapers (Corning-Costar , Fisher Scientific; Pit tsburgh, P A ) . F o r the invasion and reporter assays, cells were cultured overnight in 96-wel l plates, wh ich were either completely clear (Falcon Microtest III tissue culture plates; Becton Dick inson Canada; Chapter 2 40 Mississauga, ON), or contained clear wells with white grids between each well (Immunoware 8-Well EIA strip plates; Pierce, Rockford, IL), or contained black wells with clear bottoms (Costar; Fisher Scientific). The EIA plates were sterilized with 70% ethanol prior to seeding with cells; all other tissue culture plates used were previously sterilized by gamma irradiation. For the B-galactosidase assays, samples containing chloroform were transferred to Falcon Microtest III Flexible Assay Plates (Becton Dickinson Canada) for incubation, since the polyvinylchloride (PVC) plastic was more tolerant to chloroform than the polystyrene plastic of the other plates. Bacterial optical density was determined using an Ultraspec Plus Spectrophotometer (Pharmacia Biotech; Uppsala, Sweden). Miscellaneous equipment such as Eppendorf tips, disposable pipettes, and sterilizing filters were purchased from VWR Scientific Canada, Ltd. (London, ON), Millipore Canada, Ltd. (Nepean, ON) and Amicon Division, W.R. Grace & Co.- Conn. (Beverly, MA). 2.3. S t r a i n s a n d P l a s m i d s 2.3.1. C e l l s l i n e s Non-phagocytic cell lines used included Madin-Darby canine kidney cells MDCK (ATCC number: CCL-34) and the human epithelial cells HeLa (ATCC number: CCL-2). Phagocytic cells used included the BALB/c mouse macrophage-like cells J774A.1 (ATCC number: TIB-67) (255), and the mouse bone marrow-derived macrophage cell line B ALB .BMI. For the initial studies, the MDCK and HeLa cells were grown in MEM at 37°C in an atmosphere of 95% air-5% CO2, however for later studies, all cells were grown in DMEM++. For most studies, the cell lines were used between passages 5 and 20, although for the results obtained in Chapter 3 the cells may have been passaged up to 30 times. 2.3.2. B a c t e r i a The bacteria used in this study are listed in Table 2. The bacterium S. typhimurium SL1344 (158) was used throughout for all manipulations. All Salmonella strains were tested by agglutination to antiserum specific for S. typhimurium group B, O-antigen factors 1, 4, 5, 12 (Difco Labs). Chapter 2 41 Table 2: Bacterial strains Stra in Bacter ia l T y p e Ant ib iot ic Resis tance Other Features References D H 5 a Escherichia coli Nalad ix ic acid r-m+, recA Sambrook et ai, 1989 S. dublin L a n e S. dublin C m (<6 pg/ml) W i l d type; virulence plasmid G u i n e y et al.., 1990 L D 8 4 2 S. dublin L a n e C m (<6 pg/ml) Cured of virulence plasmid Fang etal. 1991 S L 1 3 4 4 S. typhimurium WRAY from S2337/65 parent Str U s e d as w i l d type; hisGA6; virulence plasmid Hoise th&Stocker 1981 A 1 A 1 S L 1 3 4 4 Tet & Str sigD:duxAB T h i s work D 1 1 H 5 S L 1 3 4 4 Tet & Str ssaR::luxAB This work E 1 2 A 2 S L 1 3 4 4 Tet & Str sigF::luxAB This work G 7 H 1 S L 1 3 4 4 Tet & Str UcAr.luxAB This work 2 . 3 . 3 . B a c t e r i o p h a g e The methods for isolat ing, storing, and us ing the bacteriophage P 2 2 are described in (62, 63, 226, 278-282, 300, 336). The phage P 2 2 H T m f was used as a vehic le to transfer both plasmids and chromosomal insertions from one Salmonella to the next, whi le P 2 2 H 3 was used for cross-streaking experiments in order to determine whether bacteria were phage-sensitive (true transductants wi th no remaining phage lysogens) or resistant (lysogens) after transduction. Chapter 2 42 a. P r e p a r a t i o n o f p h a g e P22 s t o c k Bacteria were grown overnight in LB broth at 37°C shaking at 200 rpm. To 1 ml of this culture was added 4 ml of a P22 lysate containing 5x l0 6 plaque-forming units (pfu) per ml. This mixture was further incubated with aeration overnight at 37°C. The next day, a few drops of chloroform were added to the culture and incubation continued for another 10 min. The cells and debris were removed with centrifugation (e.g. 10 min at 10000 rpm). The clarified supernatant was then placed in a sterile tube and stored at 4°C. A couple drops of chloroform were added to this phage lysate stock to prevent bacterial growth during long-term storage. Original phage P22 stocks of P22HTmr and P22H3 were made using the host Salmonella strain SL 1344. The phage P22HT z'rcf stock containing the plasmid pFUSLUX was then made in a similar way by making a lysate from the host bacteria SL1344 pFUSLUX, into which the pFUSLUX had originally been electroporated. The phage P22HTint was used because it cannot lysogenize within the host bacterium and also has the ability to mobilize plasmid DNA. The stocks typically contained 10 1 0 pfu/ml, which was diluted one-hundred fold for use during transduction. b. T r a n s d u c t i o n o f Salmonella w i t h p h a g e P22 Bacteria were grown overnight in LB broth at 37°C shaking at 200 rpm. From the overnight culture, a 1:100 dilution of the bacteria was made into fresh LB media and grown to approximately l -5x l0 8 colony-forming units (cfu) per ml. One volume of bacteria was then mixed with a one-tenth volume of the appropriate P22HTint phage (at 1x10s pfu/ml), and held at room temperature for 10 min. One hundred microliter aliquots were then plated onto LB plates, containing the appropriate antibiotics, and the plates incubated overnight at 37°C. The resulting colonies were then purified on "green plates" to ensure they were not undergoing active infection, nor contained lysogenized phage. As stated earlier, on these specialized plates the infected bacterial colonies turned dark green, while uninfected colonies Chapter 2 43 remained a pale green color . The pale green colonies were further tested by cross-streaking them against a l ine o f P 2 2 H 3 phage lysate, wh ich resulted in infected dark green colonies only i f the or iginal co lony was lysogen-free. 2 . 3 . 4 . P l a s m i d P r e p a r a t i o n Table 3: Bacterial plasmids Plasmid Antibiotic Resistance Other Features References p A C Y C 1 8 4 C m (>12 ug/ml) & T e t L o w copy number p lasmid C h a n g & C o h e n 1978 p F F 1 4 C m (>12 ug/ml) s/?vZ?.-:/acZtranslational fusion (spvRAB::lacZ) Fang etal. 1991 p S P L U X C m (>12 ug/ml) spvB::luxAB transcriptional fusion (spvRAB::luxAB::lacZ) Pfe i f e r&Fin lay 1995 p T F 4 2 1 A m p R N A 1 overproduction G u z z o & D u B o w 1991 p F U S L U X Tet Tn5::luxAB::tet gene cassette; C o l E I or igin of replication G u z z o & D u B o w 1991 The plasmids used in this study are listed in Table 3. The p lasmid p F F 1 4 containing an spvBr.lacZ translational fusion was previously described by Fang et al. (72). The plasmid p S P L U X was made by inserting the luxAB gene cassette (251) into the BamHI site of p F F 1 4 , as shown in F igure 11, to create a transcriptional fusion between spvB and luxA. B o t h p F F 1 4 and p S P L U X are l o w copy number plasmids derived from p A C Y C 1 8 4 (39). The plasmids pTF421 and p F U S L U X were used in concert (as described below) to randomly insert luxAB into the Salmonella chromosome (138). Chapter 2 44 2.4 . M o l e c u l a r B i o l o g y 2 . 4 . 1 . D N A I s o l a t i o n a . P l a s m i d P r e p a r a t i o n Plasmid preparations were made using the alkaline lysis technique described in Sambrook et al. (275), with an extra phenolxhloroform step added. Bacterial plasmid DNA preparations were made from 2 ml overnight cultures. Isolated plasmid DNA was resuspended in either 5 mM TE or sterile water. Qiagen plasmid kits (Qiagen Inc.; Mississauga, ON) were also used to isolate plasmids from bacterial preparations, according to manufacturer's directions. b . C h r o m o s o m e P r e p a r a t i o n A cesium chloride preparation of chromosomal DNA from S. typhimurium SL1344 was made previously by Dr. Murry Stein. Chromosomal DNA preparations from Salmonella mutants were made as described below (9). First, a stock solution of protease (Streptomyces greisus Type XXI) was made at 10 mg/ml in distilled H2O and incubated at 37°C for 1.5 h. Then 2 ml of overnight bacterial culture was pelleted at full speed in an Eppendorf microfuge (Fisher Scientific) for 5 min. The pellet was resuspended in 300 pl 50 mM TE using the flat end of a sterile toothpick, and the bacteria were then mixed with 100 pl of 20 mg/ml lysozyme for 15 min at room temperature. After this incubation, 20 ul of 10% SDS was added and the tube was mixed by gently inverting it. Then 100 pl of the predigested protease were added and the mixture was incubated at 37°C for 1 h with occasional mixing. In the next step, 400 pl phenol was added and the tube was again mixed by inverting it. This was incubated at 37°C for 1 h with occasional mixing. After 1 h, 600 pl chloroform was added, gently mixed and the phases separated by centrifuging for 5 min at 8000 rpm. The upper aqueous phase containing the DNA was transferred into a new Eppendorf tube and respun. To transfer the upper phase, a modified 1 ml Eppendorf tip was used. This tip had about 1/4 cm of its end chopped off to enlarge the tip opening and thereby minimize shear Chapter 2 45 forces on the DNA. After the second centrifugation, the DNA was again transferred to a new tube and a one tenth volume of 3 M sodium acetate was added to the sample. The tube was then filled with -20°C 95% ethanol (about 1 ml or 2.2 volume of ethanol). The sample was not mixed, but instead the DNA globs were fished out with a hooked glass rod (melted Pasteur pipette). The 'glob' was briefly transferred to a new tube containing 1 ml 95% ethanol and then moved to an empty tube. The sample was dried at 37°C for about 1 h. The DNA was resuspended in 150 pi 50 mM TE and left to stand at 4°C overnight to achieve complete rehydration of the DNA. The next day, the purity of the DNA was checked by absorbance measurement with OD260 ;OD280 ratio. 2.4.2. B a s i c M e t h o d o f D N A P r e c i p i t a t i o n w i t h E t h a n o l Ethanol-precipitation of DNA has been previously described (23). Briefly, a one-tenth volume of 3 M sodium acetate was added to the sample containing the DNA and then 2.5 volumes of 100% ethanol was added to the sample. This mixture was allowed to cool for a minimum of 15 min at -70°C, and then spun at 13000 rpm for 20 min. The pellet was washed once with 70% ethanol. The resulting pellet was then dried and the DNA was rehydrated in 5 mM T E . 2.4.3. I s o l a t i o n o f D N A f r o m A g a r o s e Three separate methods were used to isolate bands of DNA from agarose gels. All three methods were equally effective and resulted in the recovery of more than 80% of the DNA in a band. a. S e p h a g l a s B a n d p r e p K i t The Sephaglas™ Bandprep Kit from Pharmacia Biotech was used to extract DNA from agarose gels, as directed by the manufacturer. b . F r e e z e S q u e e z e M e t h o d f o r I s o l a t i n g D N A F r a g m e n t s The DNA band of interest was cut out from the agarose gel, then wrapped in parafilm and set on dry ice or in -70°C to freeze (about 20 min). Once frozen, the liquid was squeezed Chapter 2 46 out from parafilm as band melted leaving a residue of agarose in the parafilm pocket. The DNA was then recovered using ethanol precipitation and resuspended in 5 mM TE or sterile distilled H 2 0 . c. S p i n - C o l u m n M e t h o d f o r I s o l a t i n g D N A F r a g m e n t s A hot straight wire was used to poke a tiny hole in the bottom of a 500 pl Eppendorf tube. A small plug of loosely wound glass wool (about 0.5 0.75 cm deep) was then inserted into the end of the small Eppendorf tube (i.e. the column). This small tube was then placed into a larger 1.5 ml Eppendorf tube (i.e. the collection unit). The agarose gel plug containing the DNA was then placed on top of the glass wool in the small tube and the tubes were centrifuged for 45 sec at 6000 rpm in microfuge. This allowed maximum DNA recovery with little agarose contamination. The DNA was then recovered using ethanol precipitation and resuspended in 5 mM TE or sterile distilled H 2 0 (144). 2 .4 .4 . E l e c t r o p o r a t i o n o f b a c t e r i a a . P r e p a r a t i o n o f e l e c t r o c o m p e t e n t b a c t e r i a Bacteria were grown overnight in LB broth at 3 7 ° C shaking at 200 rpm. From the overnight culture, a 1:100 dilution of the bacteria was made into fresh LB media and grown to an OD600 of 0.3-0.5. Once bacteria reached proper O D , the culture flask was chilled on ice for 30 min and all subsequent steps were performed on ice or in refrigerated units. The bacteria were transferred to centrifuge bottles and spun at 10,000 rpm for 10 min in a Beckman Model J2-21 centrifuge (Beckman Instruments (Canada) Inc.; Mississauga, ON). The pellet was resuspended in 1 volume of chilled 10% glycerol for first wash. The sample was then recentrifuged as before. Subsequent washing of the bacteria was carried out in reduced volumes of 10% glycerol (1:2, 1:50, 1:100, and 1:500) in order to decrease the ionic strength of the sample and concentrate the bacteria to about 1 0 9 to 1 0 1 0 bacteria per 40 pl aliquot. Aliquots were placed into Eppendorf tubes and flash frozen using dry ice and ethanol. They were stored at - 7 0 ° C for up to 6 months. Chapter 2 47 b . E l e c t r o p o r a t i o n Electrocompetent bacterial aliquots were thawed on ice only immediately before us ing . One to two microl i ters o f D N A preparation was added to the thawed bacteria and the mix was incubated on ice for about 1 min . The sample was then transferred to a 0.2 c m electroporation cuvette and using the Gene P u l s e r ™ from B i o R a d (R ichmond , C A ) , the sample was pulsed at 2.5 k V wi th a 25 p F capacitance and 4 0 0 Q parallel resistance. The samples were immediately resuspended in 1 m l o f S O C broth containing no antibiotics and incubated for 1 h at 3 7 ° C , shaking 200 rpm. After this recovery period, the sample was diluted and plated out onto selective med ium to quantitate the transformants per pg of D N A . 2 .4 .5 . T w o p l a s m i d c o m p e t i t i o n s y s t e m A modif ied version o f a two-plasmid competition system (138) was used to obtain random insertions o f a promoterless luciferase gene cassette throughout the Salmonella chromosome. Competent S. typhimurium S L 1 3 4 4 bacteria were initially transformed using electroporation wi th either the p lasmid pTF421 or p F U S L U X . Resul t ing transformants were selected on L B plates containing either ampici l l in or tetracycline, respectively. A phage P 2 2 H T m ? lysate was then made o f S L 1 3 4 4 p F U S L U X , and this lysate used to transfect S L 1 3 4 4 p T F 4 2 1 . The p S P L U X p lasmid was transferred into S L 1 3 4 4 pTF421 about 100 fold more efficiently by the phage P22 compared with electroporation. S L 1 3 4 4 bacteria transfected wi th both plasmids were then grown for an extended period of time on L B plates containing both ampici l l in and tetracycline. The plasmid p F U S L U X has a C o l E l origin o f replication (184) and contains a gene cassette encoding a promoterless luxAB gene operon and a tetracycline resistance gene surrounded by insertional sequences from the transposon Tn5 (Figure 6) (138, 166). The plasmid pTF421 carries an ampici l l in resistance gene and also encodes for the production o f R N A 1 , wh ich inhibits the replication o f plasmids wi th C o l E l origins (131). Th i s extended incubation a l lowed the bacteria to enter a hypermutability state that a l lowed for the random insertion o f the Z«xA5-conta in ing gene cassette into the Chapter 2 48 chromosome of the bacteria. Previous testing with E. coli showed that single chromosomal insertions resulted from this technique (138). 2 .4 .6 . I n v e r s e P C R Inverse PCR was used for amplifying the regions either upstream or downstream from the site of reporter gene insertion (164, 237, 315). Chromosomal DNA was first cut with either the restriction enzyme Haelll or Rsal at 37°C for a minimum of 1 h. After cutting, the restriction enzymes were removed using phenol:chloroform extraction and ethanol precipitation as described previously, and the D N A resuspended in 20 pl distilled H2O. Five microliters of this cut D N A was placed in a 500 pl microfuge tube containing 1 pl ligase (5 U), 10 pl 5X ligase buffer and 34 pl distilled water, and incubated for 10 min in a sonicating waterbath set between 12°C and 16°C. Inverse PCR reaction tubes contained: 5 pl ligated DNA, 5 pl of each of two primers (10 pmol stock), 5 pl 25 mM magnesium sulfate, 5 pl 10X AmpliTaq buffer, 4 pl dNTPs (2.5 mM stock), 20 pl distilled sterile water, and 1 pl AmpliTaq or AmpliTaq Gold (5 U) . The PCR program consisted of an initial cycle of 94°C for 5 min and then 30 cycles of 94°C for 45 sec, 60°C for 45 sec, and 72°C for 2 min 30 sec. The reaction was then subjected to electrophoresis on a 1% agarose gel with IX T A E buffer. 2 . 4 . 7 . S e q u e n c i n g For sequencing, 90 ng purified template DNA was added to 3.2 pmol of primer and mixed with 8 pl of the FS Taq terminator premix reagent for a final volume of 20 pl. Each sequencing reaction consisted of 25 cycles of 96°C for 30 sec, 50°C for 15 sec, and 60°C for 4 min. Unused terminators (fluorescently pre-dyed dNTPs) were removed using Centrisep columns, as directed by the manufacturer. Sequencing gels were run by the NAPS Unit (UBC, Vancouver, BC). Sequences were further analyzed using NCBI's BLAST program and the GenBank database . Chapter 2 49 2.5. I n v a s i o n a n d S u r v i v a l A s s a y s F o r the studies invo lv ing the development o f the intracellular reporter system, and for the ini t ial screening o f bacteria wi th cultured cells, the Salmonella were g rown overnight in L B broth at 3 7 ° C , wi th no shaking (159). F o r later studies invo lv ing the characterization of the mutants, S. typhimurium S L 1 3 4 4 and mutants A 1 A 1 , D 1 1 H 5 , E 1 2 A 2 , and G 7 H 1 were first g rown in 1 m l L B broth in culture tubes overnight, wi th shaking at 200 rpm. The next day the bacteria were subcultured at a 1:100 dilution in prewarmed, pre-equilibrated (CO2 buffered) D M E M + + and g rown for another 3 h, wi th shaking. Invasion assays were done using a modi f ied version o f the gentamicin protection assay described by T a n g et al. (310). Sterile 96-wel l plates were seeded with cultured cells 18 h in advance wi th 100 pi o f 0 . 5 - 1 . 0 x l 0 5 ce l l s /ml (depending on cell type), in order to obtain 9 0 % confluency by the next day. The cultured cells were then infected wi th 2 pi o f bacterial cultures (mult ipl ici ty of infection ( M O I ) was, 50-100 bacteria per cel l ) . Bacteria were al lowed to invade phagocytic cells for 30 minutes and non-phagocytic cells for 1 h. (Note that bacterial invasion o f host cells took place in the presence o f serum.) F o l l o w i n g internalization of the bacteria, the cells were washed wi th P B S + + and incubated wi th 100 p i D M E M + + containing 100 pg /ml gentamicin. Where bacterial growth was studied for longer than 4 h , the gentamicin concentration was reduced to 10 pg /ml at 4 h in order to reduce any toxic effects of gentamicin on both the cells and intracellular bacteria. Ce l l s were then lysed in 2 0 p i P B S containing 1% T r i t o n X - 1 0 0 + 0 . 1 % S D S . Dilut ions o f bacteria were made in P B S — and then plated onto L B agar. Bacterial colony-forming units (cfu) were enumerated by serial dilutions and plat ing. A l l counts were obtained from duplicates wel ls wi th in triplicate experiments, and error bars represent standard error o f the means, P<(0.5) (228). Chapter 2 50 2 .6 . R e p o r t e r gene assays The reporter assays were set up in the same manner as the invasion assays. Bo th bacterial and cell cultures were set-up the day prior to the assay and g rown as indicated. Bacter ia were a l lowed to invade the cells as described in section 2.5, and intracellular bacteria were differentiated from extracellular bacteria by their resistance to gentamicin whi le inside the host cells. E a c h experiment was performed in triplicate or quadruplicate, and each mutant was tested in duplicate in each experiment. Er ror bars represent standard error o f the means, P<(0.5) (228). 2 . 6 . 1 . B - g a l a c t o s i d a s e a s says F o r determination o f bacterial B-galactosidase activity, assays were done in 96-wel l microtiter plates. Act iv i ty f rom extracellular bacteria was determined f rom the bacteria remaining in the supernatant above the cells, prior to the addition of gentamicin. Act iv i ty from intracellular bacteria was determined after treatment of the cells wi th gentamicin. Note that the cells were washed once wi th P B S + + to remove gentamicin prior to assaying for enzyme activity. F o r B-galactosidase assays, separate wel ls were used for the determination of enzyme activity and for viable bacterial counts. Over the course o f this study, two different substrates were used to determine B-galactosidase activity from bacteria: a fluorescent and a chemiluminescent substrate. a . f i - g a l a c t o s i d a s e assay u s i n g f l u o r e s c e n t s u b s t r a t e T o each sample, 20 pl o f 0.1% S D S was added and incubated for 5 10 m i n at 3 7 ° C . The wel ls were made up to approximately 100 p l wi th P B S + + , mixed w e l l , and the contents transferred to a 96-wel l P V C (polyvinylchlor ide) plate. One drop (-10 pl) chloroform was mixed wi th the 100 p l sample in flexible P V C plate and the plate was put on ice for at least 5 m i n . (Tri ton X-100 was not used for lysis since the two detergents (Tri ton X-100 and S D S ) formed a precipitate formed when were combined wi th the substrate.) T w o microl i ters of a 50 m M stock o f F D G were then added to each wel l and the plate was incubated at 3 7 ° C for 1 h. T o stop the reaction, the plate was again put on ice and 2 pl o f a 50 m M stock o f P E T G Chapter 2 51 were added to each w e l l . The reagents were light sensitive, therefore all sample manipulations were done wi th m i n i m a l exposure to light. T o determine the enzyme activity, the samples were transferred to a F luo r i con assay plate and the fluorescence at 535 n m emission (485 n m excitation) was measured in a P A N D E X Fluorescence Concentration Ana lyzer ( J D E X X ; Port land, O R ) . Duplicate wel ls for bacterial counts were run under the same conditions as wel ls for the 6-galactosidase assay, and viable bacteria were enumerated by serial dilutions and plat ing. Fluorescence was then correlated wi th viable counts to calculate 6-galactosidase activity as fluorescent units/cfu. b . f i - g a l a c t o s i d a s e a s say u s i n g c h e m i l u m i n e s c e n t s u b s t r a t e F o r the chemiluminescent assay, the bacteria and cultured cells were treated wi th S D S and ch lo roform as above, however the Galac toStor ™ K i t (Tropix Pe rk in E l m e r ) was used to detect B-galactosidase activity. One hundred microliters o f detection reagent (diluted 1:50 of stock solution as per instructions) was added to the 100 pl o f sample, and then incubated at r oom temperature for 30 m i n . The chemiluminescence (or light output) o f the sample was determined us ing the Luminograph L B 9 8 0 photon imager. Duplicate wel ls were used for viable counts, and B-galactosidase activity was correlated as photons/cfu. 2 . 6 .2 . L u c i f e r a s e a s s a y F o r the luciferase assay, the aldehyde substrate was added directly to the sample of either intact cells or bacteria alone, and then viable bacterial counts were obtained f rom the sample w e l l . A c t i v i t y from extracellular bacteria was determined from the bacteria remaining in the supernatant above the cells , prior to the addition o f gentamicin. Act iv i ty f rom intracellular bacteria was determined after treatment of the cells wi th gentamicin. In order to minimize any toxic effects during the assay, the media containing gentamicin was replaced wi th fresh media before adding aldehyde solution. T o determine light production (i.e. luciferase activity) f rom bacteria, 10 p l o f the aldehyde solut ion was added to the 100 pl sample. Immediately the light production was read over the course of 1 m i n at m a x i m u m sensitivity on the Luminograph L B 9 8 0 . L i g h t emissions Chapter 2 52 were obtained as photons/well . L i g h t production was also assessed in microtiter plates with a 1 m i n exposure to X - r a y f i l m , and the results were quantitated us ing a computing densitometer. After the light production was determined, the host cells were lysed with 1% Tr i t on X - 1 0 0 and 0 . 1 % S D S , and the bacteria plated out onto L B plates as indicated for the invasion assays. Luciferase activity was defined as photons/cfu (when using the L B 9 8 0 ) . 2 . 7 . S c r e e n f o r T r a n s f o r m e d Salmonella E x h i b i t i n g L o w L u c i f e r a s e A c t i v i t y O u t s i d e H o s t C e l l s 2.7.1. E x t r a c e l l u l a r B a c t e r i a l S c r e e n The Salmonella colonies obtained from the two-plasmid competition system (described above) were screened for l ow light production on L B plates. Approximately 1 . 5 x l 0 5 bacterial colonies, resistant to both ampic i l l i n and tetracycline, resulted from the transformations. These colonies were exposed to vapors o f n-decanal (e.g. substrate was streaked onto the l i d o f the petri dish) and the resulting light output measured with a Luminograph L B 9 8 0 l o w light video imaging system (Siemens) (251). Colonies showing little to no light production were then restreaked onto fresh L B plates containing both antibiotics, and retested for both growth and low light product ion. Colonies passing through the second stage o f the screen were then transferred to 96-wel l plates containing L B broth with both antibiotics; each we l l contained a separate co lony . The bacterial mutants were also streaked onto green plates to ensure that the bacteria were not chronically infected wi th P 2 2 phage (300). 2 .7 .2 . I n t r a c e l l u l a r versus E x t r a c e l l u l a r B a c t e r i a l S c r e e n The selected bacterial mutants were further tested for l ow light production outside cells in broth, and tested for an induction of light production from within cel ls . Bacteria were initially g rown in 100 pi o f L B broth in 96-wel l plates sealed wi th paraf i lm, at 3 7 ° C and shaking at 150 rpm overnight. Sterile 96-wel l plates wi th black wal ls and clear bottoms (Costar) were used to grow tissue culture cel ls . W e l l s were seeded wi th 100 pi o f Chapter 2 53 l x l O 5 ce l l s /ml in order to obtain 9 0 % confluency by the next day. B o t h macrophage cell lines ( J774A.1 and B A L B . B M 1 ) were used for the initial screen. The overnight bacterial plates were used to inoculate al l other plates, and served as the extracellular stationary phase bacterial controls. F o r the intracellular test plates, 2 p l o f each bacterial mutant f rom the stationary phase plates were used to inoculate the plates containing the cell l ines. Bacteria were al lowed to invade the cells for 1 h and then were washed off wi th P B S and the media replaced with D M E M + + containing l O O p g / m l gentamicin. L igh t from the intracellular bacteria was first determined at 2 h post inoculat ion and again at 4 h. The 2 h time point a l lowed the gentamicin to act on extracellular bacteria for at least 1 h, wh ich has been found to be sufficient time to k i l l the bacteria and eliminate light product ion. Once the activity at the 2 h time point had been determined, the aldehyde-containing media was removed and replaced with fresh D M E M + + wi th gentamicin for another 2 h. A t the 4 h time point, the intracellular activity was determined again. A t 4 h , the viable bacteria were plated out on L B plates to determine bacterial numbers /wel l . Dur ing the screen for bacterial genes w h i c h were upregulated intracellularly, the cfu 's were not determined for each sample. Instead, two to three samples per plate were used as an estimate o f bacterial numbers and applied to the rest o f the plate for convenience. Fo r characterization of the mutants in later studies, actual cfu 's were determined for each sample F o r the extracellular logari thmic phase control plates, 2 pl from each o f the wel ls in the stationary phase plates was first diluted in 200 p l P B S (1:100 dilution) and then 2 p l of this was added to 100 pl D M E M + + in a new plate. The plates were then incubated under similar conditions to the tissue culture cel ls , i.e. not shaking at 3 7 ° C in 5% CO2. The light from the extracellular bacteria (both stationary and log phage bacteria) was detected at 4 h postinoculation o f the log phase plate. Viab le bacteria were plated out f rom the wells after luciferase activity was determined. Chapter 2 54 2.11. M o u s e S t u d i e s 2.11.1. T y p h o i d M o u s e M o d e l Salmonella suspensions were grown at 3 7 ° C in L B broth overnight, wi th shaking at 200 rpm. The next day, bacteria were diluted 1:100 into fresh L B broth and incubated wi th shaking. Af ter 4 h , bacteria were washed once wi th P B S and resuspended in P B S containing 2 % glucose. Cont ro l mice were given P B S wi th glucose only. B A L B / c female mice, aged 6 to 10 weeks, were inoculated oral ly wi th bacterial suspensions (195, 299), after being deprived of water for 4 h. In the first experiment, the inoculat ion size was 25 p i . The dose o f wild- type S L 1 3 4 4 was 2 . 5 x 1 0 6 cfu/mouse, (approximately twice the reported LD50 (195, 299)). The Salmonella mutants were given at 200 times this dose ( 5 x l 0 8 cfu/mouse). A c t u a l counts per mouse (per 25 pi) were as fo l lows ( ± 10% error): for D I 1H5 , 4 . 2 x l 0 8 cfu; for A l A l , 4 . 1 x l 0 8 cfu; for E 1 2 A 2 , 3 . 3 x l 0 8 c f u ; and for G 7 H 1 , 5 . 0 x l 0 8 cfu. F o u r mice were used per group. In the second experiment, three doses were used o f approximately l x l O 8 , l x l O 7 , and l x l O 6 cfu/mouse, and the inoculat ion size was 10 p i . . A c t u a l counts per mouse (per 10 pi) were 10-fold di lut ions o f the f o l l o w i n g (± 10% error): 7 . 6 x l 0 7 cfu o f S L 1 3 4 4 ; 9 . 6 x l 0 7 cfu of A 1 A 1 ; 1 . 3 x l 0 8 o f E 1 2 A 2 ; and l . O x l O 8 cfu o f G 7 H 1 . F i v e mice were used per group. M i c e surv iv ing after 28 days were sacrificed, and their l ivers and spleens were harvested, the organs homogenized and the resulting slurry spread onto M a c C o n k e y plates to obtain bacterial colony counts. The day at w h i c h 5 0 % of the mice in each group had died was determined using the statistical calculations o f Reed and M e u n c h (259,260) and the median survival t ime (228). Chapter 3 55 C h a p t e r 3: D e v e l o p m e n t o f a n I n t r a c e l l u l a r R e p o r t e r S y s t e m Chapter 3 describes the development o f an assay system for gene expression from intracellular bacteria. Specif ica l ly , the benefits and challenges of w o r k i n g wi th two different reporters, B-galactosidase and luciferase, are discussed. The ability o f Salmonella to survive and grow within cells is crucial to its ability to cause disease wi th in a host (27, 199). M a n y mutants wh ich are defective in their ability to invade or replicate wi th in cells are also avirulent in the host (4, 49 , 76, 85). The development o f cell-mediated immuni ty in addition to a humoral immune response against Salmonella infection, is also indicative o f a host response to intracellular pathogens (271, 326). F o r Salmonella to propagate wi th in the intracellular environment, the bacteria must adapt by regulating the expression o f proteins necessary for growth wi th in that environment. This global regulation has been observed by us ing two-dimensional protein gels, where bacteria have been shown to regulate the expression o f proteins upon infection o f cultured macrophages (1, 30, 33 , 34). H o w e v e r , to study the expression o f individual genes, fusion o f the gene of interest to a reporter gene provides a simpler method to monitor expression. The detection o f reporter gene expression often uses bacteria grown as colonies on agar plates or g rown to h igh density in broth cultures. The number o f Salmonella wi th in host cells after invasion w i l l be 10-100 fold lower, therefore the suitability o f us ing specific reporter genes to determine gene expression from bacteria wi th in host cells was examined. 3.1. R e s u l t s 3.1.1. I n v a s i o n assay to d e t e r m i n e i n t r a c e l l u l a r b a c t e r i a l n u m b e r s A gentamicin-protection assay (310) was used to define the bacteria wh ich had invaded a host cell and thus become intracellular. Salmonella were added to medium containing cultured cells and a l lowed to invade the cells for a set period of t ime. The extracellular bacteria were then removed, the cells washed, and the new med ium containing the antibiotic gentamicin was added. Bacteria residing inside the host cells were protected from the k i l l i ng effects of gentamicin, whi le extracellular bacteria were k i l l ed (310). T o enumerate the intracellular Chapter 3 56 bacteria, the infected host cells were then lysed wi th 1% Tr i ton X - 1 0 0 , w h i c h destabilized the membrane o f the host cel l without impair ing the bacterial membrane, a l lowing the bacteria to be released f rom the ce l l . The bacteria were then diluted and colony-forming units (cfu) were determined f o l l o w i n g growth on agar plates. Cel lular 'ghosts' resulting from host cell lysis were observed under the microscope, and bacteria were seen s w i m m i n g through the lysis buffer. A possible concern wi th the method was that some o f the bacteria might c lump or associate wi th cellular debris, or that the intracellular vacuoles might not lyse wi th the same efficiency as the outer cell membrane. The result w o u l d be an underestimation o f the number of intracellular bacteria. Therefore, the addition o f the ionic detergent S D S was added to the lysis buffer in an effort to disrupt any remaining cell/bacterial interactions. Further experiments showed that 0 . 1 % S D S was not damaging to Salmonella g rown in media alone, i.e. cfu's were not reduced significantly by the presence of 0.1 % S D S . A combination o f 1 % T r i t o n X - 1 0 0 and 0 . 1 % S D S was tested and resulted in the recovery of 10-fold more bacteria f rom infected cells than wi th T r i t on X - 1 0 0 alone (Figure 4). A s the estimation o f intracellular bacterial numbers is very important for the determination o f specific activity o f a reporter enzyme, the combination o f 1% T r i t o n X - 1 0 0 and 0 . 1 % S D S in the lysis buffer was used for the enumeration o f intracellular bacteria in all further experiments. Chapter 3 57 Figure 4: The effect o f the detergents Tri ton X - 1 0 0 (1.0%) and S D S (0.1%) on the recovery of intracellular S. typhimurium f rom H e L a cells . The experiments were done in triplicate and the error bars represent the standard deviation o f the means. Chapter 3 58 3.1.2. B - g a l a c t o s i d a s e as a r e p o r t e r o f i n t r a c e l l u l a r b a c t e r i a l e x p r e s s i o n The p lasmid p F F 14 (11.2 kbp) is a p A C Y C 1 8 4 - b a s e d (39) low-copy number plasmid containing a translational fusion between the S. dublin spvB gene and the lacZ reporter gene (72, 77) (Table 2; F igure 5). It was previously demonstrated that the copy number of the plasmid p F F 1 4 remained constant throughout the growth cycle of S. dublin (72), indicating that an increase in the expression of genes encoded on this p lasmid was not due to growth cyc le but to a change i n regulation. The spv operon has previously been shown to be regulated by S p v R (72, 77, 129), and spvB::lacZ expression increased more than ten-fold upon bacterial entry into stationary phase or under conditions of carbon or nitrogen starvation (72, 125). Note that the regulation o f the spvB gene remained under the control o f S p v R wi th both the p F F 1 4 and p S P L U X constructs. The p lasmid p F F 1 4 was used to examine the production o f 6-galactosidase resulting from expression of the spvBr.lacZ fusion, from Salmonella wi th in epithelial cells ( M D C K and H e L a cells). After incubation o f bacteria and host cells together as described above, the sample was treated wi th 0 . 1 % S D S and chloroform to fully release the reporter enzyme. Act iv i ty of B-galactosidase was assayed us ing a fluorescence assay based on the substrate F D G , which had previously been shown to be more sensitive than the colorimetric assay based on the substrate O M P G (28, 108). After a set per iod o f incubation, the reaction was stopped and the fluorescence o f the sample was measured using a Pandex fluorimeter. Th is reading was converted into specific activity (fluorescence/cfu) in combination wi th the number of colony forming units (cfu) or viable bacteria recovered from a parallel sample. Note that chloroform was not added to samples used for determining cfu's. The data shown in Figure 6 A represents the expression f rom the spvBr.lacZ gene fusion from S. dublin w h i c h were either extracellular (i.e. wi th in the culture supernatant taken from above the cultured cells) or intracellular (i.e. inside cells and thus protected from gentamicin). The amount o f 6-galactosidase activity increased per bacterium when the bacteria were intracellular as compared to those which were Chapter 3 59 Figure 5: P l a s m i d maps o f p F F 1 4 and p S P L U X . The 11.3 kbp p l a smid p F F 1 4 (circular map) which contains spvR, spv A, and a translational spvB::lacZ fusion, was used for the B-galactosidase studies. The 14.55 kbp plasmid p S P L U X was made by inserting a 3.25 kbp promoterless luxAB gene cassette into the BamHI site between spvB and lacZ, thus placing luxAB under the transcriptional control o f the spvB gene. The plasmid p F F 1 4 was used for the B-galactosidase studies, whi le p S P L U X was used for the luciferase studies. Chapter 3 60 u c/j < ••!=: c cn ro O CD CD cn CD 1E+0 1E-1 1E-2 1E-3 Extracellular Intracellular B 1 0 > 3 £ 2 6 w CO .2 3 € = 4 CO CD CC o CO - 2 HeLa MDCK Figure 6: Express ion o f spvBr.lacZ by bacteria inside non-phagocytic cel ls . A ) 6-galactosidase activity from bacteria wh ich are either intracellular (inside H e L a or M D C K cells) or extracellular (remaining in the supernatant above the cel ls) . B ) Rat io of induction shown in (A) where intracellular activity is divided by extracellular activity. Chapter 3 61 extracellular. A s shown in Figure 6 B , this induction in activity was greater than f ive-fold within both epithelial cel l types ( M D C K and H e L a ) tested. A l though B-galactosidase was deemed to be a sensitive reporter o f intracellular bacterial gene expression, there were problems associated wi th its use. First , the host cells had varying background levels o f activity. Th is reduced the sensitivity o f the assay when using these cells and made comparisons between cell types more difficult . Second, both the host cells and the bacteria needed to be completely lysed to accurately assay the enzymatic activity. Since chloroform was used in combination with l ow levels o f detergent, the samples had to be transferred to special P V C plates as the chloroform reacted with the polystyrene components o f regular mul t iwe l l plates. A s w e l l , higher amounts o f detergent could not be used for lysis as it formed a precipitate in the substrate buffer, wh ich interfered wi th the determination of fluourescence in the sample. T h i r d , the F D G substrate was sensitive to light and therefore samples had to be kept in the dark as much as possible. Four th , it was impossible to separate l ive bacteria (i.e. metabolically active bacteria that were able to be cultured) f rom those that had been k i l l e d by the host ce l l , therefore B-galactosidase activity resulted from both l ive and dead bacteria wi th in a sample. This was especially important for the determination o f specific enzyme activity (defined as the number of fluorescent units per viable bacterium). The state o f the Salmonella wi th in cells has been reported to be in a constant flux wh ich can be divided into two populations, one w h i c h is dy ing and the other wh ich is g rowing (2, 32). Another reporter, bacterial luciferase, was k n o w n to be detectable from only live bacteria due to its requirement for the bacterial energy F M N H 2 for activity. It was therefore tested to determine whether it cou ld be used as a sensitive reporter o f intracellular gene expression. A promoterless 3.25 kbp luxAB gene cassette from Vibrio harveyi (138) was ligated into the BamHI o f the p lasmid p F F 1 4 creating an spvB::luxAB transcriptional fus ion, resulting in the isogenic p lasmid p S P L U X (14.5 kbp) (Figure 5) (251). Chapter 3 62 3.1.3. L u c i f e r a s e as a r e p o r t e r o f i n t r a c e l l u l a r b a c t e r i a l gene e x p r e s s i o n a . M e a s u r e m e n t o f l i g h t p r o d u c t i o n . The product o f luciferase is light, wh ich is produced specifically at a wavelength of 490 n m f rom V. h a r v e y i luciferase (210). Note that the bacterial substrate, n-decanal, was able to diffuse across both bacterial and host ce l l membranes, therefore lysis of bacterial and host cells was unnecessary for the determination o f enzyme activity. Furthermore, the number of bacteria cou ld be determined on the same sample from wh ich the enzyme activity was detected (180). These details are discussed more ful ly in the fo l lowing sections. L i g h t production from bacteria was detected and quantitated with the use of different systems, including a tube luminometer, X - r a y f i l m combined wi th a densitometer, or a photon-imaging camera combined wi th a computer processor. The sensitivity and linearity of the different light-detection methods was analyzed. A single tube luminometer was initially used for determining light production from bacteria free in culture, however , difficulties arose in determining light production from intracellular bacteria. First , the host cells cou ld not be grown in the luminometer tubes, so the invasion portion o f the assay had to be performed in a separate dish and then the host cells lysed in order to free the intracellular bacteria for transfer to the light assay tube. This introduced another variable into the system and raised the concern that detergents and other buffer components may alter the activity of the enzyme. It also resulted in the assaying of live bacteria w h i c h were no longer intracellular, and which could poss ibly adapt their gene expression. On ly the host cells were completely lysed and the bacteria were exposed to not only l o w levels o f detergents but to host cell contents containing lysosomal degradative proteins, etc. Since there was often a delay o f 5-10 m i n during cell lys i s , there was enough time for the bacteria to react to the 'new extracellular' environment, and perhaps exhibit an altered form of gene expression from that seen from truly intracellular bacteria. Th is differed from the assay for 6-galactosidase activity where both bacterial and host cells were completely Chapter 3 63 lysed at the same time. F ina l ly , the luminometer could only measure one sample at a time and was not conducive to screening many samples. X - r a y f i l m was tested and found to be capable o f determining bacterial light production from both extracellular and intracellular bacteria. B o t h bacteria and host cells cou ld be grown in 96-wel l microtiter plates, wh ich were then exposed directly to the X - r a y f i l m for a set period of t ime, and the spots appearing on the X - r a y f i lm quantitated us ing a densitometer. This a l lowed for the testing o f many samples at one time. Howeve r , a new set o f problems was encountered. First , regular 96-wel l microtiter plates were made o f clear plastic wh ich al lowed the light to sp i l l over into neighbouring wel ls , altering the true reading o f those wel l s . The cross-contamination o f light between wells was eliminated with the use o f 96-wel l white plastic gr id plate wi th 8-wel l strips wh ich could be snapped into place wi th in the gr id (Pierce Immunoware 8 -Wel l E I A Strip Plates). The second problem was that whi le light could be detected from over a 1000-fold range, it was only linear over a 10-fold range as determined by densitometry scans of the X - r a y f i l m . T h i r d , due to the small numbers of bacteria inside cells , the light produced from intracellular bacteria was often at the lower l imit of detectable activity. . W i t h the Luminograph L B 9 8 0 photon-imaging camera/computer system it was possible to detect and quantitate light production over a 10000-fold range, wh ich was linear within a 100-fold range (about 1 . 0 x l 0 3 - 1 . 0 x l 0 5 photons/well) . This system was also amenable to screening in a number o f different formats, e.g. 96-wel l microtiter plates or colonies on agar plates. It was found to be more sensitive than X - r a y f i lm by at least f ive-fold (Figure 7) . H o w e v e r , as shown in F igure 7, the white plastic from the microtiter plates produced a l ow level phosphorescence and was highly reflective. Th is problem was later reduced with the use o f black microtiter plates wi th clear bottoms (Costar). The black plastic was not phosphorescent and less reflective, whi le the clear bottoms o f the wel l a l lowed for the analysis o f the host cell monolayer . The background level from the gr id plates made o f white polystyrene was about 1 . 0 - 4 . 0 x l 0 3 photons/wel l , whi le f rom the plates made of black polystyrene, it was reduced to about 2 .0 -5 .0x10 2 photons/wel l . This l o w background level of Chapter 3 64 Method of Light Detection Bacteria 1/5 | 1/25 Dilutions 1/125 1/625 LB980 Luminograph i i w m w X-ray Film 1 Figure 7: Compar i son o f two different methods to detect bacterial light production: L u m i n o g r a p h L B 9 8 0 versus X - r a y f i l m . S. dublin p S P L U X were g rown to stationary phase to induce spvB::luxAB expression and then diluted in P B S within 96-wel l gridded microtiter plates. The substrate rc-decanal was added to a final concentration o f 0 .0022%. (Note that in order to directly compare the two methods, the picture obtained from the luminograph is shown as a negative image wi th the light output appearing as a dark image on a l ight background.) Chapter 3 65 light (i.e. f rom static electricity, dark noise from the camera) was subtracted from the calculations. b . E f f e c t s o f a l d e h y d e c o n c e n t r a t i o n o n b a c t e r i a a n d l u c i f e r a s e a c t i v i t y . A s mentioned previous ly , to produce a blue-green light (490 nm), bacterial luciferase required an energy source (reduced flavin mononucleotide - FMNH2), oxygen , and a long chain aldehyde (210). Therefore, when l ive bacteria were used, only the aldehyde substrate had to be added exogenously. Previous studies indicated that h igh aldehyde concentrations may be inhibi tory to light production (194), therefore a range of concentrations was prepared to determine the optimal aldehyde concentration needed to measure light output f rom both intracellular and extracellular Salmonella. The final concentration o f rc-decanal in the assay wel l s ranged f rom 0 .00022% to 0 .0099%. The effects o f different aldehyde concentrations on both extracellular and intracellular S. dublin L D 8 4 2 p S P L U X are shown i n F igure 8. The results us ing 5\ typhimurium S L 1 3 4 4 p S P L U X were s imi lar . In F igure 8 A , a decrease in total light output f rom the wel ls was seen for the two highest concentrations o f n-decanal (0 .00495% and 0 .0099%). Further investigation revealed that these concentrations o f aldehyde were actually toxic to bacteria (Figure 8 B ) . Viab le counts (cfu's) were reduced by 5 to 10 fold in the presence o f the two highest concentrations o f aldehyde, whereas cfu's were unaffected for aldehyde concentrations of 0 .0022% or less. T h e aldehyde concentrations w h i c h caused toxicity were similar for both intracellular and extracellular bacteria. Interestingly, the decrease in the number of viable bacteria was greater than the decline in light production per w e l l . Therefore, the light output per viable bacterium appeared to rise as the aldehyde concentration increased (Figure 8C) . Chapter 3 66 Figure 8: Effect of aldehyde concentration on bacterial viabil i ty and light product ion. M D C K cells were g rown to confluency in 96-wel l microtiter plates and infected wi th S. typhimurium S L 1 3 4 4 p S P L U X as stated in the methods. Bacteria remaining wi th the cell monolayer after washing and gentamicin treatment were termed intracellular, whereas bacteria removed with the supernatant before washing were termed extracellular. This figure depicts the individual data points f rom one experiment, and the lines represent the means o f the data points. (A) Light production as photons per 100 pi we l l ; (B) Viable bacterial counts per 100 pi we l l ; (C) L igh t production as photons/cfu: (closed symbols) intracellular bacteria; (open symbols) extracellular bacteria. Chapter 3 68 Since the purpose o f us ing the luciferase reporter was to correlate the expression o f a gene to enzyme activity within a single l ive bacterium, it was crucial that the enzyme substrate not be toxic to that bacterium. Therefore, in subsequent experiments a final aldehyde concentration of 0.0022% was used, as this concentration produced the highest amount of light without a reduction in bacterial numbers. Trypan blue exclusion studies were used to determine whether tissue culture cell death occurred (9). N o n e o f the aldehyde concentrations were toxic to the mammalian cells , even over extended periods o f co-incubation o f up to one hour (data not shown) . It was further determined that gentamicin-kil led bacteria d id not produce light (Figure 9) and therefore, light produced from intracellular bacteria was not contaminated by k i l led bacteria remaining outside the cells. Th i s provided further support that bacteria had to remain alive in order for luciferase activity to be detected; i f they were k i l l ed by the ce l l , they w o u l d most l ikely cease to produce light. The induct ion of the spvB::luxAB transcriptional fusions is shown in F igure 10, where equal numbers o f bacteria were present in the extracellular and intracellular wel l s . Bacteria lacking the luxAB fusions (S. dublin or S. typhimurium) d id not produce light. Wel l s containing extracellular Salmonella p S P L U X also d id not produce much light since the bacteria were in logarithmic phase growth, and not entering into stationary phase. F igure 10 furthermore demonstrates that there is no light-producing activity detectable from the host cells . However , intracellular Salmonella p S P L U X expressing the spvB::luxAB transcriptional fusion showed increased l ight production. Chapter 3 69 Gentamicin Concentration (Mg/ml) S. dublin pSPLUX S. typhimurium pSPLUX S. dublin S. typhimurium 1 0 0 0 1 0 0 1 0 A • 0 m • * Figure 9: The action of different concentrations of gentamicin on the luciferase activity within bacteria. Salmonella were incubated for 45 min in the presence of DMEM++ plus gentamicin, at 37°C without shaking, prior to determining luciferase activity. Concentrations of gentamicin which killed more than 90% of the bacteria within the sample (>100 pg/ml) reduced the light output of the samples to near background levels. X-ray film was used to determine light output. Chapter 3 70 SL1344 SL1344 pSPLUX LD842 pSPLUX Extracellular Hi HH Intracellular DL Figure 10: Detection o f light production in Salmonella p S P L U X in an intracellular versus an extracellular environment. M D C K cells were seeded into 96 w e l l microtiter plates wi th grids and infected with Salmonella, as described in the methods. After 2 h , extracellular bacteria were removed and placed into wel ls containing no M D C K cel ls . The samples were adjusted such that all wel ls contained equal numbers o f bacteria (approximately l x l O 5 c fu /wel l ) . N o light was produced i n the absence o f the luxAB genes. A low level o f light was detected from the extracellular bacteria containing the plasmid p S P L U X {e.g. under spvB repressing condit ions) . Increased light production was detected in wells containing intracellular bacteria wi th p S P L U X , indicating an increased expression o f the spvB::luxAB transcriptional fus ion. This figure was obtained us ing the Luminograph L B 9 8 0 . Chapter 3 71 3.1.4. Comparison of B-galactosidase and luciferase as reporters of intracellular bacterial gene expression. Table 4: Induction of expression of reporter enzyme fusions by intracellular S. dublin. Reporter Enzyme Bacterial Location 3 Specific Activity1 5 Relative Increase 0 B - G a l a c t o s i d a s e Extracellular Intracellular 0.0084 +/- 0.001 0.1872 +/- 0.174 22 L u c i f e r a s e Extracellular Intracellular 0.0157 +/- 0.015 0.3820 +/- 0.130 24 a Extracellular location refers to the M D C K cells culture medium, 2 hr after initial infection by S. dublin. Intracellular location refers to the M D C K cells, 4 hr after initial infection. b Units for specific enzyme activity. B-galactosidase units are expressed as F D G fluorescence/cfu; luciferase units are expressed as photons/cfu. The error bars represent the standard deviation of the means. c Relative increase for each enzyme is the specific activity of intracellular bacteria divided by specific activity of extracellular bacteria. The sensitivity of luciferase as an intracellular reporter was compared to that of B-galactosidase through the use of the isogenic plasmid fusions to spvB (126). Activities were first normalized to activity per bacterium (cfu) for each respective enzyme. Then the relative increase in activity after invasion was determined by dividing the intracellular activity by the extracellular activity. In side-by-side experiments, the increase in luciferase activity was similar to the increase in B-galactosidase activity (24 versus 22 fold), as shown in Table 4. Therefore the luciferase was as sensitive as B-galactosidase for the detection of intracellular gene expression. Chapter 3 72 F o r both assays, the minimurn number o f bacteria required in order to detect the spvB expression varied as the gene was induced or repressed. Unde r repressing condit ions, 0 . 5 - 1 . 0 x l 0 5 bacteria were required, whi le only about l . O x l O 3 bacteria were required when spvB was induced. It was previously shown (72) that the induction o f spv gene expression was not the result of an increased p lasmid copy number wi th in the bacteria. 3.2 . Discussion Bacterial luciferases have been used in previous studies as reporters o f intracellular bacterial gene expression, however , many o f those studies were performed under conditions wh ich were not phys io logica l for mammalian cells (e.g. lower temperatures ranging from 2 2 - 3 0 ° C ) . Some examples include the monitoring of gene activation during plant-microbe interactions under conditions optimal for plant growth (187), and the induction of hydrogen peroxide-stimulated genes in 5. typhimurium upon interaction with mouse macrophages at 3 0 ° C (92). This was o f concern since temperature is the basis for induction o f numerous virulence-associated genes, over a broad range o f pathologenic bacteria (1, 30, 80, 211) . M a n y virulence factors are optimally expressed at 3 7 ° C . The luciferase f rom V. fischeri is inadequate for temperature-dependent studies since it is inactivated above 3 0 ° C (69, 92, 155), however the luxAB gene cassette from V. harveyi encodes a heterodimeric luciferase which remains active at 3 7 ° C (69, 155, 210). In the w o r k reported here, bacteria and tissue culture cells were grown and assayed at phys io logica l temperatures ( 3 7 ° C ) , wi th min ima l disruption to interactions occurr ing between the intracellular bacteria and the mammalian host cells . Al though an alternative luciferase from Xenorhabdis luminescens remains thermostable up to 4 5 ° C (155, 210), it has a lower specific activity than V. harveyi luciferase (309). A s w e l l , V. harveyi luxAB genes were contained wi th in a convenient 3.25 kbp BamHI gene cassette wh ich d id not contain its o w n endogenous promoter. N o t included wi thin this cassette were the aldehyde biosynthetic genes luxCDE, encoded by an extra 4 kbp segment o f D N A . Whi le the presence o f substrate synthesizing genes may appear to be an advantage, a fusion with high expression promoters w o u l d result in the high production o f aldehyde, and potentially Chapter 3 73 increased Salmonella mortali ty. Even though the aldehyde substrate had to be exogenously supplied, previous studies have indicated that recombinant lux products exhibit h igh activity in the presence of externally-added decyl aldehyde (210, 309). Langr idge et al. (186) had previously found that vapors f rom high decanal concentrations (10% or more) resulted in increased levels of mortality among young plantlets. Therefore, the effects o f aldehyde concentration on both bacteria and M D C K cells was addressed. N o n e o f the aldehyde concentrations tested appeared to harm the mammalian cel ls , but higher aldehyde concentrations (>0.0022%) were toxic to the bacteria (Figure 8 B ) . It was not determined whether this toxicity was a direct or indirect effect of the aldehyde. The drop in numbers o f co lony- forming bacteria was greater than the drop in total light output per wel l (Figures 8 A , B ) , indicating that higher aldehyde concentrations elicited more light production from the remaining viable bacteria (Figure 8 C ) . It may be that the concentrations o f aldehyde tested d id not reach the substrate saturation range o f the luciferase enzyme, or conversely, that the enzyme activity rendered them unculturable. The use of higher amounts o f aldehyde therefore may have resulted in more efficient activity o f the enzyme. H o w e v e r , since the mechanism of aldehyde toxicity was u n k n o w n , the concentration o f aldehyde (0.0022%) wh ich permitted the highest light production by luciferase without concomitant bacterial death was used. The use o f bacterial luciferase as a reporter of gene expression d id pose some other problems. A n article by Forsberg et al. (87) reported that an intrinsically curved segment o f D N A in the 5 ' cod ing end o f the luxA gene may influence promoter activity o f the target gene. However , the luciferase activity correlated with the previously established B-galactosidase data, indicating that the 5 ' end o f the luxA gene d id not interfere wi th spv regulation. L i k e w i s e , Gonza lez-Flecha and Demple (114) reported that luciferase activity (in the absence o f n-decanal) was associated wi th an increase in oxidative radicals wi th in the bacteria over time. H o w e v e r , since the spv operon is not influenced by the redox state o f the bacteria (72), it seems unl ikely that either the luxAB genes or the luciferase enzyme activity interfered with Chapter 3 74 spvB gene expression. Furthermore, it had been suggested by Meighen (210) that changes in intensity o f luminescence in vivo may depend not only on the amount o f functional luciferase available, but also on the availabil i ty o f substrates (FMNH2, aldehyde, and O2). Th is implied that luciferase w o u l d be an inaccurate reporter in situations where either oxygen or energy were lack ing . It appeared that the environment Salmonella encounters upon invasion o f epithelial cells contained enough oxygen to support luciferase activity (Figures 8 and 10; and Table 4 ) . A s w e l l , intracellular Salmonella remained viable, p rovid ing the aldehyde concentration was not too high. These results indicated that use of luciferase for Salmonella studies w o u l d not be l imited by either the lack of oxygen or bacterial energy available to the intracellular bacteria. Francis and Gallagher (92) showed that luciferase activity in response to oxidative stress was variably expressed wi thin infected cel ls , suggesting that the intracellular environment may differ somewhat between individual cells or wi th in a cellular subpopulation. Furthermore, there is a constant struggle between the bacteria and the host cells taking place dur ing an infection (2, 32). A s a result of the dynamic invasion process o f the bacteria, there are two populations o f Salmonella wi th in cells , one static (and possibly decreasing in number) and the other rapidly d iv id ing . These variables together may account for the variation in Table 4 . N o reporter system is ideal for all situations and each system has its advantages and disadvantages. In this study, the use o f luciferase as a reporter o f intracellular bacterial gene expression was assessed us ing conditions optimal for Salmonella invasion o f non-phagocytic mammalian cel ls . The bacterial luciferase, encoded by promoterless luxAB genes from V. harveyi, p rovided an alternative reporter system to 6-galactosidase, wi th several advantages. Luciferase was an accurate and sensitive reporter o f intracellular Salmonella spv gene expression, as confirmed by data us ing 6-galactosidase assays (77). Moreover , the luciferase assay was faster and easier to perform than the 6-galactosidase assay. Firs t , there was no need to lyse either the cells or the bacteria, and activity in the sample was measured immediately after substrate addition. F o r the 6-galactosidase assay, it was necessary to first Chapter 3 75 lyse the bacteria and then incubate the sample in the presence of the substrate for a set period of time. It was further necessary to protect the fluorescent 6-galactosidase substrate, FDG, from light. With luciferase, it was not only possible to determine bacterial gene expression from live extra-cellular bacteria but from intra-cellular bacteria as well. Another advantage of using luciferase was the absence of background activity. Neither the cells nor the bacteria had any endogenous luciferase activity and therefore any light detected resulted from the luxAB constructs, whereas endogenous 6-galactosidase activity could be detected from mammalian cells. The ability to monitor luciferase activity without physically disrupting either the bacteria or the cells would also allow for the monitoring of bacterial-cell interactions over time. Furthermore, the product (light) did not build up within the sample, and low dose applications of the aldehyde substrate were found to be non-toxic to both bacteria and tissue culture cells. Collectively, the results demonstrate that luciferase gene fusions are a sensitive way to monitor gene expression of bacterial pathogens found within mammalian host cells. Chapter 4 76 C h a p t e r 4 : D e v e l o p m e n t o f a S c r e e n f o r B a c t e r i a l G e n e s Chapter 4 describes a screening system developed to search for Salmonella genes induced after bacterial invasion o f host cel ls . Luciferase was used as the reporter o f bacterial gene expression. Th is chapter first describes transfer o f the luxAB reporter genes to the S. typhimurium chromosome, and the selection o f the resulting bacterial mutants. Genes wh ich were differentially expressed were then identified, specifically those genes induced by intracellular bacteria and repressed by extracellular bacteria. 4 . 1 . R e s u l t s 4 . 1 . 1 . T r a n s f o r m a t i o n o f Salmonella a n d S c r e e n f o r U p r e g u l a t e d B a c t e r i a l G e n e s A two p lasmid competition system, described by G u z z o and D u B o w (138), was used to obtain random insertions o f the promoterless reporter gene cassette luxAB wi th in the wild- type S. typhimurium S L 1 3 4 4 chromosome (Figure 11). The p lasmid p F U S L U X contained a C o l E l or igin of replication and a T n 5 - t o A 5 - t e t r a c y c l i n e resistance cassette, while the p lasmid pTF421 encoded for ampic i l l in resistance and the production o f R N A 1 w h i c h acted to inhibi t the replication o f p F U S L U X . B y growing the transformed bacteria in the presence o f both tetracycline and ampic i l l in , the luxAB gene cassette was forced from the replication-inhibited p F U S L U X plasmid to integrate into the bacterial chromosome, such that the tetracycline resistance could be maintained by the bacteria. The tetracycline gene contained its o w n promoter; however , the luxAB genes were not expressed unless the cassette integrated such that it was under the control of an active bacterial promoter. Chapter 4 77 Tn5L luxA luxB tet Tn5R Figure 11: Schematic of the luciferase reporter gene cassette inserted within the bacterial chromosome, and the orientation of the primer pairs used for inverse PCR and subsequent D N A sequencing. Chapter 4 78 Ini t ial ly, the two plasmids were sequentially transferred to S. typhimurium S L 1 3 4 4 using electroporation. Firs t the p F U S L U X plasmid was transferred into a S L 1 3 4 4 bacterium. Pur i f ied p T F 4 2 1 p lasmid was then electroporated into S L 1 3 4 4 p F U S L U X . H o w e v e r transformation o f the second p lasmid by electroporation was inefficient and only a few hundred colonies wi th dual antibiotic resistance resulted from multiple attempts. Therefore, the phage P 2 2 H T int was used to transfer the p lasmid pTF421 into S L 1 3 4 4 p F U S L U X . This procedure was much more efficient and approximately 15,000 S. typhimurium colonies resulted from the transductions. T o init ial ly identify extracellular bacteria expressing the integrated reporter genes, the Luminograph L B 9 8 0 photon imager was used to examine the transduced Salmonella colonies on agar plates for light product ion. The substrate was added to the colonies by streaking the aldehyde onto l ids o f the plates and a l lowing the vapors to penetrate the colonies . Those producing h igh relative amounts o f light on the plates were discarded since they represented extracellular bacteria expressing high amounts o f luciferase, whi le those producing little to no light on plates were retested (Figure 12). Each colony to be retested was streaked out onto a fresh plate to ensure the colony was truly resistant to both antibiotics and was also a single c lone. Over 3500 colonies (about 2 .4% of the original number o f transformants) displaying little to no luciferase activity were detected in this manner. Each o f these was further tested on green plates to ensure that they d id not contain active P 2 2 phage infections (i.e. lytic infections resulted in dark green colonies rather than light green colonies). Bacter ia free of lyt ic infections were then transferred to broth cultures wi th in individual wel ls o f a 96-wel l plate. Chapter 4 79 Figure 12: Luminograph images o f L B plates wi th S. typhimurium colonies transfected with both p T F 4 2 1 and p F U S L U X plasmids. B o l d , left-facing arrows point to examples of colonies producing light on L B agar alone, w h i c h were not further screened. Smaller , right-facing arrows point to examples o f colonies producing little to no light on plates alone, wh ich were p i cked for further screening. Chapter 4 80 4 « Chapter 4 81 At this point, the bacteria were stored at minus 70°C in 25% glycerol stocks within the 96-well plates and covered by a 96-well storage mat (Costar). Note that individual colonies were not pooled but placed into separate wells. 4.1.2. S c r e e n i n g f o r B a c t e r i a l G e n e I n d u c t i o n I n s i d e C u l t u r e d M a c r o p h a g e s Each colony expressing low levels of light on agar plates was tested for luciferase activity during growth both outside and inside host cells. 96-well plates containing L B broth were inoculated with bacterial mutants and grown overnight, with shaking. The next day these plates, now considered the 'extracellular stationary phase' bacteria, were used to inoculate another plate of L B broth (the 'extracellular logarithmic phase' bacteria) and two plates of cultured macrophages, J774A.1 or B A L B . B M 1 (i.e. 'intracellular' bacteria). The time at which the bacteria was added to the cells was time zero. S. typhimurium typically has a lag period about 4 hr after it invades cells, during which no increase in cfu's is seen within the cells. This lag period is thought to allow the bacteria to adapt to the intracellular environment, and after this lag, the bacteria are able to grow exponentially, at least within epithelial cells. Therefore, both 2 and 4 hr after invasion, the bacterial samples were tested for the induction of light production, which was indicative of the promoter activity of genes expressed during intracellular growth. Bacteria were incubated with the cells for 1 hr, after which the medium was removed and the cells were washed. The infected cells were then treated with gentamicin for 1 to 3 hr in order to kill any bacteria remaining extracellular. Two and four hours after the initial infection of the cultured cells, the aldehyde substrate was added to the wells and the light production measured using the Luminograph LB980 . After the 2 hr reading, the aldehyde-containing medium was replaced by fresh gentamicin-containing medium and the incubation continued for another 2 hr. The light production from the plates of extracellular bacteria (stationary and logarithmic phase) was only read 4 hr after time zero. An example of this screening procedure is shown in Figure 13, which demonstrates the light production from one 96-well plate of bacterial mutants under the six different conditions described: two extracellular (stationary and logarithmic phases of growth) and four Chapter 4 82 intracellular (2 and 4 hr inside either J774A.1 or BALB.BM1 cells). Light from each well was quantitated using the Luminograph LB980 and compared to the estimated bacterial count for that well. Bacterial numbers were estimated based on the averages of randomly picked wells within the various conditions, and generally, stationary phase bacteria averaged between 0.5-1.0xl0 9 cfu per well; logarithmic phase bacteria averaged between 0.5-l.OxlO 8 cfu per well; and intracellular bacteria averaged between 1.0-5.Ox 105 cfu per well at both 2 and 4 hr. The thirty-three plates of frozen bacterial mutants were tested under each of the separate conditions at least twice. This was considered round one of the 96-well plate screen. Mutants demonstrating the induction of light inside cells were then assembled into new 96-well plates and frozen as before. These mutants were again tested in all six conditions mentioned above, and this was considered round two. This was done to affirm that the reporter gene expression by these mutants was indeed induced and that the phenotypes were stably expressed. After screening, the mutants continuing to demonstrate the induction of light inside cells were once again assembled into new 96-well plates for round three of the screen. From this final round of testing the S. typhimurium SL1344 mutants, 10 mutants (from the original 3500 mutants) consistently upregulated light production by more than an estimated five-fold at both 2 and 4 hr inside the mouse macrophage-like cell lines (both J774A.1 a n d B A L B . B M l ) when compared to the extracellular controls (both stationary and logarithmic phase). Chapter 4 83 Figure 13: Luminograph images of bacteria expressing light from a single sample 96-wel l plate o f mutants. Bacterial growth conditions are as fo l lows: A ) inside B M I cells 2 hr after infection; B ) inside B M I cells 4 hr after infection; C ) inside J 7 7 4 A . 1 cells 2 hr after infection; D ) inside J 7 7 4 A . 1 cells 4 hr after infection; E ) extracellular bacteria in log phase growth; F ) extracellular bacteria in stationary phase growth. Chapter 4 84 Chapter 4 85 4 .1 .3 . T r a n s f e r o f G e n e s to E n s u r e I n d u c t i o n P h e n o t y p e is L i n k e d to G e n e I n s e r t i o n s T o be certain that the induction phenotype resulted from the chromosomal insertion of the gene cassette into a single gene, phage P 2 2 H T int lysates were made from each mutant. These lysates were used to transduce new S. typhimurium S L 1 3 4 4 , and the resulting transduced bacteria were then tested for a pattern o f light induction that matched the original parental mutant. S i x mutants were identified and were named A 1 A 1 , E 1 2 A 2 , D I 1H5, G 5 D 5 , G 7 H 1 , and G 8 B 1 (Figure 14). Green plates were once again used to ensure that the transductants no longer contained the P22 phage. This time however , the light green colonies o f the transductants (i.e. no lyt ic P22 infection) were also cross-streaked against P22 H 3 . The transductants were considered free o f lysogenic P 2 2 only i f they could be re-infected by the P 2 2 H 3 phage (300). 4 . 2 . D i s c u s s i o n Intracellular survival and growth of Salmonella are important for the virulence o f the bacteria, and recent evidence has suggested that many genes required for intracellular survival are not expressed by the bacteria when they are grown on regular media in the absence of cells . Therefore a screening procedure was designed to detect bacterial genes that were upregulated by intracellular environments of mammalian cells. A two-plasmid competit ion system wasused to obtain single random insertions o f a promoterless luxAB reporter gene cassette within the S. typhimurium chromosome. This ensured the luxAB genes, (encoding luciferase), were under the transcriptional control o f the natural Salmonella promoter environment, rather than from an alternate promoter or from a mult i-copy number p lasmid . The Salmonella genes were furthermore disrupted wi th this insertion and were not duplicated. W h i l e this reduced the chances o f identifying genes crucial to bacterial viabi l i ty (i.e. housekeeping genes), there was a concern that genes crucial to Salmonella survival wi th in cells (i.e. virulence genes) may not have come out o f the screen. H o w e v e r , despite this concern, genes required for Chapter 4 86 Figure 14: Compar i son o f the luciferase activity from the P22-transductional mutants with the original S. typhimurium insertional mutants. A ) 96-wel l template showing placement of the various S. typhimurium clones. Clones wi th p S P L U X were included as positive controls. S L 1 3 4 4 (no luxAB) was included as a background level control . Uncharacterized Salmonella mutants wh ich either constitutively expressed luciferase or not were further included as "on" or "off" controls, respectively. B ) Corresponding luminograph images o f the S. typhimurium clones expressing light whi le under different intracellular and extracellular condi t ions. The transduced mutants displayed luciferase activity in s imilar patterns to the corresponding original parent mutants. Chapter 4 87 CO c CC o •3 ~o CO c CC 5 CO -4—' c; CC CC c c o CO *l_ cC Q . E o O CD Q_ CO CO Q . CC CM 1 — A1A1 D11H5 E12A2 G5D5 G7H1 G8B1 "Off" Mutant "On" Mutant A1A1 D11H5 E12A2 G5D5 G7H1 G8B1 "Off" Mutant "On" Mutant O SL1344 SL1344 O) < D11 E12 LO O r~ CD CO CD LD842 pSPLUX SL1344 pSPLUX 00 < Q E12 LO CD CD CO CD LD842 pSPLUX SL1344 pSPLUX N-CO If) A1A1 D11H5 E12A2 G5D5 G7H1 G8B1 "Off" Mutant "On" Mutant A1A1 D11H5 E12A2 G5D5 G7H1 G8B1 "Off" Mutant "On" Mutant CO SL1344 SL1344 CM < D11 E12 LO CD CD G8 LD842 pSPLUX SL1344 pSPLUX < D11 E12 LO CD r--O 00 CD LD842 pSPLUX SL1344 pSPLUX < CQ O Q UJ U_ O X T3 8 £ " D CO CO 2 Cl CO -*-c c n => O 2 T 3 CP O CO c CO CO c c ro 2 O 2 Chapter 4 8 8 Intracel lular BALB.BM1 Cells J774A.1 Cells 1 2 3 4 5 . 6 7 8 9 10 11 12 A t » B 1 i C D 9 t • * E » * 9 • * * F § § i t t » G * -H Original Transduced Original Transduced Extracel lular Logarithmic Stationary 1 2 3 4 5, 6 7 8 9 10 11 12 A f 1 • B C D E F G H '<# #• Oriainal Transduced Oriainal Transduced Chapter 4 89 intra-macrophage survival were identified (refer to Chapter 5 results and discussion), indicating that the screening parameters were not reliant on the ability of the bacteria to survive within cel ls . Salmonella were initially g rown on agar plates to test for colonies not producing significant amounts of light outside host cel ls . These low-l ight producers were then retested inside cells to see whether the amount o f light could be induced by the intracellular environment. The screening effectively eliminated bacteria containing gene insertions that were constitutively expressed or induced by medium or serum alone. In retrospect, bacteria containing insertions wi th in invasion genes were most l ike ly eliminated by default as wel l since intracellular bacterial numbers wou ld be too low to detect an increase in enzyme activity. F o r the ini t ial screening phase, an estimate of bacterial numbers was used for the calculation of the specific activity o f the luciferase, but later these numbers appeared to be an overestimation o f the actual bacterial count. B y lower ing this threshold, it may be possible to observe more bacterial genes that were upregulated wi th in host cells and not just the genes described in this study. Chapter 5 90 C h a p t e r 5: C h a r a c t e r i z a t i o n o f G e n e s U p r e g u l a t e d b y I n t r a c e l l u l a r Salmonella In Chapter 5, the S. typhimurium mutants that were identified by the screen described in Chapter 4 are further characterized. The disrupted bacterial genes were sequenced and identified by comparison to k n o w n genes. The growth and pathogenicity o f the mutants were also compared to that of the wild-type bacteria. 5.1. R e s u l t s 5.1.1. I n v e r s e P C R a n d S e q u e n c i n g Inverse P C R was used to amplify regions immediately upstream from the inserted luxAB genes us ing outfacing primers L U X 7 6 and L U X 3 4 0 , wh ich were specific for the luxA gene sequence, as indicated in F i g u r e 11. F igu re 15 demonstrates the single D N A fragment resulting from inverse P C R using chromosomal D N A from each of the mutants. N o bands were seen us ing D N A from the wild-type bacteria S L 1 3 4 4 . Based on the conditions o f the inverse P C R used, it was highly unl ikely that there was more than the one identified insertion of the luxAB cassette wi th in an individual bacterial chromosome. F o r example, us ing a four-basepair cutting restriction enzyme results in frequent cutting of the chromosomal D N A , and using an extension times o f 2 min or more in the P C R should be long enough to amplify segments o f 2 to 3 kbp in length. A m p l i f i e d D N A fragments were isolated from the agarose gel and sequenced. Sequences o f the upstream regions from each of the six mutants were compared wi th k n o w n sequences in G e n B a n k . F o r the mutant E 1 2 A 2 , the primer set E P L U S / E M I N U S was used to amplify and sequence further upstream and downstream from the insertion site. S imi l a r ly , the primer set G P L U S / G M I N U S was used to identify the D N A region around the G 7 H 1 insertion site. F i g u r e 16 illustrates the positions of the insertions o f the luxAB gene cassette wi th in the S. typhimurium chromosome. D I 1H5 had the luxAB cassette inserted within the ssaR gene w h i c h is found wi th in the Salmonella Pathogenicity Island 2 (SPI-2) ((152); Chapter 5 91 (Kbp) 23.5 9 . 4 6 . 6 4 . 4 2.3 2.0 1.5 0.7 8:1 Figure 15: D N A bands resulting from inverse PCR are visualized on a 1% agarose gel. The chromosomal D N A from each mutant was cut with Haelll, and then the fragments re-ligated prior to inverse PCR reactions using the LUX76:LUX340 primer set. The presence of a single band per mutant was indicative of a single insertion of the luxAB gene cassette within the chromosome. Chapter 5 92 F igu re 16: Pos i t ion o f luciferase gene insertions within k n o w n S. typhimurium genes. Insertions are indicated by the large black arrowheads. Transcription direction is indicated by the arrows underneath the genes. Numbers underlying genes correspond to the sequence numbering wi th in the Access ion references for S. typhimurium. A ) D l 1H5 had an insertion wi th in the ssaR gene wh ich is part o f S P T 2 found between centisome 30-31 on the S. typhimurium ch romosome (Access ion #X99944) . Th is region is part o f an operon, where the open reading frames are found to overlap. B ) B o t h A 1 A 1 and E 1 2 A 2 had insertions wi th in SPI -5 located between near centisome 25 on the S. typhimurium chromosome (Access ion #AF021817) . A 1 A 1 had an insertion in sigD/sopB whi le E 1 2 A 2 had an insertion wi th in a downstream O R F pipB (S. dublin: Access ion #AF060858) . The corresponding genes in S. dublin are indicated in brackets. In S. dublin the corresponding region within SPI-5 was thought to be transcribed as one continuous m R N A , as indicated on the diagram. C ) G 7 H 1 had an insertion wi th in a previously uncharacterized region of the Salmonella chromosome (Access ion #AF164435) . The insertion appeared to be wi thin the 5 ' end o f a potential open reading frame. Chapter 5 93 4 Chapter 5 94 Access ion #X99944) . A 1 A 1 had the gene cassette inserted wi th in the sigD gene found at centisome 25 on the chromosome ((159); Access ion #AF021817) . In S. dublin there is a homologous gene called sopB, wh i ch is found at the corresponding chromosomal posi t ion, within a region recently dubbed the fifth Salmonella pathogenicity is land (SPI-5) ((338); Access ion # A F 0 6 0 8 5 8 ) . In the mutant E 1 2 A 2 , the luxAB gene cassette was inserted downstream of the sigD/sopB gene, in a region previously identified as a potential open reading frame. This gene was named pipB, as it was found to have high homology (>90% identity at the D N A level) wi th the S. dublin pipB gene, wh ich is found downstream of sopB. The mutants G 5 D 5 , G 7 H 1 and G 8 B 1 were found to have the exact same insertion site, wh ich was in a region that has not been identified yet. Therefore the potential open reading frame was named iicA for induced intracellularly A (GenBank Access ion #AF164435) . (For the sake o f brevity, G 5 D 5 / G 7 H 1 / G 8 B 1 w i l l be referred to s imply as G 7 H 1 . ) R a p i d mapping to locate the chromosomal posit ion of the insert using locked- in P22 mapping sets was unsuccessful (179). H o w e v e r , this sequence appears to be present in both S. typhimurium and S. typhi genomes and matched a number o f contigs found in the unfinished Salmonella sequencing projects at the Genome Sequencing Center at Washington Univers i ty Schoo l o f Medic ine (http://genome.wustl.edu/gsc/bacterial/salmonella.shtml) ( B _ S T M . C O N T I G . 1 6 0 7 ; B _ S T M A 2 A . C O N T I G . 3 0 9 7 ; B _ S T M A 2 A . C O N T I G . 3 0 6 8 ) and at The Sanger Centre (http:/ /www.sanger.as.uk/) ( B _ T Y P H I 2 . h b 5 6 c 0 4 . s l ) . See Append ix A for sequences o f the four mutants: A 1 A 1 , D 1 1 H 5 , E 1 2 A 2 , and G 5 D 5 / G 7 H 1 / G 8 B 1 . Chapter 5 95 5.1.2. E x t e n t o f G e n e E x p r e s s i o n b y the M u t a n t s The luciferase activity from both the intracellular and extracellular Salmonella mutants is shown in F i g u r e 17. The activity from the intracellular bacteria can be seen to be induced when compared to the extracellular controls. This image is enhanced for l o w light and demonstrates the upregulation o f bacterial gene expression wh ich occurred wi th in phagocytic cells , J 7 7 4 A . 1 , as w e l l as wi th in the non-phagocytic cells , H e L a and M D C K . The spvB gene was used as a posit ive control for intracellular gene expression by S. typhimurium. The spvB gene was actually induced by 10-fold inside cel ls , once light output and bacterial numbers had been normalized as photons/cfu. F igu re 17 differs f rom F igure 10 in that the number of extracellular bacteria had not been adjusted to the same number found intracellularly. In F i g u r e 17, the extracellular bacteria were 20-200 fold higher than the number found inside cells, as indicated by the relative bacteria numbers. Comparat ively , the four Salmonella genes described in this study displayed lower expression outside o f host cells and were more highly induced inside cells than was the spvB gene. F i g u r e 18 demonstrates the increase in luciferase activity by Salmonella wi th in the intracellular environment over those remaining extracellular. The activity seen at the 1 hr time point represents only bacteria in the extracellular supernatant, whi le the other time points (3-7 hr ) represent only bacteria wh ich were intracellular. The bacteria outside cells d id not produce any more light than those grown in media alone, indicating that neither the proteins in the media and serum nor factors secreted by the cells were sufficient to induce the genes, as had been found wi th some Shigella genes (229). The sigD/sopB gene d id not appear to be induced over time in media alone (F igure 1 8 A ) , as had been reported by H o n g and M i l l e r , 1998 (159). Instead sigD/sopB was constantly expressed at a l ow level outside cells , Chapter 5 96 Gene Insertion ixtracellular DMEM Intracellular J774A.1 HeLa MDCK spvB A 1 A 1 (sopB/sigD) D 1 1 H 5 (ssaR) E 1 2 A 2 (pipB) G 7 H 1 (iicA) 3h 5h 7h 3h 5h 7h Relative Bacterial Numbers 200 10 10 10 Figure 17: S. typhimurium mutants show increased light production inside mammalian cel ls . The image was taken us ing a Luminograph L B 9 8 0 photon detector and provides a visual demonstration of the gene induct ion. Note that the number o f extracellular bacteria are 20 to 200-fold higher than the number o f intracellular bacteria. The spvB gene is included as a positive control and has previously been shown to be induced wi th in cells by 5-20-fold compared to extracellular logari thmical ly-growing bacteria. The numbers shown at the bottom of the figure indicate the relative number o f bacteria wi th in the wel l s to further enable comparison o f bacterial gene expression between the different conditions. Chapter 5 97 Figure 18: Comparison of light production of luciferase-expressing bacterial mutants exposed to different environmental conditions. Light was measured using a Luminograph LB980 photon detector. Individual bacterial mutants are represented in the panels: A) A1A1 (sigD/sopB); B) D11H1 (ssaR); C) E12A2 (pipB ); D) G7H1 (iicA). The lines indicate bacteria grown in media alone, shaking while in LB broth and stationary while in DMEM++ media: DMEM++ (filled squares); LB broth (open circles). The bars for the 1 hr time points represent only the activity of the bacteria remaining in the supernatant outside the cells; the bars for the following time points (3, 5, and 7 hr) represent activity from only intracellular bacteria. J774A.1 (open bars); HeLa (hatched bars); MDCK (filled bars). Triplicate experiments were performed, and error bars represent standard error, P<(0.5). Chapter 5 98 Chapter 5 99 which was sl ightly higher than the other three genes described here (pipB, ssaR, iicA). A s w e l l , the other three genes were not found to be induced in media alone (Figure 1 8 B - D ) . This was not unexpected, as the screening system was set up to eliminate mutants wh ich were induced by growth phase. F igure 19 summarizes the induction ratios of luciferase activity o f the bacterial mutants in the various condi t ions. The ratios were determined by d iv id ing the activity o f the bacteria within each condit ion at the different time points (shown in F igu re 18) by the average activity of each particular mutant in D M E M + + at 1 hr . Express ion o f the genes in L B broth was s imilar to that seen in D M E M + + . The ssaR gene was upregulated wi th in cultured macrophages by about 40-100 fo ld , and wi th in cultured epithelial cells by 30-800 fo ld depending on the cell type (30-90 f o l d wi th in H e L a cells and 400-800 f o l d wi th in M D C K cel ls) . The sigD/sopB gene was induced by 10-20 fo ld inside cultured J774A.1 macrophages, by 10-70 f o l d within H e L a epithelial cel ls , and by 200-250 f o l d inside M D C K kidney epithelial cel ls . The downstream pipB gene was induced more highly wi th in J774A.1 cells (30-100 fold) than was the sigD/sopB gene, whereas induction patterns wi th in epithelial cells were s imilar . The pipB gene was induced by 20-70 fo ld inside H e L a cel ls , and by 140-260 f o l d inside M D C K cells . The iicA gene was induced by 30-120 fo ld inside J774A.1 cel ls , by 80-160 fo ld inside H e L a cel ls , and by 200-270 f o l d inside M D C K cel ls . These data re-emphasize that none o f the bacterial gene fusions were induced by the media alone, nor by cell-secreted factors; however , they do suggest that the conditions that Salmonella encounters wi th in cells vary greatly between cell types. Interestingly, the gene fusions appear to be continuously induced over time, and expression only appears to decline once the cells begin to look sick (i.e. start to round up and lose contact wi th the monolayer) . Chapter 5 100 Figure 19: Relat ive luciferase activity by extracellular and intracellular Salmonella mutants. The relative light expression is the ratio of the specific activity at each time point d ivided by the specific activity seen by bacteria grown in D M E M + + for 1 h. Bacter ia were grown in A ) D M E M + + m e d i a alone; B ) macrophage-like J774A.1 cells; C ) epithelial-l ike H e L a cells; and D ) k idney- l ike M D C K cells . Symbols represent: D I 1H5 (ssaR) (clear bars); A 1 A 1 (sigD) (speckled bars); E 1 2 A 2 (pipB) (striped bars); G 7 H 1 (iicA) (black bars). F o r panels B , C , and D , the 1 h time points represent the activity of extracellular bacteria remaining in the supernatant taken from outside the cells; the fo l lowing time points (3, 5, and 7 h) represent activity f rom the intracellular bacteria. Tripl icate experiments were performed, and error bars represent standard error o f the ratio o f two means. Chapter 5 101 o o o o o O LO O LO O CO CM CM i - i -o LO uojssajdxg 3Ajie|ay LU Q • < < o o CM "> <=> to o uojssajdxg jijBn aAjieiau 1 j !=M L o Q • Q LO (0 3 o x c CO E CO o o o o o o " * CM O o o CO o o CD o o o o T CM uojssajdxg m6\-\ 3Ajie|oy lO 3 O X c CU E CO uojssajdxg juBn 3AUB|au Chapter 5 102 H e L a cells were the most sensitive to Salmonella infections and w o u l d begin to "look sick" 5-6 hr after infect ion, whi le both M D C K and J774A.1 cells were more hardy and the cells w o u l d remain most ly intact up to 8-9 hr after infection. 5.1.3. C o m p a r i s o n o f G r o w t h R a t e o f the M u t a n t s The reporter gene insertions were targeted to genes which were upregulated upon bacterial invasion into cells; however, it was unknown whether the insertions affected bacterial functions or su rv iva l . Therefore, the relative growth of the bacteria was tested in order to determine whether the insertional mutations increased or decreased surv iva l . Bacterial growth in media alone and wi thin cells after invasion was examined, wi th the bacteria being grown in D M E M + + prior to invas ion. Table 5 shows the growth o f bacteria wi th in the various conditions at 5 and 7 hr, as compared to the 3 hr time point. The growth rates o f the four mutants d id not appear to differ greatly from the growth rate of the w i l d type S L 1 3 4 4 when tested in extracellular media ( L B broth or tissue culture medium plus serum, D M E M + + ) . W i t h i n the epithelial ce l l lines ( H e L a and M D C K ) , the growth patterns of all four mutants were also similar to the parent strain. W i t h i n the macrophage cells , the bacteria d id not appear to increase in numbers; however, both the parental strain and al l four mutants were able to survive wi th in cultured macrophages. Longer time periods of growth wi th in the cell lines were attempted, but the bacteria were v is ib ly cytotoxic to the mammalian cells over longer time periods o f incubat ion, i.e. longer than 9 hr (data not shown). Chapter 5 Table 5: Growth of Bacteria Over Time in Various Conditions E n v i r o n m e n t Salmonella G e n e R e l a t i v e G r o w t h 3 M u t a n t I n s e r t i o n 5 h r 7 h r A1 A1 sigD/sopB 1.2 0.9 J 7 7 4 A . 1 D11H5 ssaR 1.0 1.2 C e l l s E12A2 pipB 1.3 0.9 G7H1 iicA 1.1 0.9 SL1344 - 1.4 1.3 A1A1 sigD/sopB 1.2 3.4 H e L a D11H5 ssaR 1.4 1.9 C e l l s E12A2 pipB 1.5 2.5 G7H1 iicA 1.1 1.9 SL1344 - 1.9 3.6 A1A1 sigD/sopB 2.0 7.0 M D C K D11H5 ssaR 2.6 10.0 C e l l s E12A2 pipB 1.9 12.5 G7H1 iicA 1.0 4.2 SL1344 - 1.6 13.4 A1 A1 sigD/sopB 3.2 9.8 D M E M + + D11H5 ssaR 2.8 9.9 B r o t h E12A2 pipB 3.3 10.3 G7H1 iicA 2.9 9.2 SL1344 - 2.6 10.6 A1 A1 sigD/sopB 3.9 6.5 L B D11H5 ssaR 3.5 10.1 B r o t h E12A2 pipB 4.1 11.1 G7H1 iicA 3.2 6.6 SL1344 - 4.1 10.3 Relative growth is the increase in bacterial numbers over time expressed as the number of cfu at xhr divided by the number of cfu at 3hr Chapter 5 104 5.1.4. C o m p a r i s o n o f I n v a s i v e n e s s o f the M u t a n t s All four mutants, when grown in DMEM++, remained as invasive as the wild type bacteria in the epithelial cells tested (Table 6). Since previous findings indicated that a small deletion in sigD resulted in a ten-fold reduction in invasion (159), the mutants were also tested by growing them in L B broth (both shaking overnight with subculture the next day, and standing overnight) prior to infection of the cells. Bacteria grown in L B broth were found to be about 10 fold more invasive than those grown in DMEM++ (data not shown); however, no significant difference between invasion of the mutants and the parent SL1344 was seen. Note that bacterial invasion took place in the presence of serum. The observation that the ssaR mutant D l 1H5 remained invasive correlates with previous findings where mutations within the ssa region of the SPI-2 did not affect invasion (60) Instead, the SPI-2 genes are thought to be necessary for the long-term survival of the bacteria within the mouse. Table 6: Relative Invasion of Bacterial Mutants Into Cultured Cells Salmonella G e n e % I n v a s i o n a i n D i f f e r e n t C e l l T y p e s M u t a n t I n s e r t i o n J 7 7 4 H e L a M D C K A1 A1 sigD/sopB 7.3 ± 1 . 2 0.9 ± 0 . 7 1.7 ± 2 . 2 D 1 1 H 5 ssaR 8.6 ± 1 . 1 1.2 ± 0 . 7 1.4 ± 2 . 2 E 1 2 A 2 pipB 6.7 ± 1 . 4 0.9 ± 0 . 1 2.1 ± 3 . 3 G7H1 iicA 9.1 ± 3 . 0 1.0 ± 0 . 3 3.0 ± 2 . 5 S L 1 3 4 4 - 7.4 ± 0 . 3 0.9 ± 0 . 7 1.5 ± 2 . 4 % invasion equals the number of intracellular bacteria at 3 hr divided by the total number of bacteria added to the well, then multiplied by 100. Bacteria were subcultured into DMEM++ prior to invasion. Chapter 5 105 5.1.5. V i r u l e n c e o f M u t a n t s i n T y p h o i d M o u s e M o d e l Having identified genes upregulated by the intracellular environment, experiments were carried out to determine the requirement for these genes in the disease process. The insertional mutations did not affect the growth of the bacteria within cells or media, nor the invasion of the bacteria into cells. The virulence of the S. typhimurium mutants was therefore tested in a mouse infection model. The inbred mouse strain BALB/c was used for the infection model, as it has previously been shown to be susceptible to infection with a number of bacteria, including Salmonella. When infected with either S. typhimurium or S. dublin, BALB/c mice succumb to a systemic disease very similar to that of typhoid fever in humans, (which is caused by S. typhi). Signs of disease in the mouse were observed first as a lack of grooming and an overall scruffy appearance of the mice. As the disease progressed, the mice would appear more scruffy, their backs would become hunched, and they would begin to shiver uncontrollably. They would also become indifferent to the other mice in the cage. Eventually they would die. Although the disease is more systemic than the gastrointestinal disease caused by S. typhimurium in humans, this mouse model has proven very useful for identifying virulence factors. The results of two experiments where mice were inoculated orally are presented in Figure 20. In the first experiment, the mice given D11H5 (ssaR) showed no detectable signs of disease even though they received 200 times the dose at which the wild type bacteria were 100% lethal (i.e. approximately 1000-fold higher than the reported LD50 for SL1344). In the second experiment, insertion into the pipB gene (E12A2) attenuated the bacteria by reducing the mortality to approximately 60% that of wild-type, when given at a dose of 1 .OxlO 6 cfu/mouse. At this dose, the insertion within the sopB/sigD gene (A1A1) was also seen to attenuate virulence of the bacteria to approximately 80% mortality, although this is not statistically significant due to the small numbers of mice used for these experiments. The mutant G7H1 (iicA) remained as virulent as the wild type SL1344 bacteria. The possibility of polar effects caused by these insertions remains (see discussion). Chapter 5 106 D a y P o s t - I n f e c t i o n Figure 20: Virulence o f S. typhimurium mutants in an orally infected typhoid mouse model . M i c e were g iven l x l 0 6 cfu per mouse and disease progression was monitored for one month: S L 1 3 4 4 ( w i l d type) (fil led squares); A 1 A 1 (sigD) (open circles); E 1 2 A 2 (pipB) (open diamonds); G 7 H 1 (iicA) (open triangles). Chapter 5 107 Although mutations in both the sopB/sigD (Al A l ) and pipB (E12A2) regions appeared to cause attenuation, only a disruption of the pipB gene appeared to affect the rate at which disease developed in the mouse (Table 7). Generally, the higher the bacterial dose, the quicker the mice were to develop signs of disease and die. The delay in the time at which 50% of the mice in the group had died was largest when lower doses of bacteria were used (i.e. at a dose of 1x10^ cfu/mouse, 50% mouse mortality was seen on day 21 for E12A2 and on day 13 for wild-type SL1344) and was not as noticeable at higher doses (i.e. at a dose of l.OxlO 7"!.OxlO 8, 50% mouse mortality for E12A2 occurred only one to two days after that of the wild-type bacteria). A delay in mortality was also observed when the mice were inoculated intravenously with E12A2 (pipB). Mice inoculated with D11H5 (ssaR) showed no signs of disease (data not shown). Table 7: Bacterial dose affects kinetics of mortality of mice Salmonella G e n e D a y of 5 0 % M o u s e M o r t a l i t y 3 M u t a n t I n s e r t i o n at v a r i o u s b a c t e r i a d o s e s 1 0 6 1 0 7 1 0 8 S L 1 3 4 4 w i l d - t y p e 1 3 1 0 6 A 1 A 1 sigD/sopB 1 3 1 0 7 E 1 2 A 2 pipB 21 11 8 G 7 H 1 iicA 1 2 1 0 7 Day post-infection at which over half the mice in the group died After 28 days postinfection, the remaining mice were sacrificed and organ homogenates were prepared from spleens and livers. Only the mice inoculated with either the A1A1 (sopB/sigD) or the E12A2 (pipB) were tested in this manner as all mice inoculated with G7H1 (iicA) or wild type SL1344 had died. No Salmonella could be cultured from the homogenates, indicating the mice were able to clear the bacteria completely and were not chronically infected (47). Chapter 5 108 5 .2 . D i s c u s s i o n The expression of four specific bacterial genes (ssaR, sigD/sopB, pipB , and i i c A ) was shown to be induced wi th in the host cells, and this induction was not induced by rich media or by factors secreted by host cells (Figures 17, 18, and 19). These genes were also not only induced at early time points (2-4 hr), but remained upregulated after the bacteria had adapted and begun to grow wi th in the cells (5-7 hr). Interestingly, reporter activity f rom all four genes indicated that expression was induced in both phagocytic cells and non-phagocytic cells . Express ion f rom wi th in cultured phagocytes continued to escalate over time, wi th the exception of the sigD/sopB gene, whereas intracellular gene expression leveled off or declined within epithelial cells. 5 .2 .1 . D 1 1 H 5 (ssaR) The ssaR gene ( D 1 1 H 5 ) , found wi thin SPI -2 , was homologous to the Yersinia enterocolitica gene yscR, wh i ch encodes a membrane-bound subunit o f the type III secretion system (150, 152). The Y s c R forms a critical part o f the secretion apparatus. The ssaR gene was upregulated after bacterial invasion o f both cultured macrophages and epithelial cells , and continued to be expressed throughout the course of the infection. The insertion o f the reporter gene cassette into the ssaR gene abolished virulence o f S. typhimurium in the typhoid mouse model . These findings are novel for the ssaR gene but agree wi th previous reports of insertions wi th in S P I - 2 (46, 321). A ssaH r.gfp gene fusion (within another secretion apparatus-encoding gene) was induced by 400-fold within macrophages and rendered the bacteria avirulent in mice (321). S imi l a r ly , various other gfp insertions into regulatory, structural, and effector/chaperone genes o f SPI -2 were also found to be induced within macrophage cells by 3 to 100 fo ld . Specifically the insertions wi th in ssrA and sscB genes reduced the ability o f the bacteria to spread to other organs within the mouse (46). One could argue that the luxAB insertion wi th in the ssaR gene caused a polar effect on downstream genes, and therefore this gene is not directly related to virulence in mice. H o w e v e r , a previous report had indicated that an insertion wi th in ssaT, a downstream gene wi th in the same operon, Chapter 5 109 reduced bacterial invasion (152), whereas the mutant D 1 1 H 5 was not impaired in its ability to invade non-phagocytic cel ls . The insertion is mostly l ikely having a direct effect on bacterial virulence, since type III secretion has been previously implicated as important to bacterial virulence (46), and this gene potentially encodes a structural subunit o f the type III secretion system (152). 5 .2 .2 . A 1 A 1 (sigD/sopB) a n d E 1 2 A 2 (pipB ) The mutant A 1 A 1 had an insertion wi th in the sigD/sopB gene (159), wh ich is found wi th in SPI -5 . SPI-5 is located near centisome 25 in S. typhimurium (159). Previous studies have shown that S i g D is a secreted protein, and have suggested that it is secreted into the host cell v ia the SPI-1 type III secretion system (159). Furthermore, the S. typhimurium sigD/sopB gene is more than 9 5 % identical at the D N A level to the S. dublin sopB gene, wh ich has recently been shown to act as an inositol phosphate phosphatase inside host cells (235). N o r r i s et al. (235) showed that Salmonella affected the cellular signal transduction pathways and that S o p B was able to hydrolyze phosphatidylinositol 3,4,5-trisphosphate, w h i c h is k n o w n to be a direct inhibitor o f calcium-dependent chloride secretion. A s w e l l , S o p B hydro lyzed 1,3,4,5,6-pentakisphosphate to 1,4,5,6-tetrakisphosphate, wh ich is a signaling molecule that indirectly acts to increase chloride secretion. The result of these activities dur ing infection was proposed to be increased chloride secretion by the gastrointestinal cells , ult imately resulting in diarrhea in the host (235). The mutant E 1 2 A 2 had an insertion wi th in a previously unreported S. typhimurium gene, pipB. Th i s gene was located downstream o f sigD/sopB and was also contained within S P I - 5 . The hypothetical pipB gene product was homologous to that o f the S. dublin pipB gene product, w h i c h has structural similarity to proteins involved wi th g lyco l ip id biogenesis (338). A s indicated in A p p e n d i x A , where the S. dublin gene is one O R F , the corresponding S. typhimurium region may actually be div ided into two open reading frames ( O R F ) . H o w e v e r , the size o f the actual gene product has not been determined for either S. typhimurium or S. dublin pipB. Chapter 5 110 It has been suggested that within the SPI-5 of S. dublin, the sopB and genes downstream {pipC, pipB, and pipA) are contained wi th in the same transcriptional unit, based on the analysis o f the size o f m R N A transcripts (338). S imi lar studies have not been performed for the S. typhimurium SPI-5 region, although there is evidence that the sigDE genes may be transcribed from a single promoter (159). In the study described here, the sigD/sopB ( A 1 A 1 ) and pipB ( E 1 2 A 2 ) genes had a similar expression pattern wi th in epithelial cell types; however , the downstream pipB gene was more highly induced than was sigD/sopB. A s w e l l , the genes were not induced to the same extent wi th in macrophages (Figures 18 and 19), wi th the expression of pipB continuing to escalate within the intramacrophage environment, whi le the expression of sigD/sopB leveled off over time. This suggests that pipB was transcribed from a separate promoter. Further support for a second promoter was that the insertion wi th in the pipB gene greatly attenuated virulence of the S. typhimurium, w h i l e the insertion wi th in the upstream sigD/sopB gene had only a marginal effect on virulence. The fact that the downstream gene (pipB) had a greater effect on virulence than the upstream gene (sigD/sopB) l ikewise argues that the insertion o f the reporter gene cassette d id not cause a downstream polar effect on gene expression, but that the attenuation resulted directly from inactivation of the genes containing the insertions. W i t h i n cultured cell models , previous findings (159) indicated that insertions within either sigD or sigE resulted in a ten-fold reduction in invasion of epithelial cells when compared to w i l d type bacteria. In the assays described here, invasion was not significantly reduced by the insertions wi th in either the sigD/sopB or pipB gene as compared to the parental strain. The cause o f these differences is difficult to expla in , although different S. typhimurium isolates and different cell lines were used here. It is interesting to note that others have reported variabili ty in invasion efficiency of bacterial mutants depending on the point o f mutation within a gene. Hense l et al. (152) reported that only one out o f three insertions wi th in the gene ssaV (SPI-2) resulted in a 10 fo ld reduction in bacterial invas ion, whi le two other single insertions wi th in this gene d id not affect the level o f invasiveness. Furthermore, it was found that Chapter 5 111 mutations within the S. dublin gene homologues did not affect invasion (101, 338, 339). However, when the sigD/sopB mutant described here was grown overnight and subcultured in L B , shaking at 37°C, and then allowed to invade cells in the absence of serum, there was a 30% reduction in invasion (298). (Note that there was no reduction in invasion by the other mutants (ssaR, pipB, iicA) in the absence of serum.) Future experiments with deletions of these genes may help determine their ultimate effect on bacterial invasion and virulence. The insertions within the sigD/sopB and pipB genes resulted in attenuation of S. typhimurium in the typhoid mouse model, but did not totally abolish virulence of the bacteria as did the insertion within ssaR. The effect of the insertion within the pipB gene appeared to delay the onset of the disease allowing the infected mice to live longer, irrespective of dose (although the largest delay was seen with the LD50 dose). In S. dublin studies by Galyov et al. (1997) and Wood et al. (1998), mutations within the SPI-5 region reduced intestinal secretion and inflammatory responses in a calf ileal loop model (101, 338). However, in a mouse model, the S. dublin mutants were recovered from organs in the same numbers as the wild-type strain (338). Their conclusion was that mutations in SPI-5-encoded genes affect the enteropathogenicity of Salmonella, but have no major effect on the development of systemic disease in mice. However the study here suggests there was a difference seen in the development of systemic disease, although the difference was only noticed when lower doses of bacteria were used for inoculation. In the previous S. dublin study, only one dose was reportedly tested on the mice and it could be that at that dose, the mouse immune response was overwhelmed such that no significant difference was seen when comparing the mutant to the wild-type bacteria. 5.2.3. G7H1 (iicA) The mutant G7H1 contained an insertion within the previously undescribed region of the S. typhimurium chromosome which appeared to encode an open reading frame, and was thus named iicA for induced jntracellularly. This gene fusion displayed upregulated reporter activity by intracellular Salmonella, within both macrophage and epithelial cells. However, the Chapter 5 112 insertion had no effect on invasion or survival o f the bacteria within cel ls , nor d id it appear to attenuate bacterial virulence in the mouse model . N o significant similarity to any other k n o w n gene was found to this region, based on a search o f the available D N A databases, and the function o f the iicA gene and gene product remains to be determined. 5.3. O v e r a l l C o n c l u s i o n In conc lus ion , four individual genes of the S. typhimurium chromosome were identified that were upregulated in response to the intracellular environment o f mammalian cel ls . The screen described in this report a l lowed for the identification o f specific virulence factors, indicating that genes induced inside mammalian cells often play a key role in Salmonella pathogenesis. Three o f the four genes (ssaR, sigD/sopB, and pipB) identified were virulence factors, inasmuch as insertional inactivation o f these genes decreased mouse mortality in an animal model . The fourth gene, iicA, was not found to reduce mouse mortality, however its role in virulence has yet to be tested in other animal models, e.g. calf ileal loop model . The four genes identified were tightly regulated by the bacteria, wi th very little expression from extracellular bacteria. The three genes identified as virulence factors (ssaR, sigD/sopB, and pipB) were contained wi th in chromosomal regions k n o w n as islands o f pathogenicity (SPI ) , and at least two o f the gene products had previously been shown to be invo lved wi th a type III secretion system (SsaR and S i g D / S o p B ) . Pathogenicity islands have been found in numerous bacterial pathogens, on their chromosomes and extrachromosomal elements. These regions often carry genes encoding type III secretions systems and proteins for secretion, as we l l as other products w h i c h a l low the different bacteria to survive and cause disease wi th in defined niches. The further characterization of these genes and their products w i l l lead to an enhanced understanding about how Salmonella functions as an intracellular pathogen. Future experiments w i l l define the regulation of the various genes and the specific conditions required to induce their expression. F o r example, both sigD/sopB and pipB were contained wi th in SPI -5 and both were induced wi thin cells; however their expression levels varied over time indicating that they are expressed under separate and unique promoters. Chapter 5 113 Specific deletion mutations w i l l help define which regions o f the genes are important for bacterial virulence, e.g. the regions necessary for secretion o f sigD/sopB have not been defined. Furthermore, the function of both P i p B and SsaR have only been deduced by homology to other D N A sequences and the corresponding proteins have not been biochemical ly defined. F i n a l l y , other Salmonella genes may be discovered us ing this assay, and the techniques may be applied to other pathogenic bacteria to isolate virulence factors. References 114 R e f e r e n c e s 1. A b s h i r e , K . Z . , a n d F . C . 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Identification o f a pathogenicity island required for Salmonella enteropathogenicity. M o i M i c r o b i o l . 29(3):883-91. 339. W o o d , M . W . , R . R o s q v i s t , P . B . M u l l a n , M . H . E d w a r d s , a n d E . E . G a l y o v . 1996. S o p E , a secreted protein o f Salmonella dublin, is translocated into the References 151 target eukaryotic cell via a szp-dependent mechanism and promotes bacterial entry. Mol Microbiol. 22(2):327-38. 340. Z i e r l e r , M . K . , a n d J . E . G a l a n . 1995. Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ. Infect Immun. 63(10):4024-8. Appendix 152 A p p e n d i x D N A a n d P r e d i c t e d P r o t e i n S e q u e n c e s S u r r o u n d i n g I n s e r t i o n a l M u t a t i o n s The sites wh ich are offset in the D N A sequences indicate the posit ion o f the insertion of the luxAB gene cassette. The names o f the corresponding genes are given be low the start o f the predicted proteins. The sequences wh ich are underlined were actually sequenced in this study; non-underl ined sequences were obtained from previously published sequences. A: D11H5 (ssaR) A C C E S S I O N X 9 9 9 4 4 (SPI-2 S. typhimurium) 10 2 0 3 0 4 0 50 60 C C T G C A G T A A T C T A C C A C A T C A G C T A G C G T T G C A T A T T A A A T G G A C A G T T G A A G A G C A T G G G A C G T C A T T A G A T G G T G T A G T C G A T C G C A A C G T A T A A T T T A C C T G T C A A C T T C T C G T A C ..C..S..N. .L..P..H.. Q..L..A..L ..H..I..K. .W..T..V.. E..E..H..E (ssaQ) 7 0 80 90 1 0 0 1 1 0 1 2 0 A G T T C C A T A G C A T T A T T T T T A C A T G G C C A A C G G G T T T T T T G C G C A A T A T A G T C G G A G A G C T C A A G G T A T C G T A A T A A A A A T G T A C C G G T T G C C C A A A A A A C G C G T T A T A T C A G C C T C T C G ..F..H..S. . I . . I . . F . . T..W..P..T ..G..F..L. .R..N..I.. V..G..E..L 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 T T T C T G C T G A G C G A C A A C A G A T T T A T C C T G C C C C T C C T G T G G T A G T C C C T G T A T A T T C A G A A A G A C G A C T C G C T G T T G T C T A A A T A G G A C G G G G A G G A C A C C A T C A G G G A C A T A T A A G T C ..S..A..E. .R..Q..Q.. I..Y..P..A ..P..P..V. .V..V..P.. V..Y..S..G 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 G C T G G T G C C A G C T T A C A T T A A T C G A A C T T G A G T C T A T C G A A A T C G G C A T G G G C G T T C G G A C G A C C A C G G T C G A A T G T A A T T A G C T T G A A C T C A G A T A G C T T T A G C C G T A C C C G C A A G C C T ..W..C..Q. .L..T..L.. I..E..L..E ..S..I..E. .I..G..M.. G..V..R..I 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 T T C A T T G C T T C G G C G A C A T C A G A C T C G G T T T T T T T G C T A T T C A A C T A C C T G G G G G A A T C T A A G T A A C G A A G C C G C T G T A G T C T G A G C C A A A A A A A C G A T A A G T T G A T G G A C C C C C T T A G A ..H..C..F. .G..D..I.. R..L..G..F ..F..A..I. .Q..L..P.. G..G..I..Y 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0 A C G C A A G G G T G T T G C T G A C A G A G G A T A A C A C G A T G A A A T T T G A C G A A T T A G T C C A G G A T A T G C G T T C C C A C A A C G A C T G T C T C C T A T T G T G C T A C T T T A A A C T G C T T A A T C A G G T C C T A T ..A..R..V. .L..L..T.. E..D..N..T ..M..K..F. .D..E..L.. V..Q..D..I Appendix 153 370 380 390 400 410 420 TCGAAACGCT ACTTGCGTCA GGGAGCCCAA TGTCAAAGAG TGACGGAACG TCTTCAGTCG AGCTTTGCGA TGAACGCAGT CCCTCGGGTT ACAGTTTCTC ACTGCCTTGC AGAAGTCAGC ..E..T..L. .L..A..S.. G..S..P..M ..S..K..S. .D..G..T.. S..S..V..E 430 440 450 460 470 480 AACTTGAGCA GATACCACAA CAGGTGCTCT TTGAGGTCGG ACGTGCGAGT CTGGAAATTG TTGAACTCGT CTATGGTGTT GTCCACGAGA AACTCCAGCC TGCACGCTCA GACCTTTAAC ..L..E..Q. .I..P..Q.. Q..V..L..F ..E..V..G. .R..A..S.. L..E..I..G 490 500 510 520 530 540 GACAATTACG ACAACTTAAA ACGGGGGACG TTTTGCCTGT AGGTGGATGT TTTGCGCCAG CTGTTAATGC TGTTGAATTT TGCCCCCTGC AAAACGGACA TCCACCTACA AAACGCGGTC ..Q..L..R. .Q..L..K.. T..G..D..V ..L..P..V. .G..G..C. F..A..P..E 550 560 570 580 590 600 AGGTGACGAT AAGAGTAAAT GACCGTATTA TTGGGCAAGG TGAGTTGATT GCCTGTGGCA TCCACTGCTA TTCTCATTTA CTGGCATAAT AACCCGTTCC ACTCAACTAA CGGACACCGT ..V..T..I. .R..V..N.. D..R..I..I ..G..Q..G. . E . . L . . I . . A..C..G..N 610 620 630 640 650 660 ATGAATTTAT GGTGCGTATT ACACGTTGGT ATCTTTGCAA AAATACAGCG TAAACCTGAT TACTTAAATA CCACGCATAA TGTGCAACCA TAGAAACGTT TTTATGTCGC ATTTGGACTA ..E..F..M. .V..R..I.. T..R..W..Y ..L..C..K. .N..T..A.. * 670 AAGAAAAATA TTCTTTTTAT 680 ATATGCGAAC TATACGCTTG 690 AATATAATAG TTATATTATC 700 CGTTCCAGGT GCAAGGTCCA 710 CGTGTCATGA GCACAGTACT 720 GAGATACAGT CTCTATGTCA 730 740 750 760 770 780 ATGTCTTTAC CCGATTCGCC TTTGCAACTG ATTGGTATAT TGTTTCTGCT TTCAATACTG TACAGAAATG GGCTAAGCGG AAACGTTGAC TAACCATATA ACAAAGACGA AAGTTATGAC M..S..L..P ..D..S..P. .L..Q..L.. I..G..I..L ..F..L..L. .S..I..L.. {ssaR) CCTCTCATTA TCGTCATGGG AACTTCTTTC CTTAAACTGG CGGTGGTATT TTCGATTTTA GGAGAGTAAT AGCAGTACCC TTGAAGAAAG GAATTTGACC GCCACCATAA AAGCTAAAAT P . . L . . I . . I ..V..M..G. .T..S..F.. L..K..L..A ..V..V..F. .S..I..L.. 850 860 870 880 890 900 CGAAATGCTC TGGGTATTCA ACAAGTCCCC CCAAATATCG CACTGTATGG CCTTGCGCTT GCTTTACGAG ACCCATAAGT TGTTCAGGGG GGTTTATAGC GTGACATACC GGAACGCGAA R..N..A..L ..G..I..Q. .Q..V..P.. P..N..I..A ..L..Y..G. .L..A..L.. 910 920 930 940 950 960 GTACTTTCCT TATTCATTAT GGGGCCGACG CTATTAGCTG TAAAAGAGCG CTGGCATCCG CATGAAAGGA ATAAGTAATA CCCCGGCTGC GATAATCGAC ATTTTCTCGC GACCGTAGGC V..L..S..L ..F..I..M. .G..P..T.. L..L..A..V ..K..E..R. .W..H..P.. Appendix 154 970 980 990 1000 1010 1020 GTTCAGGTCG CTGGCGCTCC TTTCTGGACG T C TGAGTGGG ACAGTAAAGC ATTAGCGCCT CAAGTCCAGC GACCGCGAGG AAAGACCTGC A G ACTCACCC TGTCATTTCG TAATCGCGGA V..Q..V..A ..G..A..P. .F..W..T.. S..E..W..D ..S..K..A. .L..A..P.. 1030 1040 1050 1060 1070 1080 TATCGACAGT TTTTGCAAAA AAACTCTGAA GAGAAGGAAG CCAATTATTT TCGGAATTTG ATAGCTGTCA AAAACGTTTT TTTGAGACTT CTCTTCCTTC GGTTAATAAA AGCCTTAAAC Y..R..Q..F ..L..Q..K. .N..S..E.. E..K..E..A ..N..Y..F. .R..N..L.. 1090 1100 1110 1120 1130 1140 ATAAAACGAA CCTGGCCTGA AGACATAAAA AGAAAGATAA AACCTGATTC TTTGCTCATA TATTTTGCTT GGACCGGACT TCTGTATTTT TCTTTCTATT TTGGACTAAG AAACGAGTAT I..K..R..T ..W..P..E. .D..I..K.. R..K..I..K ..P..D..S. .L..L..I.. 1150 1160 1170 1180 1190 1200 TTAATTCCGG CATTTACGGT GAGTCAGTTA ACGCAGGCAT TTCGGATTGG ATTACTTATT AATTAAGGCC GTAAATGCCA CTCAGTCAAT TGCGTCCGTA AAGCCTAACC TAATGAATAA L..I..P..A ..F..T..V. .S..Q..L.. T..Q..A..F ..R..I..G. .L..L..I.. 1210 1220 1230 1240 1250 . 1260 TATCTTCCCT TTCTGGCTAT TGACCTGCTT ATTTCAAATA TACTGCTGGC TATGGGGATG ATAGAAGGGA AAGACCGATA ACTGGACGAA TAAAGTTTAT ATGACGACCG ATACCCCTAC Y..L..P..F ..L..A..I. .D..L..L.. I..S..N..I ..L..L..A. .M..G..M.. 1270 1280 1290 1300 1310 1320 ATGATGGTGT CGCCGATGAC CATTTCATTA CCGTTTAAGC TGCTAATATT TTTACTGGCA TACTACCACA GCGGCTACTG GTAAAGTAAT GGCAAATTCG ACGATTATAA AAATGACCGT M..M..V..S ..P..M..T. .I..S..L.. P..F..K..L ..L..I..F. .L..L..A.. 1330 1340 1350 1360 1370 1380 GGCGGTTGGG ATCTGACACT GGCGCAATTG GTACAGAGCT TTTCATGAAT GATTCTGAAT CCGCCAACCC TAGACTGTGA CCGCGTTAAC CATGTCTCGA AAAGTACTTA CTAAGACTTA G..G..W..D ..L..T..L. .A..Q..L.. V..Q..S..F ..S..* M..N.. D..S..E..L (ssaS) 1390 1400 1410 1420 1430 1440 TGACGCAATT TGTAACGCAA CTTTTATGGA TCGTCCTTTT TACGTCTATG CCGGTAGTGT ACTGCGTTAA ACATTGCGTT GAAAATACCT AGCAGGAAAA ATGCAGATAC GGCCATCACA ..T..Q..F. .V..T..Q.. L..L..W..I ..V..L..F. .T..S..M.. P..V..V..L 1450 1460 1470 1480 1490 1500 TGGTGGCATC GGTAGTTGGT GTCATCGTAA GCCTTGTTCA GGCCTTGACT CAAATACAGG ACCACCGTAG CCATCAACCA CAGTAGCATT CGGAACAAGT CCGGAACTGA GTTTATGTCC ..V..A..S. .V..V..G.. V..I..V..S ..L..V..Q. .A..L..T.. Q..I..Q..D Appendix 155 B . A1A1 (sigD/sopB) a n d E12A2 ipipB) A C C E S S I O N A F 0 2 1 8 1 7 (sigDE S. typhimurium) 10 20 30 40 50 60 TATCTGTTCA AGCATGGAAT AGGAAAAACG AATATTCTTC GTCACGGTCT TACTTGTCCG ATAGACAAGT TCGTACCTTA TCCTTTTTGC TTATAAGAAG CAGTGCCAGA ATGAACAGGC 70 80 90 100 110 120 GGGCTTTGCT GGCATACACA CACCTGTATA ACATTTGATG TAACGCCGTT ACTTTACGCA CCCGAAACGA CCGTATGTGT GTGGACATAT TGTAAACTAC ATTGCGGCAA TGAAATGCGT 130 140 150 160 170 180 GGAGTAAATC GGTGAATTTG ATCTGAGTCA AGAAGGTGGG TTTTCAATAA AAGTTGTGCC CCTCATTTAG CCACTTAAAC TAGACTCAGT TCTTCCACCC AAAAGTTATT TTCAACACGG 190 200 210 220 230 240 ATAAATTGTG AAGTTTGTAG ATTTTATGAA CATTTGATGT ACCGATCTCC CCCATGATCG TATTTAACAC TTCAAACATC TAAAATACTT GTAAACTACA TGGCTAGAGG GGGTACTAGC 250 260 270 280 290 300 CCACTACGCT ATGGACGTCA GGATGCCTCC CCGCCTGATC AGAAGCGTTT CCTCATTAAA GGTGATGCGA TACCTGCAGT CCTACGGAGG GGCGGACTAG TCTTCGCAAA GGAGTAATTT 310 320 330 340 350 360 AAGGACATTT TTTTAAAGTT CCTGGTGCAT AAAAGTCACA TCCTTTTAAA GGGTTGTTAA TTCCTGTAAA AAAATTTCAA GGACCACGTA TTTTCAGTGT AGGAAAATTT CCCAACAATT 370 380 390 400 410 420 CCCTGTTGAA TGTTCCCACT CCCCTATTCA GGAATATTAA AAACGCTATG CAAATACAGA GGGACAACTT ACAAGGGTGA GGGGATAAGT CCTTATAATT TTTGCGATAC GTTTATGTCT M.. Q..1..Q..S (sigD/sopB) 430 440 450 460 470 480 GCTTCTATCA CTCAGCTTCA CTAAAAACCC AGGAGGCTTT TAAAAGCCTA CAAAAAACCT CGAAGATAGT GAGTCGAAGT GATTTTTGGG TCCTCCGAAA ATTTTCGGAT GTTTTTTGGA ..F..Y..H. .S..A..S.. L..K..T..Q ..E..A..F. .K..S..L.. Q..K..T..L 490 500 510 520 530 540 TATACAACGG AATGCAGATT CTCTCAGGCC AGGGCAAAGC GCCGGCTAAA GCGCCCGACG ATATGTTGCC TTACGTCTAA GAGAGTCCGG TCCCGTTTCG CGGCCGATTT CGCGGGCTGC ..Y..N..G. .M..Q..I.. L..S..G..Q ..G..K..A. .P..A..K.. A..P..D..A 550 560 570 CTCGCCCGGA AATTATTGTC CTGCGAGAAC GAGCGGGCCT TTAATAACAG GACGCTCTTG ..R..P..E. .I..I..V.. L..R..E..P 580 590 600 CCGGCGCGAC ATGGGGGAAT TATCTACAGC GGCCGCGCTG TACCCCCTTA ATAGATGTCG ..G..A..T. .W..G..N.. Y..L..Q..H Appendix 156 610 620 630 640 650 660 ATCAGAAGGC GTCTAACCAC TCGCTGCATA ACCTCTATAA CTTACAGCGC GATCTTCTTA TAGTCTTCCG CAGATTGGTG AGCGACGTAT TGGAGATATT GAATGTCGCG CTAGAAGAAT ..Q..K..A. .S..N..H.. S..L..H..N ..L..Y..N. .L..Q..R.. D..L..L..T 670 680 690 700 710 720 C C G T C G C G G C AACCGTTCTG GGTAAACAAG ACCCGGTTCT AACGTCAATG GCAAACCAAA G G C A G C G C C G TTGGCAAGAC CCATTTGTTC TGGGCCAAGA TTGCAGTTAC CGTTTGGTTT ..V..A..A...T..V..L...G..K..Q..D...P..V..L...T..S..M...A..N..Q..M 730 740 750 760 770 780 TGGAGTTAGC CAAAGTTAAA GCGGACCGGC CAGCAACAAA ACAAGAAGAA GCCGCGGCAA ACCTCAATCG GTTTCAATTT CGCCTGGCCG GTCGTTGTTT TGTTCTTCTT CGGCGCCGTT ..E..L..A. .K..V..K.. A..D..R..P ..A..T..K. .Q..E..E.. A..A..A..K 790 800 810 820 830 840 AAGCATTGAA GAAAAATCTT ATCGAACTTA TTGCAGCACG CACTCAGCAG CAGGATGGCT TTCGTAACTT CTTTTTAGAA TAGCTTGAAT AACGTCGTGC GTGAGTCGTC GTCCTACCGA ..A..L..K. .K..N..L.. I . . E . . L . . I ..A..A..R. .T..Q..Q.. Q..D..G..L 850 860 870 880 890 900 TACCTGCAAA AGAAGCTCAT CGCTTTGCGG CAGTAGCGTT TAGAGATGCT CAGGTCAAGC ATGGACGTTT TCTTCGAGTA GCGAAACGCC GTCATCGCAA ATCTCTACGA GTCCAGTTCG ..P..A..K. .E..A..H.. R..F..A..A ..V..A..F. .R..D..A.. Q..V..K..Q 910 920 930 940 950 960 AGCTTAATAA CCAGCCCTGG CAAACCATAA AAAATACACT CAC GCATAAC GGGCATCACT TCGAATTATT GGTCGGGACC GTTTGGTATT TTTTATGTGA GTGCGTATTG CCCGTAGTGA ..L..N..N. .Q..P..W.. Q..T..I..K ..N..T..L. .T..H..N.. G..H..H..Y 970 980 990 1000 1010 1020 ATACCAACAC GCAGCTCCCT GCAGCAGAGA TGAAAATCGG CGCAAAAGAT ATCTTTCCCA TATGGTTGTG CGTCGAGGGA CGTCGTCTCT ACTTTTAGCC GCGTTTTCTA TAGAAAGGGT ..T..N..T. .Q..L..P.. A..A..E..M ..K..I..G. .A..K..D.. I..F..P..S 1030 1040 1050 1060 1070 1080 GTGCTTATGA GGGAAAGGGC GTATGCAGTT GGGATACCAA GAATATTCAT CACGCCAATA CACGAATACT CCCTTTCCCG CATACGTCAA CCCTATGGTT CTTATAAGTA GTGCGGTTAT ..A..Y..E. .G..K..G.. V..C..S..W ..D..T..K. .N..I..H.. H..A..N..N 1090 1100 1110 1120 1130 1140 ATTTGTGGAT GTCCACGGTG AGTGTGCATG AGGACGGTAA AGATAAAACG CTTTTTTTTG TAAACACCTA CAGGTGCCAC TCACACGTAC TCCTGCCATT TCTATTTTGC GAAAAAAAAC ..L..W..M. .S..T..V.. S..V..H..E ..D..G..K. .D..K..T.. L..F..F..D 1150 1160 1170 1180 1190 1200 ACGGGATACG TCATGGCGTG CTTTCCCCCT ATCATGAAAA AGATCCGCTT CTGCGTCACG TGCCCTATGC AGTACCGCAC GAAAGGGGGA TAGTACTTTT TCTAGGCGAA GACGCAGTGC ..G..I..R. .H..G..V.. L..S..P..Y ..H..E..K. .D..P..L.. L..R..H..V Appendix 157 1210 1220 1230 1240 1250 1260 TCGGCGCTGA AAACAAAGCC AAAGAAGTAT TAACTGCGGC ACTTTTTAGT AAACCTGAGT AGCCGCGACT TTTGTTTCGG TTTCTTCATA ATTGACGCCG TGAAAAATCA TTTGGACTCA ..G..A..E. .N..K..A.. K..E..V..L ..T..A..A. .L..F..S.. K..P..E..L 1270 1280 1290 1300 1310 1320 TGCTTAACAA AGCCTTAGCG GGCGAGGCGG TAAGCCTGAA ACTGGTATCC GTCGGGTTAC ACGAATTGTT TCGGAATCGC CCGCTCCGCC ATTCGGACTT TGACCATAGG CAGCCCAATG ..L..N..K. .A..L..A.. G..E..A..V ..S..L..K. .L..V..S.. V..G..L..L 1330 1340 1350 1360 1370 1380 TCACCGCGTC GAATATTTTC GGCAAAGAGG GAACGATGGT CGAGGACCAA ATGCGCGCAT AGTGGCGCAG CTTATAAAAG CCGTTTCTCC CTTGCTACCA GCTCCTGGTT TACGCGCGTA ..T..A..S. .N..I..F.. G..K..E..G ..T..M..V. .E..D..Q.. M..R..A..W 1390 1400 1410 1420 1430 1440 GGCAATCGTT GACCCAGCCG GGAAAAATGA TTCATTTAAA AATCCGCAAT AAAGATGGCG CCGTTAGCAA CTGGGTCGGC CCTTTTTACT AAGTAAATTT TTAGGCGTTA TTTCTACCGC ..Q..S..L. .T..Q..P.. G..K..M..I ..H..L..K. .I..R..N.. K..D..G..D 1450 1460 1470 1480 1490 1500 ATCTACAGAC GGTAAAAATA AAACCGGACG TCGTCGCCGC ATTTAATGTG GGTGTTAATG TAGATGTCTG CCATTTTTAT TTTGGCCTGC AGCAGCGGCG TAAATTACAC CCACAATTAC ..L..Q..T. .V..K..I.. K..P..D..V ..V..A..A. .F..N..V.. G..V..N..E 1510 1520 1530 1540 1550 1560 AGCTGGCGCT CAAGCTCGGC TTTGGCCTTA AGGCATCGGA TAGCTATAAT GCCGAGGCGC TCGACCGCGA GTTCGAGCCG AAACCGGAAT TCCGTAGCCT ATCGATATTA CGGCTCCGCG ..L..A..L. .K..L..G.. F..G..L..K ..A..S..D. .S..Y..N.. A..E..A..L 1570 1580 1590 1600 1610 1620 TACATCAGTT ATTAGGCAAT GATTTACGCC CTGAAGCCAG ACCAGGTGGC TGGGTTGGCG ATGTAGTCAA TAATCCGTTA CTAAATGCGG GACTTCGGTC TGGTCCACCG ACCCAACCGC ..H..Q..L. .L..G..N.. D..L..R..P ..E..A..R. .P..G..G.. W..V..G..E 1630 1640 1650 1660 1670 1680 AATGGCTGGC GCAATACCCG GATAATTATG AGGTCGTCAA TACATTAGCG CGCCAGATTA TTACCGACCG CGTTATGGGC CTATTAATAC TCCAGCAGTT ATGTAATCGC GCGGTCTAAT ..W..L..A. .Q..Y..P.. D..N..Y..E ..V..V..N. .T..L..A.. R..Q..I..K 1690 1700 1710 1720 1730 1740 AGGATATATG GAAAAATAAC CAACATCATA AAGATGGCGG CGAACCCTAT AAACTCGCAC TCCTATATAC CTTTTTATTG GTTGTAGTAT TTCTACCGCC GCTTGGGATA TTTGAGCGTG ..D..I..W. .K..N..N.. Q..H..H..K ..D..G..G. .E..P..Y.. K..L..A..Q 1750 1760 1770 1780 1790 1800 AACGCCTTGC CATGTTAGCC CATGAAATTG ACGCGGTACC CGCCTGGAAT TGTAAAAGCG TTGCGGAACG GTACAATCGG GTACTTTAAC TGCGCCATGG GCGGACCTTA ACATTTTCGC ..R..L..A. .M..L..A.. H..E..I..D ..A..V..P. .A..W..N.. C..K..S..G Appendix 158 1810 1820 1830 1840 1850 1860 GCAAAGATCG TACAGGGATG ATGGATTCAG AAATCAAGGG AGAGATCATT TCCTTACATC CGTTTCTAGC ATGTCCCTAC TACCTAAGTC TTTAGTTCCC TCTCTAGTAA AGGAATGTAG ..K..D..R. .T..G..M.. M..D..S..E ..I..K..G. . E . . I . . I . . S..L..H..Q 1870 1880 1890 1900 1910 1920 AGACCCATAT GTTAAGTGCC CCTGGTAGTC TTCCGGATAG CGGTGGACAG AAAATTTTCC TCTGGGTATA CAATTCACGG GGACCATCAG AAGGCCTATC GCCACCTGTC TTTTAAAAGG ..T..H..M. .L..S..A.. P..G..S..L ..P..D..S. .G..G..Q.. K..-I..F..Q 1930 1940 1950 1960 1970 1980 AAAAAGTATT ACTGAATAGC GGTAACCTGG AGATTCAGAA ACAAAATACG GGCGGGGCGG TTTTTCATAA TGACTTATCG CCATTGGACC TCTAAGTCTT TGTTTTATGC CCGCCCCGCC ..K..V..L. .L..N..S.. G..N..L..E ..I..Q..K. .Q..N..T.. G..G..A..G 1990 2000 2010 2020 2030 2040 GAAACAAAGT AATGAAAAAT TTATCGCCAG AGGTGCTCAA TCTTTCCTAT CAAAAACGAG CTTTGTTTCA TTACTTTTTA AATAGCGGTC TCCACGAGTT AGAAAGGATA GTTTTTGCTC ..N..K..V. .M..K..N.. L..S..P..E ..V..L..N. .L..S..Y.. Q..K..R..V 2050 2060 2070 2080 2090 2100 TTGGGGATGA AAATATTTGG CAGTCAGTAA AAGGCATTTC TTCATTAATC ACATCTTGAG AACCCCTACT TTTATAAACC GTCAGTCATT TTCCGTAAAG AAGTAATTAG TGTAGAACTC ..G..D..E. .N..I..W.. Q..S..V..K ..G..I..S. . S . . L . . I . . T..S..* 2110 2120 2130 ' 2140 2150 2160 TCTTGAGGTA ACTATATGGA AAGTCTATTA AATCGTTTAT ATGACGCGTT AGGCCTGGAT AGAACTCCAT TGATATACCT TTCAGATAAT TTAGCAAATA TACTGCGCAA TCCGGACCTA M..E. .S..L..L.. N..R..L..Y ..D..A..L. .G..L..D.. (sigE) 2170 2180 2190 2200 2210 2220 GCGCCAGAAG ATGAGCCACT GCTTATCATT GATGATGGGA TACAGGTTTA TTTTAATGAA CGCGGTCTTC TACTCGGTGA CGAATAGTAA CTACTACCCT ATGTCCAAAT AAAATTACTT A..P..E..D ..E..P..L. . L . . I . . I . . D..D..G..I ..Q..V..Y. .F..N..E.. 2230 2240 2250 2260 2270 2280 TCCGATCATA CACTGGAAAT GTGCTGTCCC TTTATGCCAT TGCCTGACGA CATCCTGACT AGGCTAGTAT GTGACCTTTA CACGACAGGG AAATACGGTA ACGGACTGCT GTAGGACTGA S..D..H..T ..L..E..M. . C . C . P . . F..M..P..L ..P..D..D. .I. . L . . T . . 2290 2300 2310 2320 2330 2340 TTGCAGCATT TTTTACGTCT GAACTACACC AGCGCCGTCA CTATCGGCGC TGACGCAGAC AACGTCGTAA AAAATGCAGA CTTGATGTGG TCGCGGCAGT GATAGCCGCG ACTGCGTCTG L..Q..H..F ..L..R..L. .N..Y..T.. S..A..V..T ..I..G..A. .D..A..D.. 2350 2360 2370 2380 2390 2400 AATACTGCTT TAGTGGCGCT TTATCGCTTG CCGCAAACCA GTACCGAAGA AGAGGCGCTC TTATGACGAA ATCACCGCGA AATAGCGAAC GGCGTTTGGT CATGGCTTCT TCTCCGCGAG N..T..A..L ..V..A..L. .Y..R..L.. P..Q..T..S ..T..E..E. .E..A..L.. Appendix 159 2410 2420 2430 ACTGGTTTTG AATTATTCAT TTCAAACGTG TGACCAAAAC TTAATAAGTA AAGTTTGCAC T..G..F..E . . L . . F . . I . .S..N..V.. 2470 2480 2490 AATACGTCAA CATACTTTCT TAATGAGATA TTATGCAGTT GTATGAAAGA ATTACTCTAT 2530 2540 - \ 2550 CAAGACCAGA ATCTTTGGTG GAAATGTAAG GTTCTGGTCT TAGAAACCAC CTTTACATTC 2590 2600 2610 TGTTTGCGGG AGCATTTTTA GTGTGTAAGT ACAAACGCCC TCGTAAAAAT CACACATTCA 2650 2660 2670 CGCGCATTTA TTCTGGTATA AGTTGAAATA GCGCGTAAAT AAGACCATAT TCAACTTTAT 2710 2720 2730 TCTTTAAGTA AATTTTCGCT GAACAAACTT AGAAATTCAT TTAAAAGCGA CTTGTTTGAA 2770 2780 2790 GCTATGCTGG AAATGAAGGA ATCAATAGCA CGATACGACC TTTACTTCCT TAGTTATCGT 2440 2450 2460 AAGCAATTGA AAGAGCATTA TGCATAATTT TTCGTTAACT TTCTCGTAAT ACGTATTAAA K..Q..L..K ..E..H..Y. .A..* 2500 2510 2520 AAACGCGATA CGTATGCCCT TTACAAGAGA TTTGCGCTAT GCATACGGGA AATGTTCTCT 2560 2570 2580 GGGCAAACGT TCATCTCTCT CATTTTGCTC CCCGTTTGCA AGTAGAGAGA GTAAAACGAG 2620 2630 2640 ATTCCTGCTC ATCAGGTTTT TACGCCATCA TAAGGACGAG TAGTCCAAAA ATGCGGTAGT 2680 2690 2700 CTGCAAAAAA TATTGGTGCT TATTATTTTT GACGTTTTTT ATAACCACGA ATAATAAAAA 2740 2750 2760 AATTGTTTAT TCAATGATGA TGAAGCGTAA TTAACAAATA AGTTACTACT ACTTCGCATT 2800 2810 2820 AGGATAATCT TATTATTCAC GGGTGATATT TCCTATTAGA ATAATAAGTG CCCACTATAA 2830 2840 2850 2860 2870 2880 ACTTCTGCTT CACCGTTATG GCAGATATCA TCGCCTCTTG TCAGATGCCA GACACCTACT TGAAGACGAA GTGGCAATAC CGTCTATAGT AGCGGAGAAC AGTCTACGGT CTGTGGATGA 2890 2900 2910 2920 2930 2940 CATACTCAAC CAAAGCTCTA AATACAAAAA TCACCTTATA TCTTTTTTTA TTATTCCTTG GTATGAGTTG GTTTCGAGAT TTATGTTTTT AGTGGAATAT AGAAAAAAAT AATAAGGAAC 2950 2960 2970 2980 2990 3000 TATAAATGTG ACTTGACTCA CACCTATAAG GAGTCGGCTC ACTTCCATAA GAAGGAATCA ATATTTACAC TGAACTGAGT GTGGATATTC CTCAGCCGAG TGAAGGTATT CTTCCTTAGT 3010 3020 3030 3040 3050 3060 AAATGCCAAT AACTAACGCG TCCCCAGAAA ATATATTAAG ATATTTGCAT GCGGCCGGTA TTTACGGTTA TTGATTGCGC AGGGGTCTTT TATATAATTC TATAAACGTA CGCCGGCCAT M..P..I. .T..N..A.. S..P..E..N ..I..L..R. .Y..L..H.. A..A..G..T {pipB) 3070 3080 3090 3100 3110 3120 CCGGTACGAA AGAAGCAATG AAAAGTGCAA CTTCACCACG CGGTATACTG GAATGGTTTG GGCCATGCTT TCTTCGTTAC TTTTCACGTT GAAGTGGTGC GCCATATGAC CTTACCAAAC ..G..T..K. .E..A..M.. K..S..A..T ..S..P..R. .G..I..L.. E..W..F..V Appendix 160 3 1 3 0 3 1 4 0 3 1 5 0 T C A A T T T T T T T A C C T G T G G T G G A G T A A G A A A G T T A A A A A A A T G G A C A C C A C C T C A T T C T T ..N..F..F. .T..C..G.. G..V..R..R 3 1 6 0 3 1 7 0 3 1 8 0 G A A G C A A T G A A A G A T G G T T T C G G G A G G T A A C T T C G T T A C T T T C T A C C A A A G C C C T C C A T T ..S..N..E. .R..W..F.. R..E..V..I 3 1 9 0 3 2 0 0 3 2 1 0 T T G G A A A A C T G A C C A C A T C A T T A T T A T A T G A A C C T T T T G A C T G G T G T A G T A A T A A T A T A C ..G..K..L. .T..T..S.. L..L..Y..V 3 2 2 0 3 2 3 0 3 2 4 0 T A A A T A A A A A T G C T T T C T T C G A T G G T A A T A A T T T A T T T T T A C G A A A G A A G C T A C C A T T A T . . N. .K. .N. . .A. . F . . F . . D..G..N..K 3 2 5 0 3 2 6 0 3 2 7 0 A A A T A T T T C T G G A G G A T G T C A A C G G G T G T T T T T A T A A A G A C C T C C T A C A G T T G C C C A C A A ..I..F..L. .E..D..V.. N..G..C..S 3 2 8 0 3 2 9 0 3 3 0 0 C T A T A T G T C T G T C A T G T G G A G C A G C A T C C G G A T A T A g j v G A C A G T A C A C C T C G T C G T A G G C . . I . . CT 7 L . . S . . C . . G . . A . . A . . S . . E 3 3 1 0 3 3 2 0 3 3 3 0 A A A A T A C G G A T C C C A T G G T C A T T A T T G A A G T T T T A T G C C T A G G G T A C C A G T A A T A A C T T C ..N..T..D. .P..M..V.. I..I..E..V 3 3 4 0 3 3 5 0 3 3 6 0 T G A A C A A A A A T G G A A A A A C T G T A A C G G A T A A C T T G T T T T T A C C T T T T T G A C A T T G C C T A T ..N..K..N. .G..K..T.. V..T..D..K 3 3 7 0 3 3 8 0 3 3 9 0 A A G T T G A T A G T G A G A G A T T T T G G A A T G T A T T T C A A C T A T C A C T C T C T A A A A C C T T A C A T A ..V..D..S. .E..R..F.. W..N..V..C 3 4 0 0 3 4 1 0 3 4 2 0 G T C G A A T G T T A A A A C T G A T G A G T A A A C A T A C A G C T T A C A A T T T T G A C T A C T C A T T T G T A T ..R..M..L. .K..L..M.. S..K..H..N 3 4 3 0 3 4 4 0 3 4 5 0 3 4 6 0 3 4 7 0 3 4 8 0 A T A T A C A A C A G C C T G A T T C A C T T A T A A C C G G A G G A T G G T T T T C T G A A C C T G C G C G G A G T A T A T A T G T T G T C G G A C T A A G T G A A T A T T G G C C T C C T A C C A A A A G A C T T G G A C G C G C C T C A T ..I..Q..Q. .P..D..S.. L..I..T..G ..G..W..F. .S..E..P.. A..R..S..K 3 4 9 0 3 5 0 0 3 5 1 0 3 5 2 0 3 5 3 0 3 5 4 0 A A C C T G G C T C A T A A A G A T T T C C A G G G G G A A G A T T T G T C A A A A A T A G A T G C T T C T A A T G C A T T G G A C C G A G T A T T T C T A A A G G T C C C C C T T C T A A A C A G T T T T T A T C T A C G A A G A T T A C G T ..P..G..S. .* M..L. (corresponding pipB gene is continuous in S. dublin) 3 5 5 0 3 5 6 0 3 5 7 0 3 5 8 0 3 5 9 0 3 6 0 0 G A T T T C C G T G A A A C A A C T T C T A T C T A A T G T A A A T T T A G T C G G T G C A A A T T T G T G T T G T G C C T A A A G G C A C T T T G T T G A A G A T A G A T T A C A T T T A A A T C A G C C A C G T T T A A A C A C A A C A C G .L..M..Q.. I..S..V..K ..Q..L..L. .S..N..V.. N..L..V..G ..A..N..L. 3 6 1 0 3 6 2 0 3 6 3 0 3 6 4 0 3 6 5 0 3 6 6 0 A A A T C T A C A C G C T G T A A A T C T A A T G G G T T C A A A C A T G A C T A A A G C A A A C C T G A C T C A C G C T T T A G A T G T G C G A C A T T T A G A T T A C C C A A G T T T G T A C T G A T T T C G T T T G G A C T G A G T G C G .C.C..A.. N..L..H..A ..V..N..L. . M . . G . . S . . N..M..T..K ..A..N..L. Appendix 161 3670 3680 AGACCTGACT TGCGCTAACA TCTGGACTGA ACGCGATTGT • T . . H . . A . . D . . L . . T . . C 3690 3700 TGTCCGGTGT AAACTTAACC ACAGGCCACA TTTGAATTGG . . A . . N . . M . . S . . G . . V . . 3710 3720 GCTGCAATTC TATTCGGCTC CGACGTTAAG ATAAGCCGAG N . . L . . T . . A . . A . . I . . L . 3730 3740 .' 3750 AGACTTAACT GACACCAAAC TAAATGGTGC TCTGAATTGA CTGTGGTTTG ATTTACCACG • F . . G . . S . . D . . L . . T . . D . . T . . K . . L . 3760 3770 3780 GAAATTAGAT AAGATAGCTC TAACTTTAGC CTTTAATCTA TTCTATCGAG ATTGAAATCG . N . . G . . A . . K . . L . . D . . K . . I . . A . . L . 3790 3800 3810 3820 3830 3840 GAAAGCATTA ACAGGAGCCG ATCTGACAGG TAGTCAACAT ACCCCTACTC CACTCCCGGA CTTTCGTAAT TGTCCTCGGC TAGACTGTCC ATCAGTTGTA TGGGGATGAG GTGAGGGCCT . T . . L . . A . . K . . A . . L . . T . . G . . A . . D . . L . . T . . G . . S . . Q . . H . . T . . P . . T . . P . 3850 3860 3870 3880 3890 3900 TTACAATGAT AGAACTCTTT TCCCCCATCC GATATTTTAG TCGAGATAAA GGGATTTTAT AATGTTACTA TCTTGAGAAA AGGGGGTAGG CTATAAAATC AGCTCTATTT CCCTAAAATA . L . . P . . D . . Y . . N . . D . . R . . T . . L . . F . . P . . H . . P . . I . . F . . * 3910 3920 AAACAAGAAG TATTCAAACA GA TTTGTTCTTC ATAAGTTTGT CT Appendix 162 C. G7H1 (iicA) A C C E S S I O N # A F 1 6 4 4 3 5 (iicA S. typhimurium) 10 20 30 40 50 60 GAATGCAAAA GAGAAGTTAC TGGATTTTGT GGAGTTAGAA GAAAACGAAT CGCTGATTTT CTTACGTTTT CTCTTCAATG ACCTAAAACA CCTCAATCTT CTTTTGCTTA GCGACTAAAA E . . C . . K . . R . . E . . V . . T . . G . . F . . C . G . . V . . R . . R . . K . . R . . I . . A . . D . . F . . ( p o t e n t i a l ORF) 70 80 90 100 110 120 GGTAAAGATA TTCGCATCGT TCTGGCATCA GCGGATTTCA GTAAAGAATT AAC GACAAC C CCATTTCTAT AAGCGTAGCA AGACCGTAGT CGCCTAAAGT CATTTCTTAA TTGCTGTTGG G . . K . . D . . I . . R . . I . . V . . L . . A . . S . . A . . D . . F . . S . . K . . E . . L . . T . . T . . T . . 130 140 150 GCAATATGGC TAAGAGATAA AGGTGTCGAT CGTTATACCG ATTCTCTATT TCCACAGCTA A . . I . . W . . L . . R . . D . . K . . G . . V . . D . . 160 170 180 ATTCGCTGTG TTCGCTTAAC GCCTTACAAC TAAGCGACAC AAGCGAATTG CGGAATGTTG I . . R . . C . . V . . R . . L . . T . . P . . Y . . N . . 190 200 210 TTTAAGGGTG AAGTGCTGAT TAATGCTGAA AAATTCCCAC TTCACGACTA ATTACGACTT F . . K . . G . . E . . V . . L . . I . . N . . A . . E . . 220 230 240 CAAATAATAC CAGTCCCTGA AC TGGAAGAA GTTTATTATG GTCAGGGACT TGACCTTCTT Q . . I . . I . . P . . V . . P . . E . . L . . E . . E . . 250 260 270 280 290 300 TATCAGGTCA GATTCAGAGA GAAACGCACG GAACAAATTA TTAGCAGTCA AAAGTCGGAG ATAGTCCAGT CTAAGTCTCT CTTTGCGTGC CTTGTTTAAT AATCGTCAGT TTTCAGCCTC Y . . Q . . V . . R . . F . . R . . E . . K . . R . . T . . E . . Q . . I . . I . . S . . S . . Q . . K . . S . . E . . 310 320 330 340 350 360 AGGGATTATT CCTTATATAA ATATAAAGGA AAAACCTTCA ATAAACGGAA GCTTGCACTT TCCCTAATAA GGAATATATT TATATTTCCT TTTTGGAAGT TATTTGCCTT CGAACGTGAA R . . D . . Y . . S . . L . . Y . . K . . Y . . K . . G . . K . . T . . F . . N . . K . . R . . K . . L . . A . . L . . 370 380 GAACTTTTCA CTGACTGGAT CTTGAAAAGT GACTGACCTA E . . L . . F . . T . . D . . W . . 1 . 390 . 400 TAATAAACAT AATCCTGCGA ATTATTTGTA TTAGGACGCT • N . . K . . H . . N . . P . . A . . N 410 420 ATATAGATGA TCTCAAGAAT TATATCTACT AGAGTTCTTA . . I . . D . . D . . L . . K . . N . . 430 440 450 460 470 480 AAATTGAGTG AAGACTTACA GAAAAGAACA GTAGCACTGG TTGAGCAGAT CCCTGAAAAA TTTAACTCAC TTCTGAATGT CTTTTCTTGT CATCGTGACC AACTCGTCTA GGGACTTTTT K . . L . . S . . E . . D . . L . . Q . . K . . R . . T . . V . . A . . L . . V . . E . . Q . . I . . P . . E . . K . . 490 500 510 520 530 540 AGGAAAAACA GATATCATAT GCAGGAAGAT GCGTTGATTG AGTTGCCGTC CGGTGAGCGT TCCTTTTTGT CTATAGTATA CGTCCTTCTA CGCAACTAAC TCAACGGCAG GCCACTCGCA R . . K . . N . . R . . Y . . H . . M . . Q . . E . . D . . A . . L . . I . . E . . L . . P . . S . . G . . E . . R . . Appendix 163 5 5 0 5 6 0 A T T G C T A T A T C G A T A A A T G G T A A C G A T A T A G C T A T T T A C C I . . A . . I . . S . . I . . N . . G . 6 1 0 6 2 0 T C A G G A T A A T T T T G T A G T T G A G T C C T A T T A A A A C A T C A A C 5 7 0 5 8 0 G G G T T A G G G A C T A T A G A A C T C C C A A T C C C T G A T A T C T T G A . G . . * 6 3 0 6 4 0 A A A A A G T A G G T T G A C A G G A A T T T T T C A T C C A A C T G T C C T T 5 9 0 6 0 0 G C T T A T A T G A T T T T G T T C G G C G A A T A T A C T A A A A C A A G C C 6 5 0 6 6 0 G T A A T A A T A A A A T A G A T C C C C A T T A T T A T T T T A T C T A G G G 6 7 0 6 8 0 6 9 0 7 0 0 7 1 0 7 2 0 A T T C A T T A A T G G G A T C T C A C G T T T C A T C C G A T A C G A A G A C C A T G G T C T C T T T G T C A G T A G T A A G T A A T T A C C C T A G A G T G C A A A G T A G G C T A T G C T T C T G G T A C C A G A G A A A C A G T C A T C 7 3 0 7 4 0 7 5 0 7 6 0 7 7 0 7 8 0 C G T C A T A A T T A C G C A A G C C T C T T T A C T T T G C T T A T C A T T T A T A T T T A A T G T A A A T A T T C A G C A G T A T T A A T G C G T T C G G A G A A A T G A A A C G A A T A G T A A A T A T A A A T T A C A T T T A T A A G T 7 9 0 8 0 0 8 1 0 8 2 0 8 3 0 8 4 0 C G C A A C A C C A T T A A A A A A T A A G A A A A A A T G G C T C A C T G T T G A A C T G A T A T T A A T A C C T G A G C G T T G T G G T A A T T T T T T A T T C T T T T T T A C C G A G T G A C A A C T T G A C T A T A A T T A T G G A C T 8 5 0 8 6 0 8 7 0 8 8 0 8 9 0 9 0 0 A C C A C T G A A T T A G A G T A A T G T G G C G C T A T T C A T A G C G T A A T T T T T T C T G T T G C G G T T A C A T G G T G A C T T A A T C T C A T T A C A C C G C G A T A A G T A T C G C A T T A A A A A A G A C A A C G C C A A T G T 9 1 0 9 2 0 9 3 0 9 4 0 9 5 0 9 6 0 G G G G G A G G A A T G C A C A C C T T T A G A C C A T A C T C A C T A A G G C A T A G C G A T C T G T T A T A T G A A C C C C C T C C T T A C G T G T G G A A A T C T Q Q T A T G A G T G A T T C C G T A T C G C T A G A C A A T A T A C T T M . . H . . T . . F . . R . . P . . Y . . S . . L . . R . . H . . S . . D . . L . . L . . Y . . E . . ( i i c A ) 9 7 0 9 8 0 9 9 0 1 0 0 0 1 0 1 0 1 0 2 0 G A T A T T C C G T T A G A A A T A C G C G A G C A A A T A A T C T T A T T G A T T A T C A A T A C G C T A G G A A A C C T A T A A G G C A A T C T T T A T G C G C T C G T T T A T T A G A A T A A C T A A T A G T T A T G C G A T C C T T T G D . . I . . P . . L . . E . . I . . R . . E . . Q . . I . . I . . L . . L . . I . . I . . N . . T . . L . . G . . N . . 1 0 3 0 1 0 4 0 1 0 5 0 T G C T C C T C T T T T T A T G A T A T G A C A T T A T A C A C G A G G A G A A A A A T A C T A T A C T G T A A T A T G C . . S . . S . . F . . Y . . D . . M . . T . . L . . Y . . 1 0 6 0 1 0 7 0 1 0 8 0 T G C T A T C A T A A T A G T C A T T C T G A C G A A G T T A C G A T A G T A T T A T C A G T A A G A C T G C T T C A A C . . Y . . H . . N . . S . . H . . S . . D . . E . . V . . 1 0 9 0 1 1 0 0 1 1 1 0 1 1 2 0 1 1 3 0 1 1 4 0 T A T C G A A G A A T A T G T A A A A C G T T G C G C A A A G A G T A T G G C T T A T T C A C C T T A T A G G C G C A T A T A G C T T C T T A T A C A T T T T G C A A C G C G T T T C T C A T A C C G A A T A A G T G G A A T A T C C G C G T A Y . . R . . R . . I . . C . . K . . T . . L . . R . . K . . E . . Y . . G . . L . . F . . T . . L Appendix 164 1150 1160 1170 1180 1190 1200 TCAACGTCAT ATCTGGATGA AATGAGTAAT CTGTTATTAA AAACAGATGA TAAAAGAAAG AGTTGCAGTA TAGACCTACT TTACTCATTA GACAATAATT TTTGTCTACT ATTTTCTTTC M . . S . . N . . L . . L . . L . . K . . T . . D . . D . . K . . R . . K . . ( p o t e n t i a l ORF2) 1210 1220 1230 1240 1250 1260 CATATTGATA CCATTGAGCT TGCTTTTAAC TATATAGATA CCTACCTTCG GACCTATGAA GTATAACTAT GGTAACTCGA ACGAAAATTG ATATATCTAT GGATGGAAGC CTGGATACTT H . . I . . D . . T . . I . . E . . L . . A . . F . . N . . Y . . I . . D . . T . . Y . . L . . R . . T . . Y . . E . . 1270 1280 1290 1300 1310 1320 GTTACGCTTG GGTTAGAACC GGATAAGGCG ATTAGTGAAT TAAATAATAT ATTTCATGAG CAATGCGAAC CCAATCTTGG CCTATTCCGC TAATCACTTA ATTTATTATA TAAAGTACTC V . . T . . L . . G . . L . . E . . P . . D . . K . . A . . I . . S . . E . . L . . N . . N . . I . . F . . H . . E . . 1330 1340 1350 1360 1370 CATAGTTTAA AATATCGATA TGAAAAATGG TTAGGATTGT TAAGGTTGCG C GTATCAAATT TTATAGCTAT ACTTTTTACC AATCCTAACA ATTCCAACGC G H . . S . . L . . K . . Y . . R . . Y . . E . . K . . W . . L . . G . . L . . L . . R . . L . . R . . 

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