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Identification of Salmonella typhimurium genes that are induced upon contact with epithelial cells Griffiths, Angela 1991

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IDENTIFICATION OF SALMONELLA TYPHIMURIUM GENES THAT ARE INDUCED UPON CONTACT WITH EPITHELIAL CELLS. ^ b y ANGELA GRIFFITHS H. B.Sc. University of Western Ontario, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard UNIVERSITY OF BRITISH COLUMBIA September 1991 © Angela Griffiths 1991 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 fH ('PO^O ) D ) Q Q , A The University of British Columbia^ ^ Vancouver, Canada Date DE-6 (2/88) Abstract Salmonella typhimurium is a facultative intracellular pathogen which is able to infect a variety of animal species and mammalian cells in culture. It has been estimated that there are in excess of 400,000 cases of Salmonellosis every year in the United States, but very little is known about the mechanisms of pathogenesis. Several bacterial proteins are synthesized upon encountering mammalian cells which may be involved in the infection process. Tn5-VB32 transposon mutagenesis was used to create transcriptional fusions in genes in the virulent S. typhimurium strain, SL1344 . Subsequent screening identified mutants that contained the neomycin phosphotransferase II gene (from Tn5-VB32) under the control of a bacterial promoter which was induced in the presence of cultured epithelial cells. Induction was measured as an increase in the resistance of the bacteria to treatment with neomycin sulfate. Eight mutants that displayed increased resistance to neomycin upon contact with Madin-Darby canine kidney cells (MDCK) cells were identified during the initial screening process. In five of the mutants, the transposon was inserted in the same place, one mutant had two transposon insertions and the other two mutants (6-30 and 20-.10) contained single insertions in unique sites. Mutant 1-12 was selected from the group of five mutants and was studied further, as were mutants 6-30 and 20-10, which contained unique single site insertions. Each of these three mutants displayed significantly higher survival rates when treated with neomycin in the presence of MDCK cells as compared to those treated with tissue culture fluid alone indicating that there were transcriptional fusions to induced genes. Mutant 1-12 survived neomycin treatment an average of 6 times better, 6-30 survived 23.7 times better and 20-10 survived 51.7 times better in the presence of MDCK cells than in tissue culture fluid. All of the mutants were motile, contained intact LPS, and grew at similar rates compared to the parental strain. These three mutants also adhered to fixed MDCK cells at parental levels and had a similar effect on the transepithelial ii resistance of an MDCK cell monolayer. Invasion of MDCK cells by 1-12 was about 65% less than the wild type parent, mutant 6-30 invaded 95% less efficiently and 20-10 about 98% less. The growth kinetics of 1-12 and 6-30 inside MDCK cells were similar to the wild type parent. Mutant 20-10 replicated more slowly, culminating in about 50% fewer intracellular bacteria than wild type after 24 hours. Mutants 1-12 and 6-30 also showed enhanced survival to neomycin treatment in the presence of extracellular matrix components including collagen, entactin, laminin and heparin sulfate proteoglycan and in agarose, indicating that the transcriptional fusions were induced by the presence of a solid support. Mutant 20-10 did not display enhanced survival under these conditions. Since 20-10 had several altered phenotypic characteristics, it is possible that the gene affected in this mutant plays a more specific role in pathogenicity than the genes induced by the presence of a solid support. Wild type Salmonella were incubated in the presence of extracellular matrix components of tissue culture fluid before assaying their invasion into MDCK cells in an attempt to determine if pre-induction of the solid support genes enhanced invasiveness. After 1 hour, invasion by bacteria from the samples pre-incubated with the extracellular matrix components was considerably better than invasion by bacteria pre-incubated in tissue culture fluid. Thus, it appears that at least two of the bacterial genes induced in the presence of MDCK cells are induced by the presence of a solid support and that activation of these genes by pre-incubation with a solid support enhances invasion into MDCK cells. The nature of the gene affected in mutant 20-10 was not determined, but the the presence of several altered phenotypic characteristics caused by the transposon insertion indicate that the gene may be involved in regulation of additional virulence factors and may be regulated by contact with epithelial cells. iii Table of Contents Abstract ii Abbreviations vi List of Figures vii List of Tables viii 1. Introduction 1 1.1 Pathogenesis of Salmonella species 2 1.1.1 Adherence 2 1.1.2 Invasion 3 1.1.3 Intracellular Survival and Replication 7 1.1.4 Dissemination 8 1.2 Regulation of Virulence Factors 9 1.3 Objectives 10 2. Materials and Methods 11 2.1 Materials and Supplies 11 2.2 Bacteria and Plasmids 11 2.3 Tn5-VB32 12 2.4 Cell Culture 12 2.5 Conjugation of Tn5-VB32 into S. typhimurium 13 2.6 Screening for Induction of Neomycin Resistance 13 2.7 Southern Blot 14 2.8 Measuring Induction of Neomycin Resistance 14 2.9 P22 Transduction 16 2.10 Motility Assay 16 2.11 Lipopolysaccaride Agglutination 17 2.12 Bacterial Adherence Assay ... 17 iv 2.13 Transepithelial Resitance Measurements 17 2.14 Invasion Assay 17 2.15 Growth Rate in Minimal Medium 18 2.16 Intracellular Growth Kinetics 18 2.17 Induction of Neomycin Resistance in Matrigel and Agarose 18 2.18 Preincubation of SL1344 in Matrigel 19 3. Results 32 3.1 Identification of S. typhimurium Tn5-VB32 Mutants Induced in the Presence of MDCK cells 23 3.2 Southern Blot 23 3.3 Induction of Neomycin Resistance 25 3.4 Motility and LPS Test 25 3.5 Adherence of Induced Mutants 27 3.6 Effects of Induced Mutants on Transepithelial Resistance 27 3.7 Invasive Capability of Induced Mutants 30 3.8 Growth Curve in Minimal Medium 30 3.9 Intracellular Growth Kinetics of Induced Mutants 30 3.10 Effects of Incubation in Matrigel and Agarose on Induction of Neomycin Resistance 34 3.12 Effects of Pre-incubation in Matrigel on Invasion of Wild Type 36 4. Discussion 38 References 43 v List of Abbreviations Amp: ampicillin ATCC: American tissue culture collection FCS: fetal calf serum His: histidine L- broth: Luria broth LB agar: Luria Bertani agar LD50: 50% lethal dose LPS: lipopolysaccaride MDCK: Madin-Darby canine kidney MEM: minimal essential medium, tissue culture fluid Neo: neomycin sulfate NPTII: neomycin phosphotransferase II OD: optical density O/N: overnight PBS: phosphate buffered saline pen: penicillin Sm: streptomycin Tc: tetracycline tetA: tetracycline resistance gene tetR: repressor of the tetracycline resistance gene vi List of Figures Figure IrSchematic diagram of transposon Tn5-VB32 20 Figure 2: Identification of Tn5-VB32 mutants in Salmonella typhimurium that are induced upon contact with (MDCK) cells 22 Figure 3: Southern analysis of the Tn5-VB32 mutants 24 Figure 4: Adherence of bacteria to fixed MDCK cells 28 Figure 5: Transepithelial resistance of polarized MDCK cell monolayers infected with S. typhimurium and Tn5-VB32 mutants 29 Figure 6: Invasion of MDCK cells by 5. typhimurium 31 Figure 7: Growth kinetics of S.typhimurium and the induced mutants in rotating cultures (150rpm) of minimal medium (plus histidine) at 37°C 32 Figure 8: Growth kinetics of S.typhimurium and the induced mutants inside MDCK cells 33 vii List of Tables Table 1: Survival of bacteria in 100 (ig/ml neomycin in the presence of MDCK cells as compared to survival in MEM alone 26 Table 2: Survival of bacteria in 100 u,g/ml neomycin in the presence of Matrigel and agarose as compared to survival in MEM alone 35 Table 3: Effects of pre-incubation in Matrigel on invasion of S.typhimurium SL1344 into MDCK cells 37 viii 1. Introduction Salmonella typhimurium is a gram negative, rod shaped organism which is a common human pathogen. 5. typhimurium has a broad host range and can cause infections in many species of animals. In 1986, Salmonella infections were responsible for 400,000 cases of food poisoning resulting in 500 deaths and over $50 million in health care costs (Cohen and Tauxe, 1986). Most Salmonella cause a self-limiting gastroenteritis with the exception of Salmonella typhi, which causes typhoid fever (Cohen and Tauxe, 1986). A Salmonella infection usually occurs following ingestion of contaminated food or water. Cases of typhoidal salmonellosis have been decreasing since the early part of this century only to be replaced with steadily increasing rates of non-typhoidal salmonellosis (Cohen and Tauxe, 1986). Treatment of salmonellosis is becoming increasingly more difficult due to the emergence of antibiotic resistant strains (Cohen and Tauxe, 1986). Clearly, salmonellosis constitutes a major health problem in North America and for this reason, it is attracting considerable interest. The first comprehensive study of Salmonella pathogenesis was carried out by Takeuchi (1967) using guinea pig ileal loops. In this model, the organisms proceeded through the stomach to the small intestine where they interacted with the epithelial cell wall, disrupting the microvilli. After several hours of infection, some bacteria were seen in the lamina propria underlying the epithelial cells (Takeuchi, 1967). Further studies have demonstrated that Salmonella infect the host primarily through the M cells (a type of epithelial cell) in the Peyers patches lining the small intestine and secondarily through the columnar epithelial cells (Finlay and Falkow, 1989). The bacteria are able to induce the epithelial cells to internalize them via an energy requiring process. Once inside the epithelial cells, the bacteria are able to replicate inside membrane bound vacuoles, eventually filling them (Finlay and Falkow, 1988). Some bacteria penetrate 1 through the intestinal epithelial cells to enter the blood stream where they invade and propagate within macrophages (Buchmeier and Heffron, 1990). The infected macrophages are cleared to the spleen and lymph nodes, thereby spreading infection to these organs. Since Takeuchi performed his pioneering study, researchers have concentrated on dissecting each of the steps of infection and determining their molecular basis. Infection of a host by an intracellular pathogen involves several steps; adherence; invasion; intracellular replication; and dissemination. I have used these steps to describe our current knowledge of Salmonella pathogenesis. 1.1 Pathogenesis of Salmonella species 1.1.1 Adherence The attachment of Salmonella to epithelial cells is biphasic consisting of an initial reversible attachment phase followed by an irreversible attachment phase (Jones et al, 1981, Lindquist et al, 1990). The reversible attachment phase is short, lasting only about 20 minutes, during which the bacteria are easily washed away (Lindquist et al, 1990). The irreversible attachment phase represents a more stable interaction which is inhibited by the addition of chloramphenicol, indicating a requirement for protein synthesis (Lindquist et al, 1990). Bacterial motility and type 1 fimbriae play a role in attachment (Ernst et al, 1990, Jones et al, 1981, Lindquistef al, 1987). Non-motile bacteria are less able to adhere simply because they are less likely to come into contact with the epithelial cells. Salmonella possessing mannose sensitive type 1 fimbriae appear to bind much more effectively to mammalian cells than bacteria without fimbriae (Ernst et al, 1990, Jones et al, 1981, Lindquist et al, 1987, Schmoll et al, 1990). In Yersinia species, the interaction between the bacteria and the mammalian cell is very specific, involving the integrin family of mammalian cell receptors (Isberg, 1991). Integrins are surface glycoproteins that are involved in cell attachment to the extracellular matrix, cell movement and cell to cell interactions (Isberg, 1991, Ruoslahti 2 and Piersbacher, 1987). The extracellular domain of the integrin molecule interacts with extracellular membrane components (fibronectin, collagen, laminin) while the inner domain probably interacts with tailin, an actin binding protein (Burridge and Fath, 1989). Several bacterial pathogens bind to extracellular membrane components including Bacteriodes, Fusobacterium, Actinobaccillus, Staphylococci, Streptococci and E. coli and they may use this as a means of interacting with the integrin receptors (Isberg, 1989, Winkler etal, 1987). Most of the intracellular pathogens that have been studied are gram-negative organisms. Listeria monocytogenes provides an interesting exception being a gram positive intracellular pathogen. It's lifestyle is remarkably similar to the gram negative intracellular pathogens in that L. monocytogenes attaches to and infects epithelial cells in mammalian hosts (Gaillard et al, 1991). Internalin is a membrane located protein of L. monocytogenes that mediates attachment and entry into cultured epithelial cells. Adherence of Salmonella to mammalian cells has not been studied rigorously and it is not known at this time what the eukaryotic receptor is. 1.1.2 Invasion Following adherence, Salmonella enter into host cells, which are often cells that do not normally phagocytose particles (non-professional phagocytes). Many enteropathogenic bacteria bind to non-phagocytic mammalian cells but only certain species are internalized (Isberg, 1991). The precise signal that induces the epithelial cells to internalize certain species of bacteria is unknown. Internalization of Salmonella is an active, energy requiring process that involves localized polymerization of actin filaments around the bacterium (Kihlstrom and Nillson, 1977, Finlay and Falkow, 1989, 1991). Shigella invasion induces a similar polymerization of actin filaments when it enters epithelial cells (Clerc and Sansonetti, 1987). Treatment with actin polymerization inhibitors, such as cytochalasin D inhibit internalization of Salmonella and Shigella (Clerc and Sansonetti, 1987, Finlay and Falkow, 1989, 1991). Efficient internalization of Salmonella is affected by the growth state and other environmental conditions. Bacteria that are grown anaerobically invade at significantly higher levels than aerobically grown bacteria (Lee and Falkow, 1990, Ernst et al, 1990, Schieman and Shope, 1991). Some researchers have reported that bacterial growth state also affects invasion levels, concluding that bacteria in log phase invade at much higher levels than those in stationary phase (Lee and Falkow, 1990, Ernst et al, 1990). However, other studies indicate that auxotrophic bacteria which cannot replicate in tissue culture fluid are still able to invade (Finlay and Leung, 1991). Treatment of Salmonella, Shigella and Neisseria species with chloramphenicol (a protein synthesis inhibitor) prior to exposure to mammalian cells inhibits invasion, indicating that protein synthesis is required (Finlay and Falkow, 1989, Headley and Payne, 1990, Chen et al, 1991). Two dimensional gel electrophoresis of attaching and invading bacteria indicated the presence of several new bacterial proteins in these species (Finlay and Falkow, 1989, Headley and Payne,1990, Chen et al, 1991). Invasion does not occur immediately after adherence; instead, there is a lag phase after attachment before the bacteria are internalized (Finlay and Falkow, 1989, Chen et al, 1991). Salmonella and N. gonnhorreae that were pre-incubated in the presence of tissue cells and then introduced to a new monolayer invaded immediately, indicating that adherence of the bacteria to a mammalian cell acts as a signal, triggering the production of gene products required for invasion (Finlay and Falkow,1989, Chen et al, 1991). Bacterial genes involved in invasion have been identified in several species including Shigella species, Yersinia enterocolitica and Y. pseudototuberculosis, Listeria monocytogenes, Salmonella typhimurium and Salmonella typhi (Clerc and Sansonetti, 1987, Isberg, 1989, Gulig and Curtis,1987, Gaillard et al, 1991). 4 The most well characterized invasion genes are the inv and ail genes of Yersinia (Isberg, 1989). The presence of either of these gene products is enough to confer invasiveness onto normally non-invasive E. coli (Miller et al, 1988). The inv gene product promotes adherence and invasion of E. coli into all cell lines studied so far, whereas ail is more restrictive, promoting invasion only into certain cell types (Isberg, 1989). The internalin gene (m/A) of L. monocytogenes is required for invasion into cultured epithelial cells (Gaillard et al, 1991). The presence of m/A in the normally non-invasive L. innocua was enough to allow this organism to enter Caco-2 cells in culture (Gaillard et al, 1991). Internalin is present on the plasma membrane of L. monocytogenes and may bind to an as yet unidentified mammalian cell receptor (Gaillard etal, 1991). The ipa genes on the 22 kb plasmid in Shigella encode for highly immunogenic proteins which participate in invasion (Clerc et al, 1988). The products of virG and viVF regulate entry of Shigella into mammalian cells. These genes are regulated by environmental cues such as temperature and osmolality (Clerc et al, 1988). Clinical isolates of Salmonella contain a large (>100 kb) plasmid which is associated with virulence (Gulig and Curtis, 1987). The presence of this plasmid is correlated with virulence and replication in spleens and lymph nodes of infected mice (Gulig and Curtis, 1987). Curing of the plasmid from wild type Salmonella does not affect adherence and invasion of Henle-407, HEp-2 and CHO cells or replication inside CHO cells. In addition, resistance to killing by human, rabbit and guinea pig serum or the intra-peritoneal LD50 values are unchanged in plasmid-cured strains (Gulig and Curtis, 1987). From these results, it appears that the virulence plasmid contains genes involved with the interaction of Salmonella typhimurium with the cells of the reticuloendothelial system and not interactions with epithelial cells. 5 The inv locus in Salmonella tyhpimurium is involved in invasion. The presence of the inv locus can confer invasiveness onto a non-invasive Salmonella strain, but not a non-invasive E. coli (HB101)(Galan and Curtis, 1989). The inv locus consists of 4 genes; inv A, B, C, and D. Mutations in mvA and mvB abolish invasion into Henle-407 cells whereas mutations in invD reduce invasion by 5 times and mutations in invC have no effect (Galan and Curtis, 1989). The presence of the inv locus is required for efficient colonization of the Peyers patches, small intestine and spleens of mice after an oral dose of Salmonella, but not for colonization of the spleen when the mutants are introduced intra-peritoneally (Galan and Curtis, 1987). From these studies, the authors concluded that the inv locus is required for invasion of mammalian cells and colonization of organs past the gut in orally induced infections (Galan and Curtis, 1987). It is important to note that these experiments were all carried out in a non-invasive Salmonella background and the mv locus did not confer invasiveness to E. coli. In addition, the nature of the defect (resulting in invasion minus phenotype) in the Salmonella strain is unknown. Therefore, the actual role of the inv locus in invasion remains unclear. The mvA promoter was fused to marker genes in an attempt to identify conditions responsible for regulation. Transcription from the inv A promoter was increased by 8 times when the bacteria were grown in high osmolality medium, independently of the osmoregulated OmpR system (Galan and Curtis, 1990). Incubation of the bacteria under the same conditions in the presence of DNA supercoiling inhibitors (Coumeromycin A and novobiocin) abolished the induction effect indicating that expression of the inv A gene is regulated by changes in DNA supercoiling (Galan and Curtis, 1990). In addition, virulent Salmonella strains that were grown in low osmolality medium were impaired in their ability to invade tissue culture cells (Galan and Curtis, 1990). The level of supercoiling is affected by a number of environmental factors, such as temperature, oxygen levels and starvation (Galan and Curtis, 1990). 6 From this data, it can be concluded that alterations in DNA supercoiling play an important role in the regulation of invasion of Salmonella (Galan and Curtis, 1990). A region of the chromosome of Salmonella.typhi has been identified that allows E. coli HB101 to invade Henle 407 cells (Elsinghorst et al, 1989). Mutagenesis with Tn5 revealed the existence of at least 4 loci involved in the invasive phenotype (Elsinghorst et al, 1989). Homologous sequences were identified in cosmid libraries of Salmonella typhimurium but these sequences were not sufficient to allow HB101 to invade tissue cells (Elsinghorst et al, 1989). The nature of the gene products produced by these genes has not been determined. From the results presented above, it is apparent that invasion of mammalian cells by intracellular pathogens is often a complex phenomenon, involving regulation of numerous genes. Current research is focussing on identifying the genes required for invasion into mammalian cells, analysing their products and regulatory mechanism. 1.1.3 Intracellular Survival and Replication Intracellular pathogens enter mammalian cells in membrane bound vacuoles. Salmonella and Yersinia species remain within the vacuoles, multiplying inside them whereas Shigella and Listeria species escape from the vacuoles and replicate in the cytoplasm (Finlay and Falkow, 1988, Portnoy et al, 1988, Gaillard et al, 1987). The intracellular environment provides unique obstacles for the bacteria to overcome. Anti-bacterial compounds are released into the vacuoles by the mammalian cells (particularly macrophages) in an effort to kill the invading organisms. In addition, the bacteria must be able obtain the appropriate nutrients in order to multiply. Intracellular growth, like invasion, requires the induction of numerous genes in Salmonella and Shigella (Headley and Payne, 1990, Buchmeier and Falkow, 1990). Mutations in the pagC gene of Salmonella typhimurium or it's regulators (phoP and phoQ, described later) result in decreased survival in cultured mouse macrophages •7 and significantly higher oral LD50's (Miller et al, 1989). The nature of the gene product of pagC has not yet been determined. The lipopolysaccaride (LPS) composition of Salmonella appears to be an important factor of intra-cellular survival (Stinavage et al, 1990). Salmonella typhimurium with intact LPS are much more resistant to non-oxidative killing by neutrophils than rough strains (Stinavage et al, 1990). In addition, purified LPS is able to bind to several species of cationic proteins (antibacterial agents released into vacuoles), inhibiting their bacteriocidal activity (Stinavage et al, 1990). Studies that are designed to provide a catalog of the conditions within the vacuole with respect to pH, oxygen levels and nutrient availability are currently being performed using B-galactosidase fusions in Salmonella (F. Del Portillo, personal communication). The results of these studies will enable us to identify the conditions that the bacteria are exposed to in the intra-cellular environment and correspondingly, the genes required for such growth. 1.1.4 Dissemination Infections with Shigella flexneri and enteroinvasive E. coli are primarily confined to the epithelial cell layer of the small intestine therefore, disseminating to neighbouring cells (Bernardini etal, 1989). However, invasive Salmonella and Yersinia species penetrate through the epithelial cell layer, infecting the cells and organs of the reticuloendothelial system and causing systemic infection (Takeuchi, 1967, Une, 1977a and b). Epithelial cells can be grown in culture such that they mimic the epithelial cell wall of the small intestine (polarized epithelial cells). The cells form a monolayer when grown on a porous filter that has intact tight junctions (Simons and Fuller, 1985). Experiments with these small intestine models demonstrate that Salmonella are able to penetrate the epithelial cell monolayer before the integrity of the monolayer is destroyed as a result of massive infection (Finlay and Falkow, 1988). The mode of exit used by 8 the bacteria has not been identified, but it is possible that the vacuolar contents are being released extracellularly as a normal step in membrane recycling (exocytosis). L. monocytogenes and Shigella flexneri have interesting modes of infecting adjacent cells. Once the bacteria are internalized by the mammalian cell, they dissolve the vacuolar membrane with hemolytic enzymes (Gaillard et al, 1987). Once in the cytoplasm, the bacteria catalyse the formation of actin "tails" which appear to push them out of the mammalian cell in a pseudopod which plunges into neighbouring cells (Tilney and Portnoy, 1989). Once the bacteria is inside the neighbouring cell, it dissolves the membrane surrounding it and begins the process again (Tilney and Portnoy, 1989). 1.2 Regulation of Virulence Factors Many bacterial virulence factors are co-ordinately regulated by two component regulatory systems (Miller, et al 1989). These systems consist of a membrane bound protein (1st component) that is able to intercept environmental signals and transmit information to an intracellular protein (2nd component)(Miller, et al 1989). The intracellular protein then interacts directly with the DNA, regulating the expression of numerous genes at the transcriptional level (Miller, et al 1989). Pathogenic bacteria must be able to survive in vastly different environments, from soil to inside a mammalian host. Two component regulatory systems allow organisms to respond rapidly to changes in the environment by activating several genes at once. The PhoP/PhoQ system of Salmonella is a two component regulatory system which is involved in the regulation of virulence factors (Miller, et al 1989). PhoQ is the membrane located environmental sensor in this system (Miller, et al 1989). After receiving a stimulus, the carboxy terminal (intracellular) portion of PhoQ catalyses phosphorylation of PhoP (in the cytoplasm)(Miller, et al 1989). The modified PhoP 9 then activates the promoters of genes involved in virulence, including pagC (mentioned above)(Miller, et al 1989). 1.3 Objectives Since Salmonellosis is becoming an increasingly important health problem in North America, research has focussed on elucidating the molecular basis of pathogenesis in order to design potential vaccine strains. Given that regulation of virulence factors is an important aspect of Salmonella pathogenesis, the identification of the genes required for virulence and their regulators will aid in vaccine developement. The objective of my project was to identify Salmonella genes that are induced upon contact with cultured epithelial cells and to try to determine their function and regulation. Identification of Salmonella genes that were induced upon contact with cultured epithelial cells were identified by assaying the activity of a marker gene transcriptionally fused to Salmonella promoters. Transcriptional fusions were created by transposon mutagenesis with a Tn5 derivative, Tn5-VB32 (Bellofatto et al, 1984). Insertion of Tn5-VB32 downstream of an active promoter results in the expression of neomycin phosphotransferase (NPTII), rendering the bacteria more resistant to neomycin sulfate. Mutants that showed enhanced survival of neomycin treatment when exposed to epithelial cells were isolated. Adherence, invasion and intracellular replication rates were determined as was induction under various conditions in an attempt toiassign functions to the affected genes. The mutants containing the transcriptional fusions were also exposed to different environmental conditions in order to identify regulatory factors. 10 2. Materials and Methods 2.1 Materials and Supplies: Chemicals were obtained from BDH chemicals, Darmstadt, Germany, unless specified. Histidine, glutaraldehyde, NH4CI, cytochalasin D and gentamycin sulfate were obtained from Sigma chemicals, St. Louis, Missouri. Fetal calf serum (FCS), minimal essential medium (MEM), penicillin, streptomycin sulfate, neomycin sulfate, yeast extract and the Bionick labelling kit were purchased from Gibco/BRL, Gaithersburg, Maryland. The restriction enzyme Clal was supplied by Boehringer/Mannheim, Laval, Quebec. Tryptone media and the Salmonella O antiserum (Group B, factors 1,4,5, 12) were obtained from Difco in Detroit, Michigan. All plastic tissue culture supplies were purchased from Falcon/Becton-Dickinson, Missisagua, Ontario. The 3u.m filter units were from Costar, Cambridge, Massachusetts (Transwell, 3415). and the transepithelial resistance meter used was the Millicell ERS from Millipore in Bedford, Massachusetts. Matrigel was obtained from Collaborative Research, California and Bluescript plasmid pKS+ from Stratagene in La Jolla, California. 2.2 Bacteria and Plasmids: Salmonella typhimurium SL1344 is His-, streptomycin resistant, and mouse virulent)(Hoiseth and Stocker, 1981). Eschericia coli SM10 (Simons et al, 1983) was used to maintain the suicide plasmid containing the transposon and to conjugate the transposon into Salmonella typhimurium . The transposon used for mutagenesis was Tn5-VB32 (see next section) (Bellofatto et al, 1984). pBF-VB32 contains Tn5-VB32 cloned into the suicide plasmid pJM703.1 (Miller and Mekalanos, 1988). Bacteria were grown as standing overnight cultures in Luria broth or M9 minimal medium (Sambrook et al, 1989) supplemented with 30 u.g/ml histidine at 37°C. 11 2.3 Tn5-VB32: Tn5-VB32 contains a tetracycline resistance gene from TniO and the neomycin resistance gene neomycin phosphotransferase II (NPTII) (Figure 1). The NPTII gene does not contain an active promoter. Tn5-VB32 was created by cloning an NPTII gene with no transcriptional start site into a Tn5 derivative that was missing the entire NPTII gene and most of the IS50L insertion sequence (Bellofatto et al, 1984). The Tn5 transposon derivative used contained an intact tetracyline resistance (tetA) gene and tetracycline repressor gene (tetR) derived from Tn/0. The resulting transposon (Tn5-VB32) retained its ability to transpose but with 1/10 th the frequency of the parental Tn5 (Bellofatto etal, 1984). Insertion of Tn5-VB32 into the genome of a bacteria results in transcriptional fusions which can be assayed by measuring NPTII expression levels. 2.4 Cell Culture: Madin Darby Canine Kidney Cells (ATCC) were grown in MEM supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin and 100 p,g/ml streptomycin at 37°C in a 5% CO2 humidified incubator (Baclarova et al, 1984). For assays done in 24 or 96 well plates, cells were seeded at 1 x 105 or 5 x 104 cells per well respectively and grown 24 hours prior to the experiment. For experiments involving monolayers grown on 3 urn pore filters, MDCK cells were seeded at 1.5 x 10^  per transwell filter in 24 well plates and incubated for 4-5 days until an electrical resistance developed across the monolayer (Finlay and Falkow, 1989). Filter grown monolayers were fed with fresh tissue culture fluid daily and resistance measurements were taken with a Millicell ERS resistance meter after 5 days. The transepithelial resistance across an intact monolayer was typically 1650-2310 Qcm2. MDCK cells were washed with MEM without antibiotics prior to addition of bacteria in all experiments. 12 2.5 Conjugation of Tn5-VB32 into S. typhimurium: Fresh agar plates of S. typhimurium SL1344 and E. coli SmlO (k pir) containing pBF-VB32 (Tn5-VB32::pJM703.1) were grown overnight on LB agar (Maniatis et al, 1989) containing 25 |!g/ml streptomycin (Sm) or 15 u.g/ml tetracycline (Tc) respectively. pJM703.1 contains an origin of replication that requires the product of the X pir gene (Miller and Mekalanos, 1988). The plasmid cannot replicate in bacteria without the X pir gene and therefore it is not maintained. One cm squares of E. coli (donor) were streaked onto fresh, warm LB agar platesfollowed by streaking of S. typhimurium (recipient) over the E. coli. The plates were incubated for 4-12 hours at 37°C followed by harvesting of the bacteria by scraping off the plates with a sterile swab and resuspending in L broth. Aliquots of the suspensions were plated onto LB + Sm + Tc plates and incubated overnight. Colonies which grew were S. typhimurium containing Tn5-VB32 integrated in their genome. 2.6 Screening for Induction of Neomycin Resistance: Single colonies of Sm and Tc resistant transconjugants were grown overnight (O/N) in L broth in 96 well plates and then dotted onto Tc + Sm plates for archiving. 2-3 ulv of of each isolate was inoculated into a 96 well plate containing MEM plus 400 u.g/ml neomycin sulfate and incubated at 37°C for 6 hrs (Figure 2). 5 U.L aliquots of each well was dotted onto a LB agar + Sm + Tc plates and grown overnight. Wells which produced no colonies were considered to contain neomycin sensitive isolates. The neomycin sensitive isolates were then screened for the induction of neomycin resistance in the presence of MDCK cells. MDCK cells were grown on 3 u,m filters as described and then fixed with 2% glutaraldehyde in phosphate buffered saline (PBS) for 3 hours to allow potential induction (Maniatis et al, 1989). The medium was then removed and replaced with MEM containing 800 Jig/ml neomycin and incubated for an additional 6 hours. The fixed filters were then scraped with a pipette tip and the medium vigorously pipetted to dislodge bound bacteria 13 and plated onto LB agar + Sm + Tc. Survivors were re-screened for neomycin sensitivity and induction. 2.7 Southern Blot: Genomic DNA was extracted from the mutants by previously described protocols (Finlay, et ai, 1988) and digested with 11 units of Clal for one hour at 37°C. The digested DNA was electrophoresed overnight at 23V through a 0.7% agarose gel (Maniatis et al, 1989) and a Southern blot performed by standard methods (Southern, 1975). The blots were probed with a biotinylated probe prepared from Bluescript pKS, containing the NPTTI gene. Nick translation, biotinylation and visual detection were carried out using the Bionick labelling kit. 2.8 Measuring Induction of Neomycin Resistance: Survival of mutants grown in 100 |ig/ml neomycin in the presence of MDCK cells was compared to that of bacteria in MEM and neomycin alone. Two strains were selected at this point to serve as positive and negative controls for induction. M M lb is a SL1344 mutant containing a mini Mu dJ transposon insertion (Castihlo, et al, 1984, Hughes and Roth, 1988). The mini Mu transposon has an intact NPTII gene and is constitutive for neomycin resistance. The negative control used was Neos, a Tn5-VB32 mutant which does not appear to be induced for neomycin resistance. MDCK cells were grown O/N in 96 well plates as described and bacterial cultures were grown overnight in L broth. Initially, the MEM covering the MDCK cells was removed and replaced with MEM containing 1 u,g/ml cytochalasin D and incubated for 30 minutes at 37°C. Cytochalasin D causes a 10-100 times reduction in the internalization of bacteria (Finlay and Falkow, 1988). Another set of wells were used to determine the levels of internalization of bacteria into MDCK cells treated with cytochalasin D throughout the experiment. 2 |iL of overnight bacterial culture was added to the wells with MDCK cells and 2 |iL of a 1/15 dilution of overnight to wells 14 containing only MEM with 1 U.g/ml cytochalasin D for 3 hours at 37°C. The supernatant was removed from wells with MDCK cells and media containing 100 u.g/ml of neomycin sulfate or gentamicin sulfate (for assaying number of bacteria internalized) and cytochalasin D was added. MEM with neomycin sulfate and cytochalasin D was added to the wells without MDCK cells to a final concentration of 100 u,g/ml neomycin and 1 u.g/ml cytochalasin D. A set of wells was maintained without antibiotic (fresh MEM with cytochalasin D was added at the same time as the neomycin was added to the other wells) to serve as the reference for survival. Plates were incubated at 37°C for an additional 4 hours. The MEM was then removed from wells with MDCK cells, wells were washed once with PBS and then 20 uL of 1% Triton X-100 was added for 5 min to release bound bacteria. The bacteria were then suspended in 80 U.L of L broth and titred by plating onto LB agar plus Sm artd Tc plates. The number of bacteria internalized was determined by washing the wells that had been treated with gentamicin, releasing the bacteria and titring them. These numbers were subtracted from the number of survivors in wells with MDCK cells. Therefore, internalized bacteria were not counted as survivors of neomycin treatment. The bacteria in the wells with MEM were also titred. The percent survival of bacteria in neomycin was calculated for bacteria in wells with and without MDCK cells. Once the percent survival was calculated, the ratio between survival with MDCK and survival in MEM was calculated (see example calculation below). This ratio gives an indication of whether the bacteria survive neomycin treatment better when they are exposed to MDCK cells than when in MEM alone. All experiments were performed with triplicate wells and the average taken to calculate percent survival. Sample Calculation: Mutant 1-12 (1 experiment: percent survival reported is the average of 3 trials) (1) Survival with MDCK cells: 19% (2) Survival in MEM: 2.7% ratio: (l)/(2) = 19/2.7 = 7 15 therefore, mutant 1-12 survived treatment with 100 |ig/ml neomycin 7 times better when in the presence of MDCK cells than it did in MEM in this experiment. 2.9 P22 Transduction of Tn5-VB32 Mutants: The transposon insertions were moved into a wild type background to verify that the transposon was responsible for the observed phenotype. 100 u,L of OVN culture of each mutant was mixed with 100 |iL of phage P22 grown on wild type S.typhimurium and incubated at 23°C for 10 min (Schmeiger, 1972). The phage and mutants were then mixed with 0.4 % agar and poured onto LB agar plate and incubated overnight. 5 ml of phage buffer (Maniatis et al, 1989) was added to the plates and the top agar layer was removed and placed in a 15 ml Corex centrifuge tube. The preparations were then centrifuged at 10,000 rpm in a Beckman JA-17 rotor to pellet bacteria and debris. After centrifugation, the supernatant was removed, 100 uX of chloroform was added to kill remaining bacteria and aliquots were plated to ensure that there were no surviving bacteria. 100 U.L of wild type S. typhimurium (SL1344) was mixed with 100 uX of 10^ , 10_1 and 10"2 dilutions of the phage preps, incubated at 23°C for 10 min and then plated onto LB + Sm + Tc plates. Colonies which grew on these antibiotics should be SL1344 containing the transduced Tn5-VB32 and flanking sequences. Several colonies were selected for each mutant and measured for induction of neomycin resistance in the presence of MDCK cells as described above. 2.10 Motility Assay: 0.4 % LB agar in glass tubes was stabbed with colonies taken from fresh plates and incubated at 37°C overnight. Motility was assayed qualitatively by examining distance of bacterial growth from the original inoculation site.The motility of the mutants was compared to that of 5. typhimurium SL1344. 16 2.11 Lipopolysaccharide Agglutination: 20 U.L of Salmonella typhimurium antiserum O (Group B, factors 1,4,5, and 12) was placed on a glass plate and a fresh bacterial colony mixed into it. Agglutination of mutants was qualitatively compared with the parental strain. 2.12 Bacterial Adherence Assay: MDCK cells were grown overnight in 24 well plates as described, fixed with 2 % glutaraldehyde and then overlayed with MEM (Finlay and Falkow, 1989). 2 u\L of overnight bacterial culture was added to each of the wells and incubated at 37°C for 2 hours. The tissue culture fluid was then removed and the cells washed with PBS. The adherent bacteria were released with Triton X-100, suspended in L broth and titred. The overnight culture was also titred. Adherence was expressed as percent of initial inoculum remaining associated with the monolayer after washing with PBS. 2.13 Transepithelial Resistance Measurements: MDCK cells were grown on 3 pm filters as described above and the transepithelial resistance was measured after 5 days with a Millicell resistance meter. Resistance was typically 1650-2310 Qcm^. 2 u_L of overnight bacterial culture was added to the monolayers and they were incubated for 4 hours at 37°C. The resistance was measured again and the average percent decrease in resistance caused by each of the mutants and wild type was calculated. Three filters were measured for each strain. 2.14 Invasion Assay: 2 u\L of overnight bacterial cultures were added to MDCK cells grown overnight in 24 well plates and incubated for 2 hours. The MEM was then removed and replaced with MEM containing 100 u.g/ml gentamicin to kill extracellular bacteria (Vaudaux and Waldvogel, 1979) and the wells incubated for a further 2 hours. The MEM was removed, 17 the cells washed with PBS and then the bacteria released by addition of Triton X-100 followed by plating on selective media. Invasion levels were calculated by determining the ratio of the internalized bacteria to the initial inoculum and are expressed in percent. 2.15 Growth Rate in Minimal Medium: Bacteria were grown overnight in M9 minimal medium supplemented with 30 u,g/ml histidine. 50 uL of the overnight cultures was inoculated into 3 mis of fresh minimal medium and the cultures were grown rotating at 150 rpm at 37°C. The cultures all had a starting OD550' of 0.089 to 0.103. At 30 minute intervals, 50 u,L aliquots were removed and the optical density measured at 550 nm. The number of bacteria was calculated from the OD value using the conversion; 4 x 10^  bacteria/ml will give an OD550 of 1 (Berger and Kimmel, 1987). 2.16 Intracellular Growth Kinetics: 2 uT of O/N bacterial cultures were added to MDCK cell monolayers grown in 24 well plates and incubated for 2 hours. The medium was then replaced with MEM containing 100 u.g/ml gentamicin and plates were incubated for a further 2 hours. One set of wells was washed, lysed and the bacteria titred (time= t4) and the medium in the other wells was replaced with MEM containing 10 u,g/ml gentamicin and incubated further. The remaining wells were washed, lysed and titred after a further 4, 8, 12 and 20 hours. Triplicate values were obtained for each time point. 2.17 Induction of Neomycin Resistance in Matrigel and Agarose: Induction of neomycin resistance was measured in Matrigel and in agarose. Matrigel consists of extracellular matrix components (primarily collagen type IV, laminin, entactin and heparin sulfate proteoglycan) isolated from cultures of Engelbreth-Holm-Swarm mouse tumours (Collaborative Research, 1987). Matrigel was stored in aliquots at 18 -20°C which were thawed at 4°C to prevent premature gel formation and then diluted 1:1 in MEM. The Matrigel was placed in wells of a 96 well plate and incubated at 37° C for 1 hour to attain gel formation. 0.075% agarose in MEM was heated to boiling and then cooled to about 45°C before adding to a 96 well plate. 2 u.1 of overnight bacterial culture was added to wells containing agarose, Matrigel or MEM and the plates incubated (after gel formation). Neomycin was then added to half of the wells at a final concentration of 100 jig/ml and MEM was added to the other half such that the volumes were equivalent. The plates were then incubated for a further 4 hours and the bacteria titred (Matrigel and agarose were dilute enough to be pipetted). Percent survival was calculated for bacteria in wells with MEM, agarose and Matrigel. The ratios of survival in agarose or Matrigel to survival in MEM were calculated. Experiments were carried out in triplicate. 2.18 Preincubation of SL1344 in Matrigel: 10 uX of an overnight SL1344 culture was innoculated into 100 uX of MEM or 100 uX of 1:1 MEM: Matrigel and incubated for 3 hours. 30uX of these cultures was then added to 500 pX of MEM covering a monolayer of MDCK cells in a 24 well plate and incubated for 1 hour. The MEM and Matrigel cultures were titred. The medium was replaced with MEM containing 100 u.g/ml gentamicin and incubated for an additional 2 hours. Bacteria were released with 1% Triton X-100 and titred. Invasion levels were calculated as the % of innoculum internalized. The ratio of invasion of bacteria pre-incubated in Matrigel to bacteria pre-incubated in MEM was then calculated. ( 19 • Tn5 insertion sequences • Original Tn 5 sequences 13 NPTII gene from Tn5 S Tetracycline resistance genes from Tn 10 Figure 1: Schematic diagram of transposon Tn5-VB32. 20 Figure 2: Identification of Tn5-VB32 mutants in Salmonella typhimurium that are induced on MDCK cell surfaces. Sm; streptomycin sulfate, Tc;tetracycline, neo; neomycin sulfate. 21 Conjugate E. coli SM10, lambda pir with pBF-VB32 (Amp resistant) S. typhimurium SL1344 (Sm resistant) NB: pBF-VB32 is maintained in E. coli only. Tn5-VB32 must integrate into Salmonella genome in order to express tetracycline resistance. Select for bacteria resistant toSm andTc (ie SL1344:: Tn5-VB32 Select for Neo sensitivity in MEM (400 ug/ml, 6 hrs) Repeat once Neomycin sensitive mutants pooled. Identify clones which survive 800 ug/ml neo for 6 hours when incubated with fixed MDCK cells 22 3. Results 3.1 Identification of S. typhimurium Tn5-VB32 mutants induced in the presence of MDCK cells: Fifty transconjugants were selected from each of 120 matings between S. typhimurium and E. coli and tested for sensitivity to neomycin. Approximately 250 mutants (out of 6000 transconjugants) were identified that were sensitive to treatment with 400 u,g/ml neomycin. These mutants were pooled and screened for induction of neomycin resistance in the presence of MDCK cells. Bacteria that survived neomycin treament were individually re-screened for sensitivity to neomycin and induction of neomycin resistance. Eight individual mutants that displayed increased resistance to neomycin in the presence of MDCK cells were identified through this screening process. 3.2 Southern analysis of induced mutants: The mutants isolated from the above described screening were analysed by Southern blot to ensure that they contained Tn5-VB32 and that they represented different insertion sites. The 8 Tn5-VB32 mutants and SL1344 were digested with Clal, electrophoresed and analysed using a Southern blot probed with the NPTII gene (Figure 3). Wild type S. typhimurium (lane b) did not contain any DNA homologous to the NPTII probe (or the pKs vector). All of the mutants tested contained one or more Clal fragments which hybridized to the probe, indicating the presence of Tn5-VB32. The pattern of Clal fragments hybridizing to the probe fell into 4 classes. Mutants 84-17 (c), 2-35 (g), 2-17 (h), 1-24 (i) and 1-12 (j) all had Clal fragments of about 16.6 and 3.8 kb which hybridized to the probe. These mutants were also resistant to ampicillin, indicating that they contained a co-integrate consisting of two copies of Tn5-VB32 flanking the suicide plasmid. Mutant 84-1 (lane d), had three bands homologous to the probe, corresponding to 25.8 kb, 12.2 23 Figure 3: Southern analysis of the Tn5-VB32 mutants. Lane a; pKS::NPTII, b; SL1344, c; 84-17, d; 84-1, e; 20-10, f; 6-30, g; 2-35, h; 2-17, i; 1-24, j; 1-12. 24 kb, and 3.8 kb. Mutants 6-30 (g) and 20-10 (0 had single bands corresponding to about 24.7 and 6.2 kb respectively. Based on the results of this Southern analysis, mutants 1-12,6-30 and 20-10 were selected to represent three mutant classes. 3.3 Induction of Neomycin Resistance: The level of induction of neomycin resistance in the presence of MDCK cells compared to that in MEM alone was determined for the mutants as described in the materials and methods.Table 1 presents the ratio of bacterial survival in 100 u,g/ml neomycin in the presence of MDCK cells to survival without MDCK cells (calculations shown in Materials and Methods). The percent survival values obtained in one representative experiment are also presented. Mutant 1-12 survived neomycin treatment an average of 6 times better when in the presence of MDCK cells compared to survival in MEM. Mutant 6-30 survived an average of 23.7 times better and 20-10 an average of 51.7 times better in the presence of MDCK cells than without. Mulb (a constitutive NPT control) showed about 100% survival regardless of the presence of MDCK cells, resulting in a ratio of survival with MDCK to in MEM close to 1. Neos showed very low survival rates with or without MDCK cells, again resulting in a survival ratio close to 1 (0.7). Student t tests were performed on the percent survival ratios for each of the mutants in comparison to the survival ratio of the positive control, Mulb. The survival ratio obtained for 1-12 is significantly different than the ratio of Mulb at p=0.1, and 6-30 and 20-10 are significantly different than Mu 1 b at p=0.05. The experiment was repeated after the transposons (plus flanking DNA) had been transduced from the mutants into a clean SL1344 background. The survival ratios observed with the transductants were comparable to those obtained for the original mutants (Table 1). 3.4 Motility and LPS tests: 25 % survival of bacteria (representative experiment) ratio of survival with MDCK to survival in MEM Strain % survival with MDCK % survival in MEM original mutants P22 trans-ductants 1-12 19 2.7 6.0 +/- 2.7 5.1 6-30 7.3 0.25 23.7 +/- 8.2 13.8 20-10 4.4 0.09 51.7 +/-23.5 34 Mulb (NPT+) 95 93 1.0+/-0.1 -Neos (NPT-) 0.07 0.16 0.7 +/- 0.4 -Table 1: Survival of bacteria of 100 |lg/ml neomycin in the presence of MDCK cells compared to survival in MEM. The percent survivals obtained in one representative experiment are shown. The average percent survival ratios from three experiments consisting of three trials each are also presented as are survival ratios of P22 transductants of the original mutants. 26 Motility and LPS components play a significant role in Salmonella infection, primarily at the adherence step (Finlay et al, 1988, Jones et al, 1981). A motility test was performed on each of the mutants and the results compared qualitatively to motility of the parental strain. All three mutants had similar motility compared to the parental strain. In addition, all of the mutants agglutinated when exposed to Salmonella O antiserum, indicating that they probably contain intact LPS. 3.5 Adherence of Induced Mutants: Bacterial adherence to mammalian cells is one of the first steps in a Salmonella infection. An adherence assay was carried out with the mutants to determine if adherence was affected by the transposon insertion. Approximately 5 x 10^  bacteria were added to a fixed MDCK cell monolayer. After 2 hours incubation, 6.2% of the initial inoculum of SL1344 adhered to the MDCK cells (Figure 4). Under the same conditions, 4.6% of 1-12, 6% of 6-30 and 5.6% of 20-10 adhered. Statistical analysis (student t tests) of the data indicated that adherence to MDCK cells was not significantly affected in the mutants. 3.6 Effects of Induced Mutants on Transepithelial Resistance: A polarized monolayer of MDCK cells infected with S. typhimurium loses its transepithelial resistance after 4 hours of infection (Finlay et al, 1988). The effects of SL1344 and the Tn5-VB32 mutants on the transepithelial resistance of infected polarized monolayers of MDCK cells was examined (Figure 5). Filter grown MDCK cell monolayers retained an average of 17.3% of their original transepithelial resistance 4 hours after infection with SL1344. Monolayers infected with 1-12 retained 25% of their original resistance, 17.7% with 6-30 and 21.3% with 20-10. According to the results of a t test analysis, there is no difference between the effects of infection with 1-12, 6-30, 20-10 and 27 SL1344 on the transepithelial resistance across an MDCK cell monolayer. The average 10 Stra ins Figure 4: Adherence of bacteria to fixed MDCK cells. Values shown are the averages three seperate experiments with two or three trials per experiment. 28 30 H SL1344 1-12 6-30 20-10 Strai ns Figure 5: Transepithelial resistance of polarized MDCK cell monolayers infected with S. typhimurium and Tn5-VB32 induced mutants.Values are the average of three filters. 29 transepithelial resistance remaining across the monolayers infected with 1-12 was higher than those infected with SL1344 and the other mutants, but there was considerable variation between values. Therefore, the difference in effects on transepithelial resistance between 1-12 and SL1344 was not significant after t test analysis. 3.7 Invasive Capability of Induced Mutants: The proportion of bacteria added to an MDCK cell monolayer that were able to invade was determined for SL1344 and the three mutants (Figure 6). 6.6 % of SL1344 were able to invade MDCK cells after 2 hours of infection, 2.3% of 1-12,0.31% of 6-30 and 0.12% of 20-10. All of the mutants were affected in their ability to invade, showing significantly lower invasion levels (oc=0.05) than the parental strain (SL1344). 3.8 Growth Curve in Minimal Medium: 5. typhimurium strains that are auxotrophs may be deficient in intracellular replication (Hoiseth and Stocker, 1981, Leung and Finlay, 1991). The growth kinetics of SL1344, 1-12, 6-30 and 20-10 in minimal medium were examined to determine if any of the mutants are auxotrophs (Figure 7). The growth kinetics of all three mutants were essentially identical to SL1344. 3.9 Intracellular Growth Kinetics of Induced Mutants: S. typhimurium is capable of multiplying inside culture cells. Intracellular replication rates of the induced mutants and SL1344 inside MDCK cells were determined (Figure 8). All strains had a long initial lag phase of 12 hours before they began replicating proficiently. Mutant 20-10 replicated somewhat more slowly than SL1344, culminating in only about half the number of intracellular bacteria after 24 hours. 30 1 0 S L 1 3 4 4 1 - 1 2 6 - 3 0 2 0 - 1 0 Stra ins Figure 6: Log of percent invasion of MDCK cells by Salmonella typhimurium. Values shown are the averages of three separate experiments with two or three samples per experiment. 31 -CD SL1344 -• 1-12 -fl 6-30 -0 20-10 Time (hrs) Figure 7: Growth kinetics of Salmonella typhimurium and the induced mutants in rotating cultures (150 rpm) of minimal medium (plus histidine) at 37°C. 32 8 —r— 1 0 —i— 20 30 time (hrs) Figure 8: Growth kinetics of Salmonella typhimurium and induced mutants inside MDCK cells. 33 3.10 Effects of Incubation in Matrigel and Agarose on Induction of Neomycin Resistance: Several species of bacteria bind to extracellular matrix components as a means of facilitating entry into mammalian cells (Isberg, 1991, Ruoslahti and Piersbacher, 1987, Winkler et al, 1987). An experiment was carried out in order to determine if the presence of extracellular matrix components alone was sufficient to cause induction of the promoters controlling NPTII in the mutants (Table 2). Extracellular matrix components are available from Collaborative Research under the name Matrigel. Matrigel contains primarily entactin, collagen type IV, heparin sulfate proteoglycan and laminin isolated from cultures of Engelbreth-Holm-Swarm mouse tumour cells. Agarose was included in this experiment in an attempt to duplicate the environmental conditions created by the Matrigel (providing solid support) but without the specific components. The survival ratios of the mutants were compared to the positive control (Mulb) using student t tests at oc=0.05. Mutant 1-12 and 6-30 showed significantly higher survival levels in Matrigel than they did in MEM (5.8 and 2.1 times better respectively). Mutant 20-10 did not show significantly enhanced survival in Matrigel, resulting in an average survival ratio close to 1 (1.4). Mulb and Neos survived at similar levels in Matrigel and MEM, also resulting in survival ratios close to 1 (0.8 and 1.1). 1-12 also survived significantly better in agarose than it did in MEM, resulting in a average survival ratio of 5.8. The three experiments which measured induction of neomycin resistance of 6-30 in agarose displayed considerable variation with regards to the survival ratios. For this reason, t test analysis indicated that the survival ratio of this mutant was not significantly different than that of the positive control even though the average survival ratio of agarose to MEM was 5.2. Mutant 20-10 did not survive any better in agarose than it did in MEM, resulting in an average survival ratio of 0.8. Mulb and Neos again displayed survival ratios near 1 (1.1 and 1.1). t 34 Strain ratio of % survival in Matrigel to % survival in MEM Mean =/- SD ratio of % survival in Agarose to % survival in MEM Mean =/- SD 1-12 6.9 4.8 5.8 +/- 2 4.3 5.8 =/- 1.4 4.6 6.3 8.1 6-30 2 3.8 2.4 2.1 +/- 0.3 2.7 5.2 +/- 3.5 1.8 9.2 20-10 1.28 0.75 0.8 +/- 0.4 2.2 1.4+/-0.7 0.36 0.92 1.23 Mulb 1.25 1.1 1.1 +/-0.4 0.32 0.8 +/- 0.5 0.6 0.77 1.45 Neos 0.46 0.62 1.1 +/-0.6 1.2 1.1 +/- 0.6 1.8 1.7 0.82 Table 2: Survival of bacteria in 100 |ig/ml neomycin in the presence of Matrigel and agarose compared to survival in MEM. Numbers shown represent three separate experiments consisting of two trials each. 35 3.11 Effects of Pre-incubation in Matrigel on S. typhimurium Invasion: Pre-incubation of some species of E. coli with fibronectin enhances the adherence of the bacteria to mammalian cells and may also facilitate entry (Isberg, 1991). An experiment was carried out to determine if pre-incubation of Salmonella in Matrigel would enhance invasiveness (Table 3). SL1344 that were pre-incubated in Matrigel invaded MDCK cells an average of 6.4 times better than bacteria pre-incubated in MEM in a 1 hour invasion assay. 36 ratio of: Experiment # invasion after preincubion in Matrigel to invasion after preincubion in MEM 1 4 2 10 3 5.4 Table 3: Effects of pre-incubation in Matrigel on invasion of Salmonella typhimurium SL1344 into MDCK cells. Results of three separate experiments with two trials per experiment are presented. 37 4. Discussion I have used transposon mutagenesis to identify Salmonella typhimurium genes which are induced when the bacteria come into contact with epithelial cells. Salmonella produce several new proteins when they encounter mammalian cells, but the nature of these proteins and their function in pathogenesis is unknown (Finlay et al, 1989). Since epithelial cells do not normally ingest particles as large as a bacterium, it is thought that the proteins produced by the bacteria may act as signals, inducing the epithelial cells to phagocytose them. The induced genes were identified using transposon mutagenesis with Tn5-VB32 to create transcriptional fusions in genes which were induced when the bacteria encountered epithelial cells. Insertion of Tn5-VB32 downstream of an active bacterial promoter results in expression of the NPTII gene, rendering the bacteria more resistant to neomycin sulfate than the parent strain (Bellofatto et al, 1984). The transposon did not insert randomly, since about 75% of the transconjugants were resistant to neomycin, indicating that they had inserted downstream of an active promoter. If the insertion event was truly random, many transposons would have inserted in an orientation, or site that did not allow for expression of the NPT gene. Bacteria that contained transcriptional fusions in induced genes became more resistant to neomycin when in the presence of epithelial cells. Eight Tn5-VB32 mutants in Salmonella typhimurium SL1344 were identified that appeared to be induced for neomycin resistance in the presence of epithelial cells. These mutants were examined by Southern analysis and found to represent 4 different Tn5-VB32 insertion patterns (Figure 3). One of the mutants contained two insertions, 5 contained a co-integrate of the suicide pasmid (used to introduce the transposon) and Tn5-VB32 and the other two contained unique, single transposon insertions. Three mutants were selected for further study, one of the co-38 integrates and the two unique insertions. These three mutants showed significantly higher survival in neomycin when they were in the presence of fixed epithelial cells than in tissue culture fluid (Table 1). It is important to note that the numerical ratios assigned to induction levels in these experiments do not necessarily correlate directly with gene expression. Survival of bacteria in increasing antibiotic concentration follows a sigmoid curve (Bryan, 1984). If the antibiotic concentration chosen for testing lies in the linear part of the percent survival curve, then the differences in survival between induced and uninduced bacteria will be greater than if a concentration in one of the flat parts of the curve is chosen. In order to get an accurate picture of the level of induction occurring, survival of the bacteria at various antibiotic concentrations in the presence and absence of mammalian cells could be assayed. Levels of antibiotic resistance are affected by a number of environmental conditions such as oxygen levels, pH and nutrients (Bryan, 1984, Phillips and Shannon, 1984). Bacteria can display "phenotypic resistance" to antibiotics under certain conditions. The inclusion of the uninduced Tn5-VB32 control (Neos) in the induction experiments indicates that factors such as pH and oxygen levels are not responsible for the increased resistance to neomycin observed in the mutants exposed to epithelial cells. Pathogenesis of Salmonella involves several steps including adherence, invasion and replication inside epithelial cells which can be duplicated in tissue culture cells. The Tn5-VB32 mutants were analysed to determine if any of the steps in pathogenesis had been affected by the insertion of the transposon. LPS components and motility contribute to bacterial adherence to epithelial cells and consequently also to invasion. All three of the mutants appear to contain complete LPS and functional motility. Some rough mutants of Salmonella do agglutinate LPS, but most do not. In addition all three mutants adhered to the epithelial cells at levels similar to the wild type (Figure 4). This result was expected due to the nature of the screening procedure. The bacteria were allowed to interact with the epithelial cells for three hours at which time the 39 medium was removed and replaced with medium containing neomycin. At this step, any bacteria that were deficient in adherence would have been removed with the medium. The next step in pathogenesis involves invasion of mammalian cells (Finlay and Falkow, 1988). Invasion levels in vitro are sometimes correlated to virulence in mice (Finlay et al, 1988). Invasion of mutant 1-12 into epithelial cells was reduced by about 35% in comparison to the wild type parent. Mutants 6-30 and 20-10 invaded at only 5% and 2% of wild type levels. Clearly, the insertion of the transposon in induced genes also affected genes involved in internalization in these three cases. A polarized monolayer of epithelial cells in vitro will lose its transepithelial resistance when infected with Salmonella typhimurium. Infection with the transposon mutants also caused a reduction in the transepithelial resistance of a monolayer. Another factor of Salmonella pathogenesis is the ability of the bacteria to survive and replicate inside epithelial cells. Mutants 1-12 and 6-30 appeared to replicate at levels similar to wild type when inside MDCK cells. However, mutant 20-10 replicated more slowly than the wild type strain culminating in 50% fewer bacteria after 24 hours. Auxotrophic bacteria are often unable to replicate inside mammalian cells (Leung and Leung, 1991). The Tn5-VB32 mutants duplicate wild type growth kinetics in minimal medium, indicating that they are not auxotrophs. From my results, it can be concluded that some of the Salmonella genes induced upon contact with epithelial cells are also involved with other aspects of pathogenesis. The most profoundly affected mutant was 20-10. This mutant was deficient in its effects on transepithelial resistance across an epithelial cell monolayer, invasion and intracellular replication in epithelial cells. The gene affected in 20-10 may be a regulatory gene that is responsible for activating genes required later on for invasion and intracellular replication. Recent reports in the literature discuss the possible role of extracellular matrix components in bacterial pathogenesis (Isberg, 1991, Winkler et al, 1987, Ruoslahti and Piersbacher, 1987). Some bacterial pathogens coat themselves in extracellular matrix 40 components as a means of attaching to and entering mammalian cells (Isberg, 1991). It has not been determined if Salmonella species interact with the extracellular matrix. I performed an experiment to determine if the genes affected in the Tn5-VB32 mutants were induced in the presence of extracellular matrix components. The mutants were incubated in Matrigel (purified extracellular matrix components) and agarose and assayed for survival in neomycin. Mutant 1-12 exhibited enhanced survival in both Matrigel and agarose. Thus the gene affected in 1-12 appears to be induced by the presence of a solid support. Mutant 6-30 was induced slightly in the presence of Matrigel. 6-30 also appeared to be induced in the presence of agarose, but since there was considerable variation between the three experiments performed, the level of induction was not statistically significant in a student t test. Mutant 20-10 was not induced in agarose or Matrigel; therefore the gene affected in this mutant is not induced merely by the presence of a solid support. To determine if the induction of these solid support genes actually played a role in invasion of wild type bacteria, I pre-incubated wild type Salmonella (SL1344) in Matrigel prior to an invasion assay. Invasion of the bacteria into epithelial cells increased an average of 6.5 times by pre-incubation in Matrigel (as opposed to tissue culture fluid). This result suggests that genes induced by the presence of a solid support may be involved in invasion. Electron microscopic studies performed by Finlay and Falkow (1989) showed that Salmonella produce "hair-like" structures linking the bacteria to each other when bound to epithelial cells. Structures such as these could be induced in response to any solid support as a means of strengthening attachment. Further studies with these mutants could include two dimensional electrophoresis to ascertain which, if any, of the induced proteins are no longer produced. If the gene affected in 20-10 is a regulatory gene, several induced proteins may be absent. Electron microscopy studies could be performed to determine if the "hair-like" structures normally seen on adherent Salmonella are still produced by the mutants when bound to epithelial cells. 41 Cloning and sequencing of the genes affected in the mutants would give insight into the function of the gene products and their role in pathogenesis. Analysis of the sequence may allow us to classify the predicted protein as one which is located in the membrane, cytoplasm, or if it interacts with DNA directly. The precise localization of the gene products in the bacteria could be determined by using antibodies to immune-precipitate the proteins from various cellular fractions. If the gene products proved to be membrane proteins, experiments could be carried out to determine if these proteins interact with anything on the surface of the epithelial cells. Antibodies could be made by over-expressing the protein in E. coli (by placing the gene under the control of a strong promoter), purifying it and injecting it into rabbits. The genes could also could be cloned into non- invasiveZT. coli to determine if their expression is sufficient to confer invasiveness. In conclusion, three Salmonella genes were identified which were induced when the bacteria came into contact with epithelial cells. Bacteria with mutations in these genes were defective in invasion. One of the mutants was also defective in its effects on transepithelial resistance across an epithelial cell monolayer and intracellular replication. Two of the three genes identified appeared to be induced by the presence of a solid support matrix. Pre-incubation of wild type Salmonella with the solid support matrix enhanced invasion, indicating that these genes might play a role in pathogenesis. 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