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The utilization of Caenorhabditis briggsae as a host model to study bacterial pathogenesis Price, Nancy Lee 2002

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THE UTILIZATION OF CAENORHABDITIS BRIGGSAE AS A HOST M O D E L TO STUDY B A C T E R I A L PATHOGENESIS by N A N C Y LEE PRICE B. Sc. Hons. Biochemistry, University of Regina, 2000 B. Sc. Biology, University of Regina, 2000 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Genetics Graduate Program We accept this thesis as conforming to the required standards THE UNIVERSITY OF BRITISH C O L U M B I A November, 2002 © Nancy Lee Price, 2002 Thesis Authorization Form In presenting this thesis "The utilization of Caenorhabditis briggsae as a host model to study bacterial pathogenesis" in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I, Nancy Lee Price, 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. Genetics Graduate Program University of British Columbia Vancouver, Canada November 19tn, 2002 ABSTRACT Caenorhabditis elegans has now been established as a host model for studies of infectivity by bacterial pathogens including Pseudomonas aeruginosa, Salmonella typhimurium, Burkholderia pseudomallei, and Enterococcus faecalis. However, virulence determinants of bacterial pathogens are regulated by temperature and environmental conditions, thereby limiting the use of C. elegans which cannot survive in temperatures higher than 25°C. This study shows that the related species, C. briggsae survives better than C. elegans on bacterial culture media at higher temperatures and describes the effects on C. briggsae of mammalian enteric pathogens, primarily Yersinia enterocolitica. C. briggsae grown on Y. enterocolitica accumulated bacteria in the gastrointestinal tract of the worm, resulting in decreased life-span and progeny fitness in a depleted-calcium environment. The mechanisms of the interaction are as yet unknown as both virulent and avirulent strains of Y. enterocolitica displayed the similar results. Investigations were undertaken to examine whether the shortened life span could be attributed to starvation, toxicity, or possible infection. Starvation effects was determined not to be the sole cause of pre-mature worm death as C. briggsae grown on Y. enterocolitica survived several days longer than starved worms. A bacterial mixing experiment using both Y. enterocolitica and E. coli OP50 shortened the worm life span compared to feeding on Y. enterocolitica alone, suggesting a possible toxic effect. However worms feeding on Y. enterocolitica and later shifted to E. coli OP50 resulted in reversion of the survival curve to that of worms feeding on E. coli OP50 alone, indicating that the detrimental effect of Y. enterocolitica was reversible. Fluorescent labeling of bacterial strains with a /acZ-GFP reporter gene demonstrated that Y. enterocolitica, but not E. coli OP50, is retained in the gut, a result which is compatible with a molecular interaction between Y. enterocolitica and nematode cellular components. These findings suggest that Y. enterocolitica may cause an infection within the nematode gastrointestinal tract and provide an assay for genetic dissection of the molecular basis of pathogenesis. ii T A B L E O F C O N T E N T S A B S T R A C T i T A B L E O F C O N T E N T S i i i L I S T O F T A B L E S vi i i L I S T O F F I G U R E S ix D E D I C A T I O N xi A C K N O W L E D G E M E N T S xi i C H A P T E R I 1.0 I N T R O D U C T I O N 1 1.1 C. elegans as a Genetic Model Organism 3 1.1.1 C. briggsae: A Close Relative of C. elegans 4 1.2 C. elegans as a Model Host for Pathogenesis 5 1.3 Established Infections in C. elegans 6 1.3.1 Visualized Infections in C. elegans 6 1.3.2 Infection or Toxicity Models? 9 1.3.3 C. elegans Pathogenesis Model with Mammalian Pathogens 14 1.4 Host Defense Strategies in C. elegans 16 1.4.1 The Innate Immune System 17 1.4.2 Antimicrobial Agents 18 1.5 Yersinia spp. and Mechanisms of Pathogenesis 19 1.5.1 Yersinosis in British Columbia 21 1.6 Scope and Nature of This Work 23 iii CHAPTER II 2.0 METHODS AND MATERIALS 24 2.1 Organism Strains and Plasmids 24 2.1.1 Nematode Strains 24 2.1.2 Bacterial Strains 24 2.1.3 Plasmids 24 2.2 Media and Growth Conditions 24 2.2.1 Nematode Media and Growth Conditions 24 2.2.2 Bacteria Media and Growth Conditions 29 2.3 Organism Frozen Stocks 29 2.3.1 Caenorhabditis Frozen Stocks 29 2.3.2 Bacterial Frozen Stocks 30 2.4 Microscopy 30 2.4.1 Light Microscopy 30 2.4.2 Fluorescent Microscopy 30 2.4.2.1 Fixing and Mounting Procedure 30 2.5 Molecular Genetic Techniques 31 2.5.1 Plasmid Construction 31 2.5.2 Transformation 32 2.5.2.1 Preparation of Electro-Competent Cells 32 2.5.2.2 Transformation Methods 32 2.5.3 Caenorhabditis Lysis for PCR 32 2.5.4 Polymerase Chain Reaction 33 iv 2.6 Hypochlorite Extraction of Caenorhabditis Embryos 33 2.7 Thermotolerant Testing 34 2.8 Caenorhabditis - Bacterial Assay 35 2.8.1 C. elegans - Bacterial Assay 35 2.8.2 C. briggsae - Bacterial Assay 35 2.8.3 C. briggsae - Bacterial Assay Using E G T A 36 2.9 Creation of the Virulence Gene Subset Database 36 2.9.1 Analysis for C. elegans Homologs 37 CHAPTER III 3.0 RESULTS 38 3.1 Assessment of C. elegans as a Host for Bacterial Enteric Pathogens 38 3.2 C. briggsae as a Host for Bacterial Enteric Pathogens 39 3.2.1 C. briggsae Survives in Bacterial Incubation Environment '. .39 3.2.2 Growth of C. briggsae on Bacterial Enteric Pathogens 43 3.2.3 Growth of C. briggsae on Y. enterocolitica in Ca2+-Depleted Environment 46 3.3 Characterization of C. briggsae - Y. enterocolitica Pathogenesis Model 48 3.3.1 Effect on C. briggsae Progeny Fitness 48 3.3.2 Effect of Starvation 48 3.3.3 Effect of E G T A Concentration 50 3.3.4 Effect of Virulent vs. Avirulent Y. enterocolitica Strains 54 3.3.5 Visualization of Y. enterocolitica in C. briggsae 56 3.3.6 Visualization of the Effect of E G T A on Bacterial Retention in C. briggsae 62 3.3.7 Shifting of C. briggsae from Y. enterocolitica to E. coli OP50 67 3.3.7.1 Visualization of Shifting C. briggsae from Y. enterocolitica to E. coli 69 3.3.8 C. briggsae on Mixed Bacterial Lawns of Y. enterocolitica and E. coli 69 3.3.8.1 Visualization of Y. enterocolitica - E. coli mix in C. briggsae 73 3.4 Re-examination of the Effect of C. briggsae on S. typhimurium 78 3.4.1 Visualization of S. typhimurium in C. briggsae 78 3.5 Virulence Gene Subset Database 82 3.5.1 Compilation of VGS Data 82 3.5.2 B L A S T Analysis and Identification of Putative Protein Homologs 84 C H A P T E R IV 4.0 DISCUSSION 86 4.1 Re-Examining C. elegans as a Host Model 86 4.2 General Assessment of Mammalian Infection Environment 88 4.2.1 Relevance of Nematode and Bacteria Media 88 4.2.2 Relevance of the Incubation Temperature 90 4.2.3 Utilization of Thermotolerant Caenorhabditis Strains 91 4.3 C. briggsae as a Host Model for Mammalian Enteric Pathogens 92 4.4 C. briggsae - Y. enterocolitica Infection Assay in a Low Ca Environment 93 4.4.1 The Role of the Yersinia Virulence Plasmid 95 4.5 Visualization of Y. enterocolitica in C. briggsae 97 4.5.1 Calcium Role in the Retention of Y. enterocolitica in C. briggsae 99 4.6 Persistence of Y. enterocolitica has a Deleterious Effect on C. briggsae 99 vi 4.7 Possible Mechanisms of C. briggsae Killing 101 4.7.1 The Possible Protective Role of Calcium 101 4.7.2 Implications of the VGS Database 102 4.8 Conclusions 102 CHAPTER V 5.0 FUTURE WORK 104 5.1 Characterization of Genes Involved with Worm Defense System 104 5.2 A GFP Genetic Screen 104 LITERATURE CITED 105 APPENDIX A 116 vii LIST OF TABLES Table 1. Reported infection models between C. elegans and various pathogens 7 Table 2. List of Caenorhabditis strains utilized 25 Table 3. List of laboratory bacterial strains employed 26 Table 4. List of mobile vectors and plasmid construct utilized 28 Table 5. Results of thermotolerant testing with Caenorhabditis species 41 Table 6. C. briggsae progeny yield with E. coli or Y. enterocolitica as a food source ...49 Table 7. A selection of data entries from the VGS database 83 Table 8. Summarized selection of high C. elegans - pathogen homology hits 85 viii LIST OF F I G U R E S Figure 1. Recorded rate of various notifiable diseases by year, 1988 - 2001 22 Figure 2. Determination of Caenorhabditis fatality rate on N G M plates at 25°C and 30°C temperatures 42 Figure 3. Determination of Caenorhabditis fatality rate on BHI plates at 25°C and 30°C temperatures 44 Figure 4. Infection assay results of the incidence of C. briggsae death with the Y. enterocolitica in the presence of E G T A (5mM) in BHI culture media 47 Figure 5. Infection assay results investigating possible signs of C. briggsae starvation when supplied with a foreign food source 51 Figure 6. Infection assay results investigating the effect of E G T A concentration (0 m M to 20 mM) on the incidence of C. briggsae death after exposure to E. coli OP50 as a food source 52 Figure 7. Infection assay results investigating the effect of E G T A concentration (0 m M to 20 mM) on the incidence of C. briggsae death after exposure to Y. enterocolitica as a food source 53 Figure 8. Infection assay results investigating the effect Y. enterocolitica strains of serotype 0:8 as a food source for C. briggsae 55 Figure 9. Visualization of Y. enterocolitica-GF? WA-314, W A - C , and E40 strains in C. briggsae after 24 h of exposure 57 Figure 10. Whole worm and head visualization of Y. enterocolitica-GF? WA-314, W A - C , and E40 strains in C briggsae after 48 h of exposure 59 Figure 11. Whole worm visualization of GFP expressing bacteria strains grown in the presence and absence of 5 m M E G T A 63 Figure 12. Infection assay results investigating the effect of shifting C. briggsae from Y. enterocolitica strains to E. coli OP50 strain as food source 68 Figure 13. Whole worm visualization from bacterial shifting experiments 70 Figure 14. Infection assay results investigating the effect of presenting C. briggsae with a mixture of E. coli and Yersinia strains together as a food source 72 ix LIST O F F I G U R E S (cont'd) Figure 15. Whole worm visualization of Y. enterocolitica-GF? strains mixed with E. coli OP50 after 24 h and 48 h of exposure 74 Figure 16. Infection assay results using S. typhimurium SL1344 strain grown in BHI and BHI E G T A 5 m M liquid and solid media 79 Figure 17. Whole worm visualization of S. typhimurium-GYV SL1344 strain grown in the presence and absence of E G T A 5mM 80 x D E D I C A T I O N For my Granny, an incredible soul who truly taught me to never give up. xi A C K N O W L E D G E M E N T S To my supervisor Dr. Ann Rose, who believed in this project and in me from the very beginning. Thank you for your guidance, your confidence, and encouraging smiles. I am indebted to my thesis committee (Dr. Fiona Brinkman, Dr. Brett Finlay, and Dr. Steven Jones) and all past and present members of the Pathogenomics Network for their continuous support, fruitful discussions, and helpful hints. To the Rose lab rats, Risa, Lois, Kim, and many more, thank you for embracing a bug geek in a worm lab. Special thanks to two Finlay lab rats, Carrie and Armick, for helping out a bug geek in a worm lab. To my greatest fans: Mom and Grandma, thank you for enduring another degree with me and for being the tender wind that carries me. Whether I'm near or far away, your endless love and support are always felt close to my heart. Sherry, thank you for being my closest colleague and my best friend (you're awesome!). M y world is a better place because of you. The Iatridis', thank you for all the encouragement, hugs, and visits. You make me realize that I am the luckiest person on earth (evhairisto for the soup!). To my Shepherd, thank you for watching over me and giving me the strength to see this journey to the end (Galatians 6:9). This project was funded in part by the Peter Wall Institute for Advanced Studies at the University of British Columbia, Vancouver, B C , Canada. xii C H A P T E R I 1.0 I N T R O D U C T I O N Despite advances in the technological and medical fields of science, public health is still challenged by pathogenic microorganisms. The onslaught of outbreaks from contaminated food and water supplies, as well as nosocomial infections has posed an escalating health concern in the public, advocating the need to find solutions. The first arsenal of defense to combat outbreaks of infection was focused solely on targeting the pathogen using antibiotics which resulted in several decades of improved health and well-being of countless individuals. Although antibiotics are still used today to combat microbial infections, the overall success of antibiotics has been hampered as microorganisms demonstrated their remarkable ability to adapt to their environment and developed mechanisms to reduce or avoid the action of antibiotics (Gold and Moellering, 1996). As a result, the continued overuse and misuse of antibiotics have forced the emergence of multi-drug resistant bacterial strains, leaving researchers and health officials scrambling to develop novel methods to combat microorganisms. To aid in the fight and to better understand the pathogenesis process, the research approaches utilized to identify bacterial virulence factors have increasingly become genome-orientated. Current methods utilize newly developed tools from the emerging fields of genomics, biotechnology, and bioinformatics to examine and compare genomes between host and pathogens (Rosamond and Allsop, 2000; Weinstock, 2000). Several of these techniques include D N A microarrays, genome 1 sequence comparison, mutagenesis analysis, and development of software and databases that identify new virulence determinants, anti-microbial drug targets, or key biological processes that are shared between pathogen and host that could be exploited during pathogenesis (Strauss and Falkow, 1997; Cummings and Relman, 2000; Kotra et al, 2000; Rosamond and Allsop, 2000; Diehn and Relman, 2001). In addition, the genomic and bioinformatic approaches to study host-pathogen interaction have led to the development of several new research groups such as the pathogenomics research group. The pathogenomics project (http://www.pathogenomics.bc.ca/) utilizes a combination of informatics, evolutionary biology, microbiology, and eukaryotic genetics to identify pathogen genes that are more similar to host genes than expected, and likely to interact with, or mimic, the host's gene functions. Currently the project has been divided into two complementary fields of bioinformatics and laboratory-based functional analysis. Within the informatic analysis, software has been developed that aids in the identification of genes with unusually high similarity between pathogens and hosts with the hypothesis that this approach may enable researchers to identify new potential virulence factors. The goals achieved from the informatic analysis need to be tested using a genetically tractable model. It was proposed that Caenorhabditis elegans could be used as a host model for the functional analysis of candidate virulence factors identified by the informatics approach and potentially provide new insight into the mechanisms of microbial pathogenesis. 1.1 C. elegans as a Genetic Model Organism The use of C. elegans has proven to be a valuable laboratory model organism and has provided valuable scientific insight into numerous biological questions. C. elegans is a free-living, non-parasitic nematode. C. elegans exists as either a self-fertilizing hermaphrodite or a male which arises from non-disjunction events of the sex chromosome at a frequency of 0.2 %. Since hermaphrodites cannot fertilize each other, the existence of the two sexes enables cross-breeding between strains. The developmental cycle of C. elegans involves four larva stages (LI, L2, L3, and L4) before reaching a mature adult. If the worm encounters unfavorable environmental conditions (e.g., limited food supply) during the L2/L3 development stage, C. elegans can enter the dauer larva stage, a specialized L3 stage in which the worm does not feed, is resistant to desiccation, and can survive for up to 3 months (Wood, 1988). Once environmental conditions become favourable, C. elegans matures to L4 larva and continues on with its development. C. elegans offers numerous experimental advantages for genetic analysis including its small size, rapid development time, large brood size (one hermaphrodite worm can produce 250 - 300 progeny), completed cell lineage maps, inexpensive and ease of maintenance (both short- and long-term), and transparent morphology. From C. elegans completed sequenced genome in 1998 (C. elegans Sequencing Consortium, 1998), researchers are able obtain any cosmid or Y A C sequenced by the C. elegans Genome Sequencing Consortium, as well as open access to C. elegans mutant strains from the Caenorhabditis Genetics Center (CGC, University of 3 Minnesota) and C. elegans Gene Knock-Out Consortium (http://elegans.bcgsc.bc.ca/knockout.shtml). Other research advantages using C. elegans include the availability of various newly developed molecular data collections such as gene expression profile maps obtained from D N A microarrays (Kim et al, 2001), databanks of expressed sequence tags (ESTs) from C. elegans (Reboul et al, 2001), and R N A interference (RNAi) libraries (Ahringer lab, U K H G M P Resource Centre). The large availability of well-developed genetic and molecular tools combined with comprehensive genetic maps allows for the easy genetic manipulation and position cloning within C. elegans unlike any other genetic model. 1.1.1 C. briggsae: A Close Relative of C. elegans In addition to the achievement of the sequenced C. elegans genome in 1998 (C. elegans Sequencing Consortium, 1998), researchers have nearly completed the genome sequence of another Caenorhabditis species, C. briggsae (http://www.sanger.ac.uk/Projects/C_briggsae/). Sharing the same genus, C. elegans and C. briggsae are closely related nematodes which are estimated to have diverged 2 5 - 120 million years ago (Kent and Zahler, 2000; Coghlan and Wolfe, 2002). Preliminary sequence analyses have revealed that sequences encoding proteins are highly conserved (~ 55 % to 72 %) between the two species (Shabalina and Kondrashov, 1999; Kent and Zahler, 2000). Morphologically, C. briggsae is almost indistinguishable from C. elegans with relative similar size, life-cycle, and life-span. In addition, the experimental advantages including the genetic techniques and molecular tools utilized with C. elegans are equally applicable to C. briggsae. 4 However, as nematode research groups have extensively focused on the C. elegans model, only a handful of papers have been published within the last four decades regarding research on C. briggsae and therefore specific biological information of C. briggsae remains limited. 1.2 C. elegans as a Model Host to Study Pathogenesis Although great strides have been accomplished within the molecular biology and microbiology fields regarding the identification and characterization of pathogenic mechanisms within a vast array bacterial species, the molecular and genetic analysis of host-pathogen interactions has been constrained due to the usage current host models. The limited complexity of tissue cultured cells hampers the analysis of pathogen interaction in a multi-cellular host and the utilization of live mammals brings forward concerns of associated high costs, ethical issues, and extensive laborious maintenance. A feasible alternative is to establish a pathogenesis model utilizing a highly organized yet simple host organism such as C. elegans. The previously described advantages of C. elegans as a genetic model are equally amenable for the usage of C. elegans as a host model in elucidating host genetics during pathogenesis. Although bacterial host range may challenge the utilization of C. elegans to study mammalian bacterial pathogens (Ewbank, 2002), protein and genome comparisons have demonstrated that various microorganisms utilize common virulence factors and mechanisms that enables pathogens to invade and infect a wide range of hosts (reviewed by Finlay and Falkow, 1997). Additional issues with using C. elegans as a 5 host model for mammalian bacterial pathogens surround concerns of proper incubation temperature and nutritional content of the media; however, the issues with the infection environment can be addressed by including these factors into the experimental design. Furthermore, within the past few years, established infection models have been developed using C. elegans and several pathogens (Table 1). The potential gains of utilizing C. elegans in a pathogenesis model include the identification of host genes involved in pathogenesis and the elucidation of the biological mechanisms C. elegans utilizes in defense against pathogens. 1.3 Established Infections in C. elegans In the past few years several infection models involving C. elegans have been reported within the literature and continue to be the subject of active research. Below contains a summary of the established pathogenesis models. 1.3.1 Visualized Infections in C. elegans An ideal approach to studying host genetics in pathogenesis is establishing an infection model that results in a defined and well-characterized infection phenotype which can be easily visualized within the worm. Visual phenotypes such as abnormal tissue growths, swollen cellular masses, or premature death are often hallmark signs of infection and can be quantified to determine the presence of an infection within the worm. In addition, during a genetic screen a visible infection phenotype could be easily utilized to determine mutants that are either resistant or more susceptible to pathogens. However, only a few pathogens have been reported to cause a visual infection phenotype in C. elegans. 6 Table 1. Reported infection models between C. elegans and various pathogens. Pathogen Type Pathogen Reference(s) Gram-negative Agrobacterium tumefaciens Aeromonas hydrophila Burkholderia pseudomallei Burkholderia thailandesis Burkholderia cepacia Erwinia chrysanthemi Erwinia carotovora Pseudomonas aeruginosa Pseudomonas fluorescens Salmonella typhimurium Serratia marcescens Shewanella frigidimarina Shewanella massilia Couillault and Ewbank, 2002 Couillault and Ewbank, 2002 O'Quinn etal, 2001 Ganet al, 2002 O'Quinn etal, 2001 O'Quinn etal, 2001 Couillault and Ewbank, 2002 Couillault and Ewbank, 2002 Tan etal, 1999a Darby etal, 1999 Tan and Ausubel, 2000 Aballay et al, 2000 Labrousse et al, 2000 Pujol etal,20Q\ Kurz and Ewbank, 2000 Couillault and Ewbank, 2002 Couillault and Ewbank, 2002 Gram-positive Andrew and Nicholas, 1976 Leyns etal, 1995 Garsin et al, 2001 Bacillus megaterium Bacillus thuringiensis Enter ococcus fae calls Microbacterium nematophilum Hodgkin et al., 2000 Staphylococcus aureus Garsin et al. ,2001 Streptococcus pneumoniae Garsin et al., 2001 Streptococcus pyogenes Jansen et al., 2002 Fungi Drechmeria coniospora Jansson et al, 1994 7 The first described visual infection phenotype in C. elegans from a pathogenic organism was a fungal infection caused by Drechmeria coniospora (originally designated as Meria coniospora) (Jansson and Nordbring-Hertz, 1983; Jansson et al, 1994). The described pathogenesis of D. coniospora in C. elegans begins at the head of the worm where the spores adhere. The progress of the infection can be observed visually as the as the hyphae extend into the worm through chemosensory neurons (Jansson and Nordbring- Hertz, 1983; Jansson, 1994). Worm death occurs within two days from initial exposure as hyphae sprout outward from the inside and the worm is fully engulfed by the fungus. Another visible infection phenotype within C. elegans is the Dar (deformed anal region) phenotype described by Hodgkin et al, 2000. The Dar phenotype was first observed in a genetic cross when a variant strain of C. elegans spontaneously displayed a swollen tail. Originally, Dar was characterized as a phenotype from a genetic alteration. However, subsequent genetic test-crosses, segregation patterns, and linkage-mapping suggested that the observed Dar phenotype was not the result of a genetic mutation but rather the result of an infection (Hodgkin et al., 2000). Further studies revealed the presence of a bacterial contaminant within the worm culture medium which was later identified as a Gram-positive coryneform, Microbacterium nematophilum (Hodgkin et al, 2000). Instead of adhering to the head as seen with D. coniospora, the pathogenesis of M. nematophilum involves attachment to the rectal and post-anal cuticle of C. elegans which results in swelling of hypodermal tissues. The infection is non-lethal to C. elegans; however, worms infected with M. 8 nematophilum suffer from constipation which slows growth and development (Hodgkin etal, 2000). Subsequent directions with the C. elegans - M. nematophilum pathogenesis model have already tested C. elegans mutants for hypersensitivity or resistance to M. nematophilum infection. In a preliminary screen of 200 genes, three surface antigen mutants (srf-2, srf-3, and srf-5) and eight novel mutants arising from a newly defined genetic loci named bus (bacterially un-swollen) were found to be resistant M. nematophilum infection (Hodgkin et al, 2000 and Hodgkin, personal communication). Each of the mutants failed to produce a Dar phenotype after prolonged exposure to M. nematophilum. The observed immunity of the srf-2, srf-3, and srf-5 mutants suggests that changes in surface properties within the cuticle alter bacterial attachment to C. elegans and prevents infection. The roles of the bus gene products have yet to be determined; however, the authours suggest that Bus may be also involved with the surface properties of the cuticle or directly affect various stages of infection and induced swelling (Hodgkin, personal communication). The established infections of D. coniospora and M. nematophilum within C. elegans clearly demonstrated that C. elegans is susceptible to parasitic infections, hence presenting the opportunity to examine pathogenesis using C. elegans as the host organism and to characterize novel host genes involved with infection. 1.3.2 Infection or Toxicity Models? Much of the attention surrounding the C. elegans pathogenesis model has been focused on the established infection model by Tan et al. (1999a). Using a clinical P. aeruginosa isolate, PA 14, Tan et al devised the first successful C. elegans 9 - P. aeruginosa infection assay. Depending on the experimental conditions, P. aeruginosa was demonstrated to effectively kil l C. elegans within 4 - 72 h from the time of initial exposure (Tan et al, 1999a). In a high osmolarity medium the incidence of worm death was observed within 4 - 24 h (fast-killing model), when the bacteria were grown in minimal media worm fatality occurred within a few days from initial exposure (slow-killing model). In the fast-killing model, the elevated fatality rates of C. elegans were found to be directly mediated by a diffusible phenazine toxin produced by the P. aeruginosa into the medium (Tan et al, 1999a). The differences of fatality rates were associated to the difference of the bacterial growth medium, as high osmolarity conditions would result in the increased secretion rates of toxins into the medium compared to low-osmolarity conditions. Evidence that the phenazine toxin mediated the fast-killing model was confirmed with mutational studies in C. elegans. The phenazine toxin in P. aeruginosa has been shown to produce elevated levels of free-radicals which induce oxidative stress within cells (Sorensen and Joseph, 1993). C. elegans age-1 (ageing abnormal) mutant, which is highly resistant to free-radicals (Johnson, 1990), displayed resistance to fast-killing assay compared to wild-type worms. Conversely, C. elegans mev-1 (methyl viologen resistance abnormal, Ishii et al, 1990) and rad-8 (radiation sensitivity abnormal, Hartman and Herman, 1982; Ishii et al, 1993) mutants, which have been documented to be extremely sensitive to oxidative stress, showed increased hypersensitivity to fast-killing (Mahajan-Miklos et al, 1999). Hypersensitivity was also reported when a C. elegans pgp-1 pgp-3 (P-glyconrotein) double mutant was utilized in fast-killing assays (Mahajan-Miklos et al, 1999). C. 10 elegans pgp genes encode ATP-binding cassette transporter proteins that are involved in translocating specific substrates across the membrane, including toxins (Broeks et al, 1995). No data was reported using C. elegans pgp or oxidative mutants (i.e., age-1, mev-1, and rad-8) in slow-killing assays and therefore it is unclear whether the mechanism of the slow-killing model is also toxin-mediated or the result of a true infection. However, the authours endorse that slow-killing model is the result of an infection opposed to a toxicity effect (Mahajan-Miklos et al, 2000). In addition to the work performed by Ausubel and colleagues (Mahajan-Miklos et al, 1999; Tan et al, 1999a), another independent research group utilizing the well-characterized P. aeruginosa strain, P A O l , was able to establish a pathogenesis model with C. elegans (Darby et al, 1999). Using different media conditions, Darby et al. (1999) demonstrated that P A O l secretes a neuromuscular toxin that results in lethal paralysis in C. elegans. The paralytic toxin was unable to be identified; however, mutational studies of P A O l revealed that the toxin was involved with the quorum-sensing regulators LasR and RhlR (Darby et al, 1999). The relevance of the quorum-sensing systems may affect C. elegans host defenses as a LasR mutation in the P A H strain resulted in attenuation during slow-killing assays performed by Tan et al. (1999b). Further mutational investigations by Darby et al. (1999) revealed that the C. elegans egl-9 (egg laying defective) mutant was resistant to lethal paralysis. Elg-9 has been documented as a nonessential modulator for muscle contraction in C. elegans (Trent et al, 1983); however, the role of Elg-9 in pathogenesis is unclear. The authours hypothesize that Elg-9 is involved with the activation or transport of the paralytic toxin within C. elegans (Darby et al, 1999). 11 Other toxin-mediated killings of C. elegans by various bacterial species have also been reported in the literature. A few independent groups have documented a lethal interaction between C. elegans and Bacillus spp. In examining the dispersal and behaviour of C. elegans when exposed to a variety of bacterial species, Andrew and Nicholas (1976) reported that B. megaterium not only repelled C. elegans but also was toxic as worm death was observed within 15 min of exposure. Unfortunately, the identification of the potential nematicidal toxin secreted by B. megaterium was not investigated. It has been reported that B. megaterium produces toxins that are lethal to several species of fungus (Bhattacharyya and Purkayastha, 1989); however, it has not been identified whether these fungitoxins are toxic to higher organisms. A couple of research groups have focused on another species of Bacillus, B. thuringiensis, which produces multiple 5-endotoxins. These 5/-toxins have been widely utilized as an insecticide for agriculture (Cannon, 1996; Schnepf et al, 1998). In addition to its lethal effects on insects, the B. thuringiensis 8-endotoxins have been shown to be toxic to C. elegans, causing decreased fertility and severe damage to the intestinal lumen (Leyns et al, 1995; Marroquin et al, 2000). Coomans and colleagues (Borgonie et al, 1996a, b, and c) published a series of papers detailing the observed biological processes of the B. thuringiensis nematicidal activity in C. elegans. The authours show that the toxin is limited to the intestinal lumen, where cells undergo dramatic biological changes including reduction in cell volume and regression of microvilli. As pathogenesis progresses, the intestinal cells are eventually destroyed; however, no cell lysis occurs as the apical intestinal cell 1 2 membranes remain intact (Borgonie et al, 1996b). Mutational studies in C. elegans by Marroquin et al (2000) have isolated five bre (Bacillus-toxin resistant) mutants that were shown to be resistant to a certain B. thuringiensis toxin, Cry5B. However, the resistance of the bre mutants was not observed for another unrelated B. thuringiensis nematicidal, namely Cry6A (Marroqui et al, 2000). The authours hypothesize that multiple B. thuringiensis 5-endotoxins utilize various toxicity pathways, resulting in different modes of pathogenesis (Marroqui et al., 2000). In a follow-up paper, Griffitts et al. (2001) demonstrated that bre-5 encodes a pM,3-galactosyltransferase which transfers galactose residues to proteins and lipids. As C. elegans bre-5 mutants were unable to uptake toxins into gut cells, the group hypothesized that carbohydrate modifications to proteins and/or lipids within the cell membrane mediates the toxicity action of the Z?Moxin. Three species of Burkholderia, B. pseudomallei, B. thailandensis, and B. cepacia, have also been reported to be lethal to C. elegans (Green and Hooke, personal communication; O'Quinn et al, 2002). Each considered as a human opportunistic pathogen which causes melioidosis (Dance, 2002), B. cepacia is commonly known to infect the lungs of cystic fibrosis patients (reviewed by Mahenthiralingam et al., 2002), whereas B. pseudomallei is regarded as a general public health risk but its use as a potential biological warfare agent has also been examined (O'Quinn et al, 2001). B. thailandensis is closely related to B. pseudomallei; however, changes in its ability to assimilate arabinose have rendered this species almost completely avirulent (Wuthiekanun et al, 1996; Brett et al, 1997; Brett et al, 1998). In C. elegans - Burkholderia spp. pathogenesis assays, worm 13 death from exposure to each of the Burkholderia species appeared to be mediated by secreted toxins (O'Quinn et al, 2001). The nematicidal activity was observed to cause neuromuscular paralysis in C. elegans by affecting L-type voltage-gated C a 2 + signal transduction as unc-36 (uncoordinated) and elg-19 mutants were observed to be hypersensitive to Burkholderia infection (O'Quinn et al, 2001). Further evidence of a toxin-mediated killing was supported by B. pseudomallei mutational studies that revealed that worm death did not require the presence of classic pathogencity factors such as type II secretion exoenzymes, aminoglycoside/macrolide efflux pumps, or lipopolysaccharide O-antigens (O'Quinn et al, 2001). As previously stated, classic traits of an infection usually provoke a characterized swelling or growth of tissue masses within the host which can cause cellular necrosis or premature death. However, the above described pathogenesis models have utilized bacterial species that actively secrete toxins into the media. The observed elevated worm fatality rates are clearly the result from toxicity and not by an infection per se., which questions successful establishment of a C. elegans "infection" model. 1.3.3 C. elegans Pathogenesis Model with Mammalian Pathogens Setting aside the toxicity debate, subsequent studies have utilized the framework of the C. elegans - P. aeruginosa infection assay for pathogenesis models involving mammalian pathogens such as Salmonella enterica serovar Typhimurium (notated as Salmonella typhimurium) and Enterococcus faecalis. The C. elegans - S. typhimurium pathogenesis model has been reported from the laboratories of Ausubel and Ewbank (Aballay et al, 2000; Labrousse et al, 14 2000). Although 6 - 8 days of exposure are required to promote worm death, both groups observed proliferation of S. typhimurium within the gastrointestinal tract of C. elegans. It is suggested that the accumulation of bacteria within the gut leads to increased levels of cellular necrosis that results in worm fatality. Cellular apoptosis of C. elegans is regulated by the programmed cell death (PCD) pathway involving ced (cell death abnormal) genes. The involvement of PCD with pathogenesis was demonstrated by Aballay and Aususbel (2001) as C. elegans ced-3 and ced-4 mutants were shown to be hypersensitive to S. typhimurium infection. Loss-of-function mutations in ced-3 and ced-4 (which encodes a caspase and an apoptotic protease-activating factor (Apaf)-1 human homolog, respectively) disrupt the progression of cell apoptosis (Xue et al, 1996; Gumienny et al, 1999; Seiffert et al, 2002). Thus, PCD may play a role in C. elegans host defenses. PCD has been demonstrated to act a host defense mechanism in plant and higher animals (reviewed by Weinrauch and Zychlinsky, 1999). In addition, the ced-3-hke caspase Dredd found in Drosophila has been demonstrated to be essential for resistance to Gram-negative pathogens (Leulier et al, 2000). Recently, Ausubel and colleagues (Garsin et al, 2001) have established a C. elegans pathogenesis model with three Gram-positive mammalian pathogens, E. faecalis, Staphylococcus aureus, and Streptococcus pneumoniae. Detailed examination of the C. elegans - E. faecalis model suggests a similar mechanism of pathogenesis as seen with the C. elegans - S. typhimurium model. Proliferation of E. faecalis was observed within the gastrointestinal lumen and worm death occurred within 2 - 4 days from initial exposure (Garsin et al, 2001; Sifri et al, 2002). In 15 addition, mutational studies of E. faecalis revealed the requirement of the quorum-sensing system in order to establish an infection in C. elegans. Worms exposed to the E. faecalis fsrB mutant strain survived longer than worms exposed to the wild-type strain (Garsin, et al, 2001; Sifri et al, 2002). As the importance of the quorum-sensing system was also shown in the C. elegans - P. aeruginosa model (Darby et al, 1999; Tan et al, 1999b), the data from mutational studies of E. faecalis further implies an important connection between the bacterial quorum-sensing systems and the C. elegans host defense mechanisms. Although pathogenesis investigations have been performed on numerous bacterial species, studies with other prominent mammalian bacterial pathogens such as enteric pathogenic strains of Escherichia coli, Shigella flexneri, or Yersinia spp. have yet to be thoroughly investigated. Only one brief communication has been published concerning the establishment of C. elegans - Yersinia pathogenesis model. The authours show that Y. pseudotuberculosis forms a biofilm around the head of C. elegans which prevents feeding and slows worm growth. However, the model failed to observe systemic infection within C. elegans growing on Y. pseudotuberculosis suggesting the need for additional investigations on the interaction between C. elegans and Yersinia spp. (Darby et al, 2002). 1.4 Host Defense Strategies in C. elegans C. elegans is a free-living soil organism and therefore it is reasonable to assume that C. elegans has developed defense mechanisms to combat potential 16 pathogens encountered within its natural environment. However, the biological process by which C. elegans defends against pathogens remains uncharacterized. 1.4.1 The Innate Immune System Much of the study on primitive immunity mechanisms has been focused on the innate immune system of Drosophila, the Toll-pathway, which is a highly conserved signal-transduction system (Imler and Hoffmann, 2000a). The Toll-pathway plays a dual role during the life span of Drosophila, being involved with both development and defense (reviewed by Belvin and Anderson, 1996). During development, the Toll-pathway is found to be essential for dorsal-ventral polarity regulation; however, in adult flies, the Toll-pathway is responsible for the production of antimicrobial peptides. The activation of antimicrobial peptide production has been shown to be induced by the presence of a pathogenic organism (bacterial or fungal) that triggers a cascade of cellular processes leading to the expression of the antimicrobial peptide genes (Imler and Hoffmann, 2000b). Interestingly, studies have shown that Drosophila can discriminate between various groups of microorganisms in which the Toll-signaling pathway induces gene expression of antimicrobial peptides specific for the type of infection (i.e., bacterial vs. fungal infection) (Lemaitre etal, 1997). Recently researchers have identified C. elegans genes expressing Toll-like factors (Pujol et al, 2001). Four C. elegans genes, tol-1, trf-l,pik-l, and ikb-1, were identified to be homologous to Drosophila genes Toll, dTraf,pelle, and cactus, respectively. Unfortunately, mutational analysis of each C. elegans Toll-like genes revealed that the gene products were not involved with microbial resistance or 17 antimicrobial peptide production (Pujol et al, 2001). Furthermore, Pujol et al (2001) showed that C. elegans tol-1 gene product played a role in worm development and pathogen recognition whereas the other three Toll-like genes have no apparent function in C. elegans. The developmental role of the C. elegans tol-1 gene product may reflect the connection to the Drosophila Toll-pathway since Toll is involved with the dorsal-ventral polarity development in fly embryos (Hashimoto et al, 1988). However, further analysis of the coding region of the C. elegans tol-1 gene revealed that the TIR domain, which is essential for proper function of Drosophila Toll, is virtually absent (Pujol et al, 2001; Ewbank, 2002). 1.4.2 Antimicrobial Agents Other host defenses of C. elegans may involve antimicrobial agents which are produced within the gastrointestinal lumen. C. elegans intestinal cells have been reported contain to copious amounts of lysosomes, lectins, and proteases which would result in a hostile environment for microbes (Ewbank, 2002). The search for gene induction as a response to a bacterial infection has recently implicated the involvement of the DBL- l /TGFp pathway in C. elegans (Mallo et al, 2002). Using cDNA microarrays and the C. elegans - S. marcescens pathogenesis model, Mallo et al. (2002) demonstrated the induction of several genes encoding lectins and lysozymes are known to be involved with immune responses in other organisms. The findings were confirmed with analysis of dhl-1 (dpp, bone morphogenetic protein-like) and lys-1 (lysozyme) mutants where null dhl-1 mutants showed hypersensitivity to S. marcescens and overexpression of lys-1, which encodes a nematode lysozyme, 18 augmented C. elegans resistance to the pathogen (Mallo et al, 2002). These results suggested that C. elegans does possess inducible pathways for antimicrobial action. In addition, A S A B F (Ascaris suum antibacterial factor)-type antimicrobial factors (Abf-1 and Abf-2), which are short peptides expressed within the pharyngeal lumen, have been found in vitro to be lethal to a wide range of microbes, including Gram-negative, Gram-positive pathogens, and several types of pathogenic yeast (Kato et al, 2002). However, it remains unclear whether the ASABF-type antimicrobial peptides act as a host defense in vivo. 1.5 Yersinia spp. and Mechanisms of Pathogenesis The Yersinia bacterium has eleven identified species, including three species that have been reported to be pathogenic to humans. Y. pestis is the causative agent of the plague; however, more present-day Yersinia infections are caused by the food-borne pathogens Y. pseudotuberculosis and Y. enterocolitica (Cornells and Wolf-Watz, 1997). Y. pestis is inoculated by a flea bite, whereas transmission of Y. enterocolitica and Y. pseudotuberculosis occurs from contaminated food or water consumption resulting in clinical manifestations of severe gastroenteritis and pseudoappendicitis (Putzker et al, 2001). Regardless of the mode of transmission, all three Yersinia species have a common tropism for lymphoid tissues (Cornells et al., 1998; Cornells, 2002). The pathogenic nature of Yersinia spp. is dependent upon the presence of a large (-70 kb) virulence plasmid, p Y V and various key chromosomally encoded virulence factors (Cornelis et al, 1998; Revell and Miller, 2001). The p Y V encodes 19 an array of virulence proteins including Yersinia adhesion protein YadA, Yersinia outer proteins (Yops), specific yop chaperones (Sycs), members of a type III secretion system (Yscs), and low calcium response proteins (Lcrs) (Michiels and Cornelis, 1989; Michiels etal., 1990; Koornhof etal., 1999; Snellings etal., 2001). However, early studies on Yersinia pathogenesis revealed that the pYV was not solely responsible for the pathogenic nature of Yersinia (Heesemann and Laufs, 1983; Heesemann et al., 1984). Several chromosomal genes encoding virulence factors have been identified including^/, inv, and a high pathogenicity island (HPI) (also referred to as the yersiniabactin locus). The genes yst and inv encode a heat-stable enterotoxin and invasin protein, respectively (Revell and Miller, 2001). The Yersinia HPI harbours the psn/JyuA gene and several irp and ybt genes, all which are involved with iron acquisition (Heesemann, 1987; Pelludat et al, 1998; Carniel, 2001). The process of Yersinia pathogenesis (reviewed by Koornhof et al, 1999) begins with the expression of the type III secretion system involving several syringe-like organelles, the Ysc injectisome, and various Yop proteins (Cornelis, 2002). The regulation and expression of the type III secretion machinery and Yop proteins is highly dependent on temperature (i.e., 37°C) and environmental cues (i.e., pH and ion concentrations). Once constructed, the Ysc injectisome is embedded within the bacterium cell membrane and several Yop proteins are capped by Syc chaperones to prevent premature association (Cornelis, 2002). The next stage involves initial attachment to the host cell which mediated by the Yersinia adhesin YadA and invasin through direct binding to fibronectins, collagens, and integrins (Isberg and Leong, 1990; Tertti et al, 1992; Roggenkamp et al, 1995). The docking of the bacterium at 20 the host cell surface opens the Ysc injectisome and the Yop proteins are exported. Subsequently, translocator proteins, YopB and YopD, form a pore in the host cell membrane which allows the effector Yop proteins (i.e., YopE, Y o p M , YopO, YopP/YopJ, and YopT) to be translocated across the membrane into the host cell (Cornells et al, 1998; Koornhof et al, 1999; Cornells, 2002). Once inside the eukaryotic cell, the Yop effectors act on various host cell machinery which results in the interruption of cytoskeleton dynamics, the disruption of phagocytosis, the induction of apoptosis, and the inhibition of proinflammatory proteins production (Cornells et al, 1998). As a result, the Yersinia bacterium overcomes the host's inflammatory responses and is allowed to establish itself in the peripheral lymphoid tissues (Cornelis et al, 1998; Koornhof er al, 1999). 1.5.1 Yersinosis in British Columbia Although Health Canada does not list Yersinia spp. infections as a reportable disease, Yersiniosis is a prevalent bacterial infection in British Columbia. In its 2000 and 2001 annual reviews, the British Columbia Centre of Disease Control reported an average of 25.8 cases per 100,000 population over a 13 year period (1988 - 2001). The average rate of Yersinosis is comparable to reported provincial and national rates of Salmonellosis (24.7 and 25.6 cases per 100,000, respectively) (Figure 1). Health officials have been unable to explain the reason for the high incidence of Yersiniosis cases in British Columbia, but it is clear that Yersinia infections are a prominent health concern for British Columbians. 21 zz Rate (per 100,000 pop.) CO o ro O SO CD p SO p ~ • W zr ca' oa' CD Imo CD o Imo o" Cfl =3 (/> (fl' CD CA' llosis 3 <2. 0 3 1 s Cfl Cfl' 1.6 Scope and Nature of This Work The purpose of this project was to investigate establishment of a pathogenesis model using Caenorhabditis spp. and mammalian enteric pathogens. This thesis describes the development of the C. briggsae - Y. enterocolitica infection assay. As part of its development, various experimental conditions of the infection assay were considered including the issues of incubation temperature and optimal media content. Establishment of a C. briggsae - Y. enterocolitica infection model was evaluated through a series of controlled experiments which reported worm fatality rates, progeny fitness, and visualization of the ingestion and retention of Y. enterocolitica within the gastrointestinal tract of C. briggsae. The establishment of the C. briggsae - Y. enterocolitica pathogenesis model can be utilized to perform functional genetic analyses to investigate the molecular processes that occur within the host during the infection process including the characterization of host genes. Additional thesis directives were undertaken to develop a virulence gene subset database that lists all known and well-characterized virulence factors from the published literature. The database can aid in the identification of gene products conserved between C. elegans and pathogenic bacteria which may potentially identify host factors that resemble pathogen proteins and are potentially being manipulated by pathogens during the infection process. 23 CHAPTER II 2.0 METHODS AND MATERIALS 2.1 Organism Strains 2.1.1 Nematode Strains Two wild-type Caenorhabditis species, C. elegans, var. Bristol N2 strain and C. briggsae, var. Gujarati G16 strain, were utilized during the course of this study and are listed in Table 2 along with mutant derivatives from these wild-type strains. 2.1.2 Bacterial Strains Various species and strains of Escherichia, Listeria, Pseudomonas, Salmonella, and Yersinia were employed during the course of this study and are listed in Table 3 along with their relevant genotypes. 2.1.3 Plasmids Cloning vectors and plasmid constructs which were utilized or constructed during the course of this study are listed in Table 4 with their significant characteristics. 2.2 Media and Growth Conditions 2.2.1 Nematode Media and Growth Conditions As described by Brenner (1974), C. elegans and C. briggsae strains were maintained on 5.0 cm petri plates with Nematode Growth Media (NGM) streaked with Escherichia coli strain OP50 as food source. Nematode strains were incubated at 20°C or ambient temperature. Table 2. List of Caenorhabditis strains utilized. Nematode Strain 3 Relevant Genotype Reference C. elegans . N2 N2 Bristol wild-type; subclone KR2693 Brenner, 1974 C. elegans DR1564 daf-2 (e41) III Riddle, PC C. briggsae G16 G l 6 Gujarati wild-type; subclone KR144 Brenner, 1974 a All strains were acquired from the laboratory collection of Ann M. Rose with the exception of DR1564 which was kindly provided by the laboratory of Don Riddle, University of Missouri-Columbia, USA. 25 Table 3. List of laboratory bacterial strains employed. Bacteria 3 Strain Relevant Genotype Reference E. coli OP50 universal strain utilized as food source for laboratory Caenorhabditis spp. Brenner, 1974 E. coli DHlOp F" mcrA A(mrr-hsdRMS-mrcBC) qp80/ocZAM15 AlacX74 deoR recAX endAl araA139 A(ara, leu)1691 gal\J galK X rpsL nupG Invitrogen, 2002 L. monocytogenes EGD wild-type; Bof297 Finlay, P C b L. monocytogenes 4b wild-type; silent Tn916 mutant of 4b 1; Serotype 4b Finlay, PC L. monocytogenes 1/2 a3 derivative of SLCC5764; Serotype 1/2 a Finlay, PC L. monocytogenes 46 Clinical isolate; Serotype 1/2 a BC CDC C L. monocytogenes 394 Clinical isolate; Serotype 4 b BC CDC L. monocytogenes 405 Clinical isolate; Serotype 1/2 a BC CDC L. monocytogenes 475 Clinical isolate; Serotype 1/2 b BC CDC P. aeruginosa P A K wild-type Hancock, PC d P. aeruginosa P A K gacS Hancock, PC S. typhimurium SL1344 wild-type Finlay, PC S. typhimurium KR1562 metA22 metE551 trpC2 ilv-452 leu-3121 xyl-404 flaA66 rpsL120 Kelln, PC e Y. enterocolitica E40 wild-type; Serotype 0:8 Finlay, PC Y. enterocolitica E40 yscN Finlay, PC (continued) 26 Table 3. List of laboratory bacterial strains employed (continued). Bacteria 3 Strain Relevant Genotype Reference Y. enterocolitica MRS40 yopE Finlay, PC Y. enterocolitica WA-314 wild-type; N a l R ; Serotype 0:8 Finlay, PC Y. enterocolitica W A - C WA-314 strain cured from the virulence plasmid; N a l R Finlay, PC Y. enterocolitica 36 Clinical isolate; Serotype 0:4 BC CDC Y. enterocolitica 56 Clinical isolate; Serotype 0:5, 27 BC CDC Y. enterocolitica 1035 Clinical isolate; Serotype 0:8 BC CDC Y. enterocolitica 1564 Clinical isolate; Serotype 0:3 BC CDC Y. enterocolitica 1613 Clinical isolate; Serotype 0:3 BC CDC Y. enterocolitica 1615 Clinical isolate; Serotype 0:6, 31 BC CDC Y. enterocolitica 1640 Clinical isolate; Serotype 0:5, 27 BC CDC Y. enterocolitica 1662 Clinical isolate; Serotype 0:6, 30 BC CDC Y. pseudotuberculosis YIII P+ wild-type Finlay, PC Y. pseudotuberculosis YIII P- YIII; cured of p Y V Finlay, PC Y. pseudotuberculosis 1425 Clinical isolate; Biotype IB BC CDC Y. pseudotuberculosis 1430 Clinical isolate; Biotype IB BC CDC Y. pseudotuberculosis 1431 Clinical isolate; Biotype IB BC CDC Y. pseudotuberculosis 1432 Clinical isolate; Biotype IB BC CDC a Strains were obtained from the laboratory collection of Ann M . Rose unless noted otherwise. b Strains were kindly obtained from the personal laboratory collection of B. Brett Finlay, UBC. c Strains graciously supplied by the British Columbia Centre for Disease Control. The strains were acquired from the personal laboratory collection of Robert E. W. Hancock, UBC. e Strain was kindly obtained from the personal laboratory collection of Rodney A . Kelln, University of Regina. 27 Table 4. List of mobile vectors and plasmid construct utilized. Plasmids Properties Reference pSMlOl pBR322 A Asel - EcoRI containing a 0.92 kbp GFP coding region under lacZ promoter control; TetR; 4.4 kbp Mills, PC pNLP8 pSMlOl A Asel - BstBI; TetR; 3.6 kbp a a pNLP8 was constructed during the course of this study. 28 2.2.2 Bacteria Media and Growth Conditions Various liquid media were utilized for overnight growth of bacterial liquid cultures, including Lennox L broth (LB) (Sambrook et al, 1989), King's B broth (King et al, 1954), and Brain Heart Infusion (BHI) broth (Becton, Dickinson, and Company). General maintenance of bacterial plate cultures were grown 10 cm petri L B agar plates solidified with 1.5 % agar. For infection assays, bacterial plate cultures were grown on 5 cm petri N G (netamode growth, modified N G M with 0.35 % peptone instead of 0.25 %) (Tan et al, 1999a) or BHI agar plates. To ensure the growth of the correct strain and/or the retention of plasmids within the bacterial strain, tetracycline (Tet), ampillicin (Amp), nalidixic acid (Nai), and carbenicillin (Car) were used as the antibiotic for selection at a concentration of 10 Lig/mL, 50 u.g/mL, 25 ug/mL, and 25 ug/mL, respectively. A l l plate cultures were grown in an incubator at 37°C. Liquid cultures were grown at 37°C on a gyrorotary shaker at 250 rpm. 2.3 Organism Frozen Stocks 2.3.1 Caenorhabditis Frozen Stocks Long-term maintenance of C. elegans and C. briggsae strains was achieved through the preparation of frozen stocks. Caenorhabditis strains were grown on five N G M plates until overcrowding, typically 6 - 8 days. Worms were concentrated by washing with 5 mL sterile freezing solution (0.1 M NaCl , 50 m M KFLPO4 pH 6.0, 30 % glycerol, 0.3 m M MgS04) with a sterilized Pasteur pipette. Worms were then aliquoted into three 1.5 mL cryogenic vials and stored at -80°C for one week. To 29 ensure successful freezing, one cryogenic vial was thawed, worms were placed on a seeded N G M plate and observed for worm survival. If freezing was successful, the remaining two vials were stored in liquid nitrogen until needed for experimental use. 2.3.2 Bacterial Frozen Stocks To ensure proper maintenance of virulent bacterial stocks, each bacterial strain was frozen in 15 % glycerol. To prepare frozen stocks, a single colony isolate of each bacterial strain was inocculated into 5 mL of LB broth and grown overnight. In a 1.5 mL cryogenic vial, 325 uL of 60 % sterile glycerol was added to 1.175 mL of overnight bacteria culture and mixed by inversion. The prepared bacterial stock was stored at -80°C until needed for experimental use. 2.4 Microscopy 2.4.1 Light Microscopy Observation of nematodes was performed using a WILD M 3 B light dissecting microscope. 2.4.2 Fluorescent Microscopy Fluorescent microscopy was conducted at the University of British Columbia Biosciences Electron Microscopy Facility, using a Zeiss Fluorescent Axioplan microscope. Specimens were prepared a few hours (2 - 3 h) prior to viewing. 2.4.2.1 Fixing and Mounting Procedure To a poly-L-lysine coated specimen microscopy slide, a series of 5 uL Mil iQ H2O droplets were placed on the slide. Worms were carefully transferred to H2O drops and the H2O was allowed to dry. The worms were then washed twice with 30 200 uL of 100 % methanol and the methanol was allowed time to evaporate. Worms were rehydrated with 100 uL phosphate-buffered saline (PBS) solution for 20-30 minutes. Next, 5 uL of 2X mounting media (20 m M Tris-HCl pH 8.0, 0.2 M 1,4-diazabicyclo-2,2,2-octance (DABCO), 90 % glycerol) was added and a coverslip was gently placed over the worms. The coverslip was allowed time to settle (1-2 min) before sealing with clear nail polish. 2.5 Molecular Genetic Techniques Various recombinant D N A techniques were adapted primarily from Sambrook et al. (1989). These procedures included small-scale rapid lysis isolation of plasmid D N A , restriction endonuclease digests, and agarose gel electrophoresis. Small-scale rapid lysis isolation of bacterial plasmids was performed using a Plasmid Miniprep Kit from QIAGEN Inc. (2002) as per manufacturer's instructions. Restriction endonucleases were supplied by various sources (i.e., New England BioLabs and Invitrogen). In addition, isolation and purification of linearized D N A fragments from 1.5% low-melting point agarose gels was performed using a QIAEX II Gel Extraction kit from QIAGEN Inc. (2002). 2.5.1 Plasmid Construction Recombinant plasmids constructed during the course of this study were previously listed in Table 4 and are further described within the text. Ligations of D N A fragments and vectors using T 4 D N A ligase followed procedures described by New England Biolabs (2002). When applicable, the Klenow fragment from E.coli D N A polymerase I was utilized to fill 5' overhangs, creating blunt-ended D N A for 31 ligations (Invitrogen, 2002). Confirmation of the nature of the plasmid constructs was performed through digestion with restriction endonucleases and analysis by 1.0 % agarose gel electrophoresis. 2.5.2 Transformation 2.5.2.1 Preparation of Electro-Competent Cells Bacterial strains were grown in 20 mL of sterile L B broth to an OD660 « 0.6. The cells were then concentrated by several centrifugation steps in 10 % glycerol and then stored in minimal amounts of 10 % glycerol (-40 uL) at -80°C until needed for transformations. 2.5.2.2 Transformation Methods Plasmid constructs (1 uL - 8 uL) were mixed with prepared electro-competent cells. Each mixture was placed into an ice-cold 0.2 cm electrocuvette and given a 1.8 kV pulse using a BioRad Micropulser Electroporation Apparatus. Immediately following the pulse, 450 uL of ice-cold recovery buffer (containing LB broth, 15 m M MgCb, and 0.2 % glucose) was added. The mixture was transferred to an 1.5 mL microcentrifuge tube and incubated with shaking for 1 h at 37°C. After incubation, the cells were plated onto L B media with an appropriate antibiotic for selection. 2.5.3 Caenorhabditis Lysis for PCR To obtain genomic D N A from C. elegans for PCR procedures, worm lysis was performed (Kitagawa, personal communication). One adult hermaphrodite was selected and placed in 0.6 mL microcentrifuge tube containing 5uL of lysis solution ( IX PCR buffer, 1.5mM M g C l 2 , 60 pg/mL Proteinase K). The tube was placed at -32 80°C for 15 min, followed by an incubation at 57°C for 1 h and a 94°C denaturation segment for 15 min. Lysis solution containing C. elegans genomic D N A was either utilized immediately for PCR or stored at -80°C until needed. 2.5.4 Polymerase Chain Reaction A l l PCR preparations were performed using procedures of an in-house protocol. The amplification reaction mixture included appropriate amounts of H2O, I X PCR buffer, 1.5 m M M g C h , 1.25 m M deoxyribonucleotide tri-phosphates (dNTPs), 0.75 u.M forward and reverse primers, template D N A , and thermal-stable D N A polymerase (i.e., Taq D N A polymerase). A l l components were kept on ice during preparation. The PCR mixture was placed in a Mastercycler thermal-cycler and subjected to repeated cycles of denaturation, annealing, and polymerization, resulting in the amplification of the D N A template. Analysis of PCR products was performed using endonuclease digests and 1.0 % agarose gel electrophoresis. 2.6 Hypochlorite Extraction of Caenorhabditis Embryos To synchronize the life cycle of worm populations, a hypochlorite extraction was performed (Lewis and Fleming, 1995). A population of C. elegans or C. briggsae was grown to gravid stage on several N G M plates (20 or more). The gravid worms were concentrated together by washing each plate with 4 mL of sterile Mill iQ H2O and transferred to a clean glass Kimax tube. The worms were centrifuged at 1100 rpm for one minute and the supernatant was discarded. To wash worms from the residual bacteria, the worm pellet was carefully layered onto 5 mL of 40 % sterile sucrose solution in a clean glass Kimax tube and centrifuged at 1100 rpm for 5 min. 33 After centrifugation, worms in the top aqueous layer were removed and transferred to 7 mL of M i l l i Q H2O in a clean glass Kimax tube. The tube was centrifuged at 1100 rpm for 5 min and the supernatant was discarded. To the remaining worm pellet, 5 mL of freshly prepared hypochlorite solution (1.5% NaOCl, 0.5 M KOH) was added, and the sample was mixed by inversion and incubated at room temperature for 10 min. After worm lysis, the solution was centrifuged at 1100 rpm for 5 min and the supertanant was discarded. The remaining worm embryos were washed three times with Mi l l iQ H2O, transferred to an unstreaked N G M plate, and incubated at room temperature to allow the embryos to hatch overnight. In the absence of food, all hatching embryos will enter and remain in LI larvae stage; thus, synchronizing all viable worms to the same life cycle stage. If required, synchronized LI worms were transferred to N G M plates with E. coli as a food source to allow the worms to mature to L4/adult stage. 2.7 Thermotolerant Testing Assessment of Caenorhabditis tolerance to temperatures higher than 25°C was performed through thermotolerant testing. Ten synchronized L4 hermaphrodites were introduced onto N G M plates and incubated at six different temperatures (i.e., ambient temperature, 28°C, 30°C, 33°C, or 37°C). Worm death, infertility, and visible mutant phenotypes were scored every 12 h over a three day period. 34 2.8 Caenorhabditis - Bacterial Assay Several procedures were attempted to establish a successful assay between Caenorhabditis and bacterial strains. Worms were placed on bacterial lawns by transfer and incubated at various temperatures for 72 h or 96 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping was observed. The E. coli OP50 strain was utilized as a negative bacterial control for all trials and for statistical purposes each trial was carried out in triplicate. 2.8.1 C elegans - Bacterial Assay Initial Caenorhabditis - bacterial assays were performed using the procedure described by Tan et al. (1999a). Overnight bacterial cultures were grown in 5 mL of L B broth with shaking at 37°C. Bacterial lawns were prepared by spreading 50 uL of overnight culture onto N G agar plates with subsequent incubation at 37°C for 24 h. The N G agar plates with bacterial lawns were brought to room temperature and seeded with 20 L4 C. elegans hermaphrodites. Plates were incubated at various temperatures (i.e., 20°C, room temperature, or 30°C) and scored for live worms every 12 h. 2.8.2 C. briggsae - Bacterial Assay Overnight bacterial cultures were grown in 2 mL of BHI broth. Overnight cultures were diluted 1:200 in BHI broth and allowed to grow for 90 min at 37°C with shaking. Bacterial lawns were prepared by spreading 10 uL of overnight culture onto BHI agar plates with subsequent incubation at 37°C for 24 h. The BHI agar plates with bacterial lawns were seeded with 20 L4 C. briggsae hermaphrodites. Plates were incubated at 30°C and scored for live worms every 12 h. 35 2.8.3 C. briggsae - Bacterial Assay Using E G T A Overnight bacterial cultures were grown in 2 mL of BHI broth. Overnight cultures were diluted 1:200 in 2 mL BHI broth with added ethylene glycol-bisfbeta-aminoethylether)-N,N'-tetraacetic acid (EGTA) mixture (5 m M E G T A , 15 m M MgCb, 0.2 % glucose) and incubated for 90 min at 37°C with shaking. Bacterial lawns were prepared by spreading 10 uL of overnight culture onto BHI E G T A 5 m M agar plates with subsequent incubation at 37°C for 24 h. Unless stated otherwise, the final concentration of E G T A within the liquid and solid culture media was 5mM. The BHI E G T A 5 m M agar plates with bacterial lawns were seeded with 20 L4 C. briggsae hermaphrodites. Plates were incubated at 30°C and scored for live worms every 12 h. 2.9 Creation of the Virulence Gene Subset Database The entries of the virulence gene subset (VGS) database were obtained by reviewing several general microbial textbooks (e.g., Groisman, 2001) and website sources, including the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nih.gov/) and the PubMed database (http://www.ncbi.nih.gov/entrez/query.fcgi?db=PubMed). Candidate entries were selected on the basis of the following criteria: (1) listed pathogencity factors are from a bacterial species with a published genome or plasmid sequences, (2) candidate pathogencity factors have published literature of performed mutagenesis studies that resulted in attenuation of the bacterial strain, and (3) attenuation due to mutagenesis was verified in an animal host model. Selected entries were cataloged and listed in a Microsoft Excel spreadsheet. 36 2.9.1 Analysis of C. elegans Homologs From the entries of the VGS database a B L A S T analysis (Altschul et al., 1990; Altschul et al., 1997) was performed to identify putative C. elegans homologs of pathogenicity factors. B L A S T searches were performed using the online website (http://www.ncbi.nih.gov/BLAST/) and further characterization of the identified C. elegans homologs was performed using the Caenorhabditis elegans WWW Server (http://elegans.swmed.edu/) and Wormbase (http://www.wormbase.org). 37 C H A P T E R III 3.0 R E S U L T S 3.1 Assessment of C. elegans as a Host for Bacterial Enteric Pathogens To test for effects on C. elegans, two Y. enterocolitica strains, Y. enterocolitica E40 wild-type and Y. enterocolitica E40 YscN mutant were cultured in L B broth, plated onto N G agar plates, and grown overnight. The gene product of yscN harbours ATP-binding motifs and is considered a necessary component to energize the Yersinia type III secretion system (Woestyn et al, 1994; Cornells et al, 1998). The utilization of both Yersinia wild-type and YscN mutant strains enabled a simple comparison of the effects of a functioning Yersinia type III system on C. elegans. Following the protocol described by Tan et al. (1999a), C. elegans N2 L4 hermaphrodites were introduced separately onto Yersinia lawns on N G agar plates and incubated at ambient temperature. The worms were examined at 24 h intervals and each trial was terminated after 120 h (5 days) from initial exposure to Yersinia strains if no considerable worm death or mutant phenotype was observed. Three separate trials were performed and each trial showed no elevated worm death or visible mutant phenotype in the parental hermaphrodites or their progeny after 120 h of exposure to either of the two Yersinia strains. In addition, assays were performed using the following bacterial strains: Y. enterocolitica MRS40 YopE mutant, L. monocytogenes EGD strain, and S. typhimurium SL1344 strain. YopE enables Yersinia to resist phagocytosis and mutant strains defective myopE are characterized to be less virulent compared to wild-type Yersinia strains (Rosqvist et al, 1990; Rosqvist et al, 1991; Cornells et al, 1998). 38 Therefore, the Y. enterocolitica YopE mutant was utilize to further examine the significance of Yersinia virulence mechanisms in C. elegans. L. monocytogenes and S. typhimurium strains were utilize to examine possible establishment of an infection model using different types of mammalian bacterial pathogens in C. elegans. In addition, the nutritional content of the solid media was varied by increasing the yeast extract content from 0.15 % to 0.25 %. However, no worm death and no visible mutant phenotypes were observed in the parental hermaphrodites or their progeny after 120 h of exposure to the bacterial strains. In all attempts, no detrimental effects were observed when C. elegans was grown on Y. enterocolitica, L. monocytogenes, or S. typhimurium. 3.2 C. briggsae as a Host for Bacterial Enteric Pathogens 3.2.1 C. briggsae Survives in Bacterial Incubation Environment Virulent gene expression in mammalian enteric pathogens is tightly temperature regulated, with the optimal temperature usually being at 37°C. Therefore, a possibility was considered that unsuccessful infection of C. elegans with a pathogenic enteric bacterium could be due to the lack of virulent gene expression at ambient temperature incubation. To examine this possibility, a thermotolerant species of Caenorhabditis was used. Two Caenorhabditis strains, C. elegans daf-2 and C. briggsae var. Gujarat G16, have been documented to be fertile at temperatures higher than 25°C (Larsen and Riddle, personal communication; Yarwood and Hansen, 1969). Experiments to determine the Caenorhabditis species ability to tolerate higher temperatures (i.e., 39 thermotolerant testing) were performed on both strains using C. elegans N2 strain as a control. The results of the thermotolerant testing are summarized in Table 5. After 72 h at 28°C, both C. elegans daf-2 and C. briggsae G16 strains remained viable and produced fertile progeny. However, the Fi generation of daf-2 mutants formed dauer larvae within 12 h of incubation at 28°C and remained in dauer stage until transferred to fresh NGM plates and incubated ambient temperature. The Fi generation of C. briggsae G16 continued to develop normally at 28°C and remained fertile. At 30°C, the C. elegans daf-2 mutant hermaphrodites produced dauer larvae which were later noted to be infertile upon reaching maturity; whereas, C. briggsae G16 hermaphrodites continued to mature normally; however, were semi-fertile as the average brood size of hermaphrodites decreased by 52 % after 72 h. The Fi generation of C. briggsae developed normally at 30°C without entering dauer stage, however the worms had a reduced brood size similar to that observed in the Po generation. At temperatures exceeding 30°C, C. elegans daf-2 mutants were unable to survive longer than 24 h even in the dauer stage. C. briggsae was able to survive for 72 h at 33°C before considerable worm death (i.e., worm fatality > 50 %) was observed, however the Po generation demonstrated infertility as unfertilized eggs were produced after 72 h. At 37°C, C. briggsae hermaphrodites were unable to survive incubation for more than 24 h. Since culture medium can also influence bacterial gene expression the use of BHI broth and agar media was considered. A series of experiments was performed to determine the effects of Caenorhabditis growth on BHI media. Figure 2 illustrates the incidence of C. briggsae G16 and C. elegans N2 fatality rate during incubation on 40 Table 5. Results of thermotolerant testing with Caenorhabditis species3. Temperature (°C) C. elegans N2 C. elegans daf-2 C. briggsae G16 A T b Fertile Fertile Fertile 28 Infertile0 Dauer, fertile Fertile 30 Infertile or deadd Dauer, infertile Semi-fertile 33 Dead Dead Infertile 37 Dead Dead Dead a Reported results are after 72 h of incubation. b A T = ambient temperature (20°C - 22°C). c Infertility described as P 0 generation produced void eggs or no eggs were laid after 72 h. d Observation described as >50% of P 0 generation unable to survive after 72 h. 41 50.0% 12 24 36 48 Time (h) 60 72 -*—C. briggsae at25°C C. briggsae at30°C C. e/egans at25°C ..... C. elegans at30°C Figure 2. Determination of Caenorhabditis fatality rate on N G M plates at 25°C and 30°C temperatures. Worms were scored for death and transferred to fresh E. coli OP50 bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 42 N G M plates at 25°C and 30°C. On N G M agar plates, the C. briggsae fatality rate was 9.8 % (± 0.2 %) at 25°C and 10.0 % (± 1.4 %) at 30°C after 72 h of incubation. For C. elegans, the fatality rate was 7.1 % (± 1.0 %) at 25°C and 26.8 % (± 1.4 %) when worms were incubated at 30°C after 72 h. The experiment was repeated using BHI plates in replacement of N G M plates as solid culture media and results are shown in Figure 3. In general, the usage of BHI agar plates resulted in a higher incidence of fatality with both C. elegans N2 and C. briggsae G16 wild-type strains. However after 72 h of incubation, C. briggsae G16 at 30°C showed a similar incidence of fatality (36.4 %, ± 1.8 %) as the fatality rate of C. elegans N2 at 25°C (38.6 %, ± 1.0 % after 72 h). The results show that C. briggsae can be cultured at higher temperatures than C. elegans in BHI media and therefore C. briggsae was selected as the host for infection assays with mammalian bacterial pathogens. 3.2.2 Growth of C. briggsae on Bacterial Enteric Pathogens Since Ausubel and colleagues (Aballay et al., 2000;Tan et al., 1999a) demonstrated successful infection of C. elegans, pathogenesis experiments were repeated using C. briggsae with P. aeruginosa or S. typhimurium. P. aeruginosa wild-type strain P A K and a P. aeruginosa GacS mutant were grown in King's B broth and BHI broth. Each culture was plated onto BHI agar plates for overnight growth. Synchronized C. briggsae G16 L4 hermaphrodites were introduced to the bacterial lawns and the assay procedure was performed as previously described. Worms were exposed to E. coli OP50 as a control. After 36 h from the initial infection, no considerable worm death or mutant progeny were observed on each 43 100.0% 80.0% S 60.0% Q I 40.0% 20.0% 0.0% C. briggsae at 25°C C. briggsae a t30°C -*— C. elegans at 25°C C. elegans a t30°C Time (h) Figure 3. Determination of Caenorhabditis fatality rate on BHI plates at 25°C and 30°C temperatures. Worms were scored for death and transferred to fresh E. coli OP50 bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 44 plate. After 96 h from the initial exposure, no worm fatality was noted and the assay procedure was terminated. S. typhimurium lab strain SL1344 was grown in L B broth and cultures were plated onto BHI agar plates. Synchronized C. briggsae G16 L4 hermaphrodites were introduced to the bacterial lawns, placed at 30°C, and scored for worm death every 12 h. After 36 h from the initial infection, no considerable worm death or mutant progeny were observed on each plate. After 96 h from the initial exposure, no considerable worm death was observed compare to worms exposed to E. coli OP50 and assay procedure was terminated. In both cases C. briggsae feeding on P. aeruginosa and S. typhimurium at 30°C did not result in elevated worm fatalities. Subsequent experiments focused on the utilization of enteric pathogens that are able to grow at 30°C or below (e.g. Listeria spp. and Yersinia spp.) (Walker et al., 1990; Goverde et al, 1994). Three L. monocytogenes laboratory strains and four L. monocytogenes isolates obtained from the British Columbia Centre for Disease Control (BC CDC) were utilized in pathogenesis assays. Each isolate was grown in BHI broth overnight and plated to BHI solid media. Infection assays were performed as described above; however, after 96 h from the initial exposure no considerable worm fatality was observed and the assays were terminated. Subsequently, five Y. enterocolitica laboratory strains, eight Y. enterocolitica BC CDC isolates, and four Y. pseudotuberculosis BC CDC isolates were tested. Each isolate was grown in BHI broth and plated to BHI solid media in triplicates and infection assays were performed as previously described. After 96 h from the initial exposure, no considerable worm death was observed and the assays were terminated. 45 Overall the data of C. briggsae feeding on L. monocytogenes and Yersinia spp. did not display an increase in worm death compared to C. briggsae feeding on E. coli OP50. 3.2.3 Growth of C. briggsae on Y. enterocolitica in Ca2+-Depleted Environment Since media composition and incubation temperature were considered as key factors for proper virulent gene expression to establish a successful pathogenesis model using C. briggsae, further investigations were undertaken to examine the low calcium response (LCR) of Yersinia. In a calcium-depleted environment, Yersinia spp. actively express and secrete key virulent factors into the extracellular fluid (Straley and Bowmer, 1986; Cornelis et al, 1989; Straley, 1991). Therefore, subsequent experiments were designed to introduce E G T A into the bacterial culture media to chelate calcium ions and activate the L C R of Yersinia. To test whether Yersinia L C R could have an effect on the C. briggsae infection assay with Y. enterocolitica, two Y. enterocolitica BC CDC isolates (1613 and 1564) and the Y. enterocolitica MRS40 YopE mutant were cultivated in BHI broth with 5 m M E G T A and plated to B H I - E G T A 5 m M agar media. After 48 h of exposure, the average observed incidence of worm fatality exposed to Yersinia strains was higher (59.8 %, ± 9.5 %) than exposure to the E. coli OP50 control (14.3 %, + 1.6 %) (Figure 4). After 72 h, an average of 87.2 % (± 4.2 %) worm fatality rate was observed on Y. enterocolitica plates compare to 62.9 % (± 7.9 %) worm death on E. coli OP50 control. During the progression of the assay, the rate of pharyngeal pumping, body size, and motility of C. briggsae exposed to Y. enterocolitica strains steadily declined until worm became unresponsive to touch and no internal movement 46 100.0% 80.0% S> 60.0% Q | 40.0% 20.0% 0.0% * -+-OP50 - • -1613 - • 1 5 6 4 MRS40 YopE-12 24 36 48 Time (h) 60 72 Figure 4. Infection assay results of the incidence of C. briggsae death with the Y. enterocolitica in the presence of EGTA (5 mM) in BHI culture media. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. E. coli OP50 was utilized as the control. 47 (i.e., internal muscle contractions) could be observed. The results indicated the possible establishment of a successful infection assay between C. briggsae and Y. enterocolitica. 3.3 Characterization of C. briggsae - Y. enterocolitica Virulence Model 3.3.1 Effect on C. briggsae Progeny Fitness To further investigate the effect Y. enterocolitica on C. briggsae the number of surviving progeny was scored when C. briggsae was exposed to Y. enterocolitica strains grown on BHI E G T A 5 m M agar media. E. coli OP50 was utilized as a control. One L4 hermaphrodite C. briggsae was introduced to the bacterial lawn and incubated at 30°C. The worm was transferred every 24 h to a fresh bacterial lawn over a three day period. Progeny hatched and grew to L3/L4 larva stage before scoring and experiments were performed in triplicate. The data is reported in Table 6. Worms grown on Y. enterocolitica strains displayed a 47 % average decrease in progeny compared to worms grown on E. coli OP50. In addition, on average, only 23 % of the Fi progeny hatched on Y. enterocolitica plates survived compared to 75 % of the Fj generation hatched on E. coli OP50. The results demonstrate a deleterious effect of Y. enterocolitica on C. briggsae reproduction and growth. 3.3.2 Effect of Starvation To determine whether the observed worm fatality on plates with Y. enterocolitica as the food source was a result of starvation, worms were grown in the absence of a bacterial lawn. Prior to being introduced to the bacterial agar plates, L4 hermaphrodite C. briggsae were washed in 40 % sucrose to clean the outer cuticle 48 Table 6. C. briggsae progeny yield with E. coli or Y. enterocolitica as a food source. Bacterial Strain Average Total Average Surviving Average Survival Progeny (per worm) Progeny Percentage E. coli OP50 (control) 98 (± 13) 73 (± 6) 74.5 % (± 5.9 %) Y. enterocolitica 1613 isolate 59 (±21) 17 (± 9) 28.8 % (± 5.9 %) Y. enterocolitica 1564 isolate 40 (± 28) 10 (±7) 25.0 % (± 3.6 %) Y. enterocolitica MRS40 (YopE") 38 (± 16) 6 (±5) 15.8 % (± 8.5 %) 49 from contaminating bacteria. Once washed, the worms were placed on BHI E G T A 5 m M agar plates containing bacterial lawns of either E. coli OP50, Y. enterocolitica 1613, Y. enterocolitica 1564, or a plate lacking a bacterial lawn (NONE plate). Each assay was performed in triplicate and the results are reported in Figure 5. After 12 h of incubation, 85.7 % (± 14.3 %) of worms with no food source died. After 36 h of incubation, 100 % (± 8.2 %) worm death was observed with worms lacking a food source compared to 46.5 % (± 5.7 %) observed worm death with C. briggsae using Y. enterocolitica as a food source. C. briggsae survived longer with Y. enterocolitica as its food source than worms lacking a food source. The results demonstrate that the observed worm death in the C. briggsae - Y. enterocolitica pathogenesis assay was not solely due to starvation, but perhaps due to malnutrition. 3.3.3 Effect of EGTA Concentration In order to test the possible role of calcium in the C. briggsae — Y. enterocolitica model, subsequent studies were performed to determine the optimal E G T A concentration in the assay. E. coli OP50, Y. enterocolitica 1613, or Y. enterocolitica 1564 were grown and plated on BHI media with various E G T A concentrations (i.e., 0 m M , 2.5 m M , 5 mM, 10 mM, 15 m M , or 20 mM). Assays were performed as previously described and results are illustrated in Figures 6 and 7. Assays using E. coli (Figure 6) or Y. enterocolitica (Figure 7) showed that when the E G T A concentration was above 5 m M an increase in the incidence of worm death was observed. The results indicate that a calcium-depleted environment promotes worm death and that the optimal EGTA concentration for the assay is 5 mM. 50 Time (h) Figure 5. Infection assay results investigating possible signs of C. briggsae starvation when supplied with a foreign food source. Food source supplied in was E. coli OP50 (control), Y. enterocolitica isolates 1613 or 1564, and no bacteria (NONE plate). Bacterial strains were grown on BHI E G T A 5 m M agar plates overnight prior to worm introduction. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 51 Effect of [EGTA] (mM) using E. coli strain OP50 100.0% 80.0% re Q 60.0% o 40.0% 20.0% 0.0% - • — 0 mM EGTA - • — 2 . 5 mM EGTA - * — 5 mM EGTA - * — 10 mM EGTA - • — 1 5 m M EGTA -o--- 20 mM EGTA 24 36 48 Time (h) 60 72 Figure 6. Infection assay results investigating the effect of E G T A concentration (0 m M to 20 mM) on the incidence of C. briggsae death after exposure to E. coli OP50 as a food source. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 52 a Effect of [EGTA] (mM) using Y. enterocolitica strain 1613 100.0% T 80.0% -x : re d> Q 60.0% -O 40 .0% -20 .0% -0.0% tt 12 24 36 48 Time (h) 60 72 — 0 mM EGTA - • — 2 . 5 mM EGTA 5 mM EGTA — — 10 mM EGTA - • — 1 5 mM EGTA •-«•-• 20 mM EGTA Effect of [EGTA] (mM) using Y. enterocolitica strain 1564 100.0% i 80.0% -. c Io Q> Q 60 .0% -£ 40 .0% -20 .0% -0.0% I 12 24 36 48 Time (h) 60 72 - * — 0 mM EGTA - • — 2 . 5 mM EGTA -A— 5 mM EGTA - * — 1 0 mM EGTA - • — 1 5 mM EGTA •<>--• 2 0 mM EGTA Figure 7. Infection assay results investigating the effect of E G T A concentration (0 m M to 20 mM) on the incidence of C. briggsae death after exposure to (a) Y. enterocolitica 1613 or (b) Y. enterocolitica 1564 as a food source. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 53 3.3.4 Effect of Virulent vs. Avirulent Y. enterocolitica Strains The Y. enterocolitica 1613 and 1564 strains isolates obtained from the BC C D C are biochemically characterized as serotype 0:3. Other laboratories performing molecular studies on Y. enterocolitica normally utilize wild-type Y. enterocolitica strains 8081, E40, and WA-314 which are serotype 0:8 (Cornelis et al, 1987). Therefore, subsequent assays were performed with Y. enterocolitica strains of r serotype 0:8 (E40, WA-314, and BC CDC isolate 1035) to determine whether differences in the serotype would result in differences of worm fatality. The results are illustrated in Figure 8. A l l three Y. enterocolitica serotype 0:8 strains show similar fatality rates as observed with Y. enterocolitica serotype 0:3 (Figure 4). Both serotypes show an average 87.5 % worm death after 72 h of incubation (87.2 %, ± 4.2 % for serotype 0:3 and 87.8 %, ± 2.3 % for serotype 0:8). In addition, the original successful assays using Y. enterocolitica strains (Figure 4) showed that the Y. enterocolitica YopE mutant displayed a similar extent of worm death compared to the pathogenic Y. enterocolitica isolates. To test whether the observed worm death was directly related to the virulent nature of Y. enterocolitica strains, the Y. enterocolitica WA -C lab strain was utilized. The Y. enterocolitica W A - C strain is fully cured of the Yersinia virulence plasmid which encodes the Yersinia type III secretion system and associated virulence factors. A n assay using the Y. enterocolitica strain W A - C was carried out and the results are also shown in Figure 8. Worms exposed to the avirulent Y. enterocolitica WA -C strain performed better than worms exposed to the virulent WA-314 strain until 60 h of incubation. After 72 h of incubation, the majority (82.2 % to 90.5 %) of C. briggsae 54 100.0% Time (h) Figure 8. Infection assay results investigating the effect Y. enterocolitica strains of serotype 0:8 as a food source for C. briggsae. E. coli OP50 was utilized as a control Bacterial strains were grown on BHI E G T A 5 m M agar plates overnight prior to worm introduction. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 5  exposed to both virulent and avirulent Y. enterocolitica strains died, which indicates that exposure to avirulent Y. enterocolitica strains was as detrimental to C. briggsae as exposure to virulent strains. Thus, the results show that worm death observed within the assay does not correlate with known mammalian virulence mechanisms. 3.3.5 Visualization of Y. enterocolitica in C. briggsae To determine whether or not Y. enterocolitica strains were being ingested and retained within the gastrointestinal tract of C. briggsae, a reporter plasmid p S M l O l was utilized (Figure 1 A , Appendix A) . The plasmid construct p S M l O l contains a 0.97 kbp GFP ORF under lacZ promoter control (Mills, personal communication). The /acZ-GFP coding region was obtained from the pGFP construct produced by Clonetech (1998). Strains habouring the pSMlOl plasmid were able to express GFP fluorescence when induced with 1 m M isopropyl-l-thio-|3-D-galactopyranoside (IPTG). pSMlOl was transformed into the E. coli OP50 strain and Y. enterocolitica E40, WA-314, and W A - C strains. Subsequently, E. coli OP50-GFP and Y. enterocolitica WA-314-GFP, WA-C-GFP, and E40-GFP strains were plated and grown onto BHI T e t 1 ( W m L E G T A 5 m M I P T G l m M plates. C. briggsae hermaphrodites were introduced to the bacterial lawns. After 24 h and 48 h of incubation, the worms were fixed and mounted onto microscope slides. The results are shown in Figures 9 and 10. After 24 h of incubation, there is observable green fluorescence within the gastrointestinal tract of the worms exposed to E. coli OP50-GFP and Y. enterocolitica-GF? strains. However, the green fluorescence in the worms exposed to E. coli OP50-GFP was not uniformly seen throughout the digestive tract but rather localized to the pharynx or immediately behind the pharynx in the anterior region of 56 Figure 9. Visualization of Y. enterocolitica-GF? WA-314, W A - C , and E40 strains in C. briggsae after 24 h of exposure. Bacterial strains were grown on BHI Tet 1 0^ g / m L E G T A 5 m M I P T G l m M agar plates overnight prior to worm introduction. E.coli OP50-GFP strain was utilized as a control. 57 Figure 10. Whole worm (a) and head (b) visualization of Y. enterocolitica-GFV WA-314, W A - C , and E40 strains in C. briggsae after 48 h of exposure. Bacterial strains were grown on BHI T e t 1 0 ^ m L E G T A 5 m M I P T G l m M agar plates overnight prior to worm introduction. E.coli OP50-GFP strain was utilized as a control. 59 09 [9 the gastrointestinal tract (Figure 9). After 48 h of incubation, little to no green fluorescence could be observed in worms exposed to E. coli OP50-GFP except within the anterior region of the gastrointestinal tract (Figure 10). In contrast, after 24 h worms feeding on Y. enterocolitica-GFF strains were fluorescent throughout the gastrointestinal tract, although not all worms exposed to Y. enterocolitica-GFF strains displayed green fluorescence within their gastrointestinal tract (Figure 9). However, after 48 h all worms exposed to Y. enterocolitica-GFF strains displayed completely distended gastrointestinal tracts (Figure 10). Clearly, Y. enterocolitica is ingested by C. briggsae and retained in the gut. 3.3.6 Visualization of the EGTA Effect on Bacterial Retention in C. briggsae A n experiment was performed to observe the effect of EGTA concentration on the retention of Y. enterocolitica-GFF strains in the C. briggsae digestive tract (Figure 11). E. coli OP50-GFP was utilized as a control and regardless of the presence of EGTA, C. briggsae exposed to E. coli OP50-GFP displays green fluorescence only in the anterior portion of the gastrointestinal tract at both 24 h and 48 h intervals (Figure 11a). In the experimental condition, C. briggsae grown on Y. enterocolitica-GFF and 5 m M E G T A displayed fluorescence throughout the intestine after 48 h (Figure 1 lb and 1 lc). However, in absence of EGTA, the green fluorescence was observed within the pharynx or the anterior area of the intestinal lumen (Figure 1 lb and 1 lc), a similar observation as seen in worm exposed to E. coli OP50-GFP (Figure 1 la). Thus, both the rate of worm fatality and the bacterial retention in the intestine are elevated with the addition of E G T A to the culture media. 62 Figure 11. Whole worm visualization of GFP expressing bacteria strains grown in the presence and absence of 5 m M EGTA. Bacterial strains utilized were (a) E. coli OP50-GFP, (b) Y. enterocolitica WA-314-GFP, and (c) Y. enterocolitica WA-C-GFP. Mounting and fixing procedures of C. briggsae were performed at 24 h and 48 h intervals. Bacterial strains were grown on BHI T e t 1 0 > l g / m L i p T G l m M agar plates with or without E G T A (5 mM) overnight prior to worm introduction. 63 S9 99 3.3.7 Shifting of C. briggsae from Y. enterocolitica to E. coli OP50 To determine whether Yersinia strains could maintain colonization within C. briggsae after the presentation of a normal food source (i.e., E. coli OP50) the following experiment was performed. C. briggsae was exposed to Y. enterocolitica WA-314 and W A - C bacterial lawns on BHI E G T A 5 m M and incubated at 30°C. After 24 h of incubation, the worms were transferred to BHI E G T A 5 m M agar plates containing E. coli OP50 and incubation was continued at 30°C for an additional 48 h. The results of the shifting experiment are shown in Figure 12. The first 36 h of the assay displayed similar fatality rates as observed in previous infection assay experiments. However, after 36 h of incubation, a plateau was observed in the fatality rates of Yersinia-'mfected worms rescued by E. coli OP50. Fatality rates of the shifted worms remained at an average of 17.1 % (± 2.9 %). After 72 h from initial exposure, worm death from the shifted experiment increased, with final fatality rates averaging 37.0 % (± 3.5 %). The average final fatality rate of shifted worms from Y. enterocolitica to E. coli strains was comparable to the observed woim death of C. briggsae constantly feeding on E. coli OP50 (36.4 %, ±1 .8 %). However, the average worm shifted fatality rates are considerably lower than the average fatality rate observed when worms were exposed to Y. enterocolitica strains for 72 h (85.8 %, ± 1.4 %). Therefore shifting C. briggsae from Y. enterocolitica to E. coli within 24 h of incubation rescued the worms from the detrimental effects of feeding on Y. enterocolitica. The results indicate that the observed worm death may be due to a reversible malnutrition illness caused by Y. enterocolitica. 67 100.0% 0 WA-314 24h; OP50 48h — - W A - C 24h; OP50 48h ^ - W A - 3 1 4 OP50 WA-C 0 12 24 36 48 60 72 Time (h) Figure 12. Infection assay results investigating the effect of shifting C. briggsae from Y. enterocolitica strains to E. coli OP50 strain as food source. Worms were subjected to Y. enterocolitica strains WA-314 and W A - C for 24 h then transferred to E. coli OP50 for 48 h. Bacterial strains were grown on BHI E G T A 5 m M agar plates overnight prior to worm introduction. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 68 3.3.7.1 Visualization of Shifting C. briggsae from Y. enterocolitica to E. coli In proceeding experiments worms were exposed to GFP-expressing Y. enterocolitica WA-314 and W A - C for 24 h and subsequently transferred to plates containing E. coli OP50 for an additional 24 h. The visualization of these results is shown in Figure 13. C. briggsae exposed to both Y. enterocolitica WA-314-GFP and W A - C-GFP strains show fluorescence in the gastrointestinal tract even after having E. coli OP50 as available food source. The observed green fluorescence of the gut was similar to the fluorescence seen in C. briggsae exposed to Y. enterocolitica strains for the full 48 h (refer back to Figure l i b and 11c). The experiments show that although Y. enterocolitica is retained within C. briggsae, the retention of Y. enterocolitica is not directly responsible for worm fatalities. 3.3.8 C. briggsae on Mixed Bacterial Lawns of Y. enterocolitica and E. coli To determine whether a competition existed between bacterial strains for ingestion and digestion, both Y. enterocolitica WA-314 and W A - C strains were each plated with E. coli OP50 on BHI E G T A 5 m M agar plates. C. briggsae was introduced to the bacterial lawns and scored for worm death every 12 h. The results are summarized in Figure 14. The results show an increase in fatality rates of worms exposed to both Y. enterocolitica and E. coli strains at the same time. After 48 h, worms exposed to a mixture of Y. enterocolitica and E. coli displayed an average worm fatality more than double (53.0 %, ± 2.3 %) the average fatality rate of worms exposed to Y. enterocolitica strains alone (22.1 %, ± 2.9 %). This increase in worm fatality continued with average fatality rates of 91.6 % (+ 4.3 %) after 72 h of exposure to Y. enterocolitica - E. coli mix compared to 85.8 % (± 1.4 %) when worms were exposed to Y. enterocolitica strains 69 Figure 13. Whole worm visualization from bacterial shifting experiments. C. briggsae was exposed to Y. enterocolitica-GF? WA-314 and WA-C for 24 h then transferred to E. coli OP50 for an additional 24 h. Bacterial strains were grown on BHI T/ et 1 0* 1 8 / m L E G T A 5 m M i P T G l m M (for Y. enterocolitica-GF? strains) and BHI E G T A 5 m M (for E. coli OP50 strain) agar plates overnight prior to worm introduction. 70 100.0% Time (h) Figure 14. Infection assay results investigating the effect of presenting C. briggsae with a mixture of E. coli and Yersinia strains together as a food source. E. coli OP50 was plated with Y. enterocolitica strains WA-314 and W A - C and strains were grown on BHI E G T A 5 m M agar plates overnight prior to worm introduction. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 72 alone. The elevated onset of fatality rates suggest that it is more detrimental for C. briggsae to feed on a mixed food source of Y. enterocolitica - E. coli than feeding on Y. enterocolitica alone. 3.3.8.1 Visualization of Y. enterocolitica - E. coli mix in C. briggsae In order to differentially visualize Y. enterocolitica within C. briggsae after exposure to a Y. enterocolitica - E. coli mix, sub-cloning of p S M l O l was required. A n 800 bp Asel - BstBl deletion within pSMlOl allowed the removal of the lacZ promoter and the GFP coding region, creating pNLP8. Strains harbouring pNLP8 would be tetracycline resistant but be unable to express GFP. Subsequently, pNLP8 was transformed into the E. coli OP50 strain. Worms were introduced to bacterial lawn of either Y. enterocolilica-GF? WA-314 or W A - C , each mixed with E. coli OP50 habouring pNLP8. Any observed green fluorescence within the worm would only indicate the presence of Y. enterocolitica strains. The results are shown in Figure 15. After 24 h of exposure, green fluorescence was observed in the pharynx and anterior portion of the gastrointestinal tract of C. briggsae (Figure 15a). In Figure 15b, different distributions were seen in living and in dead worms. For worms still alive after 48 h of incubation, half the population displayed a fully illuminated intestinal lumen while the remaining half showed no green fluorescence whatsoever (1:1 ratio, n = 72). However, the inspection of worms that died after 48 h of exposure revealed that the majority of the population (83 %, n = 60) had a fully illuminated gastrointestinal tract (Figure 15b). Since dead worms displayed a fully illuminated tract, these results suggest that worm death is a result of the presence of Y. enterocolitica within the gastrointestinal tract. 73 Figure 15. Whole worm visualization of Y. enter•ocolitica-GFP WA-314 and WA-C each mixed with E. coli OP50 after (a) 24 h and (b) 48 h of exposure. Figure 15c visualizes the head of C. briggsae after exposure to Y. enterocolitica-GF? WA-314 - E. coli OP50 mix. Bacterial strains were grown on BHI Tet 1 0 f l g / m L E G T A 5 m M l P T G l m M agar plates overnight prior to worm introduction. 74 75 77 3.4 Re-examination of the Effect of C. briggsae on S. typhimurium Exposure to Y. enterocolitica strains in a low-calcium environment appears to have a detrimental effect on C. briggsae. To determine whether this observation was limited to Yersinia species, additional experiments were performed with S. typhimurium and EGTA. In Figure 16, C. briggsae exposed to S. typhimurium in the absence of E T G A show similar fatality rates as worms exposed to E. coli OP50. However, when C. briggsae was exposed to S. typhimurium grown in BHI E G T A 5 m M media, the worm fatality rate was significantly greater (44.9 %, ± 3.2 % after 48 h and 95.9 %, ± 2.1 % after 72 h) compared to E. coli OP50 control (10.7 % ± 2.1 % after 48 h and 55.4 %, ± 2.8 % after 72 h). The observed results are similar to those recorded with C. briggsae feeding on Y. enterocolitica (refer to Figures 4 and 6), which suggests that the presence of E G T A and/or low calcium environment itself contributes to worm fatality. 3.4.1 Visualization of S. typhimurium in C. briggsae To ensure that S. typhimurium strains were being ingested by C. briggsae in low-calcium environment, pSMlOl was transformed into S. typhimurium SL1344, creating a Salmonella strain capable of GFP expression for visualization S. typhimurium SL1344 within C. briggsae. Therefore, S. typhimurium SL1344-GFP was grown and plated onto BHI Tet 1 0 M g / m L I P T G l m M plates in the presence and absence of 5 m M EGTA. C. briggsae were introduced to the S. typhimurium bacterial lawns and incubated at 30°C. Worm fixing and mounting was performed at 24 h and 48 h of incubation and results are shown in Figure 17. In a depleted calcium environment, worms exposed to S. typhimurium-GY? strains displayed a completely 78 100.0% 80.0% I 60.0% 40.0% 20.0% 0.0% A 12 24 36 48 60 72 Time (h) -•— OP50 (0 mM EGTA) • - O P 5 0 (5 mM EGTA) SL1344(0 mM EGTA) SL1344 (5 mM EGTA) Figure 16. Infection assay results using S. typhimurium SL1344 strain grown in BHI and BHI E G T A 5 m M liquid and solid media. E. coli OP50 strain was utilized as control. Worms were scored for death and transferred to fresh bacterial lawns every 12 h. A worm was considered dead when it no longer responded to touch and no pharyngeal pumping could be observed. 79 Figure 17. Whole worm visualization of S. typhimurium-GFF SL1344 strain grown in the presence and absence of E G T A 5mM. Mounting and fixing procedures of C. briggsae were performed at 24 h and 48 h intervals. Bacterial strains were grown on BHI T e t 1 ( W m L I P T G l m M agar plates with or without 5mM EGTA overnight prior to worm introduction. 80 IS distended gastrointestinal tract after 48 h, similar to that seen previously with Y. enterocolitica strains (refer to Figures l i b and 1 lc). This demonstrates that S. typhimurium is ingested by C. briggsae and retained in gut after 48 h from the time of initial exposure. However, in culture media containing free calcium, the fluorescence of S. typhimurium-GYV strain can only be visualized in anterior regions of the pharynx, which is the same observation of worms grown on E. coli OP50 (refer to Figure 11a). 3.5 Virulence Gene Subset Database Since bacteria often make use of host cellular processes in order to establish and sustain an infection, previously identified host homologs may be involved in the infection process. To assess the presence of possible bacterial protein homologs expressed within C. elegans, additional efforts were undertaken to create a V G S database which lists identified virulence proteins from various bacteria pathogens. 3.5.1 Compilation of V G S Data Through reviewing various published literature and selected microbiological textbooks, the VGS database was developed. This database lists previously characterized proteins that have been identified to be associated with virulence. The current database lists a collective 305 different pathogencity factors from 19 different bacterial pathogens. A sample of the V G S database is listed in Table 7, which displays 20 pathogencity factors from various bacterial pathogens. 82 c CD Q. CD a 5 ft 2 0 2; 0 P = £ ss- S 2* S- E* S> " 3 c O g a" f» 5 * 3 P o p 0 O . g . 1 / 1 5. S 3 3 ' • C ° -' 3 ft 3 2| § 3 8? cr CD 2 » » 3 „ . 0 n 3 •->> Cc ci ^ R- — c r ta 1 8 c 3 a 5- 0 P CD CD RT § § § _ CD era CS p . p CO "1 ff 5" & CL 3 Ocra 3 § 3 2? CT" ft C u e c r 3 ^ 2 ** 0 o g 2, a a " i o S 3 3 z & ffQR 0 2 5' 0. . g su P & s g I. o. 01 3> 3" a' K -(V (V (V 2 3 2 S' 75 O N + O i N O -J N O N O IO « "2 Co 3 CD p c r p 0 CD co S. co* 3 CD Cu CT* p o ci o 3 o to 5' 5' 2 2 S £? £3 Pi Pi TO TO Pi 3 3 3 3 <? PJ PJ P? (? ~t "1 M O O 0 0 O O O 0 0 O O O 0 0 O «~~. *-* cv S' 0 ' 0 ' 0 ' 0 a a 0 a s= 3= 3 to to tO IO IO tO —1 —J —) O O O U > t>J U J 00 o o 2 2 m IO o 00 D r 0 0 0 0 0 N O N O N O N O o N O O l O l O l O l O l O l O l O l O l O l O l O l O l O l co 4*. u> O i I O O N O N O p Ci 3 * z: a 3 o a * ta 3 3 -3 CD a ft B-CD 3» o -a 0 cr 1 3 CD CO a CD 2 0 CD ft -1 3 $ ° % s-O 3 o •CT (A O 3 CD CO •a o 3 to to o o C J J u» r Ci 0 O l O l O N O CL. o o o o o 5 ? 2 a. > O N N O O N O X co > > 3 > 2 o o cr o_ cT CD C i o o Ci R Co 3 Pi S 3 o 3 70 O N O l O l O l O N ~ J O i N O cr P CD O > 2 . N O o to o 4^ •a 3 CD c p O CD T3 O CD 5' > o o Ci Ci s 3 PJ S o 3 S' Pi •a > 3 c o ^ & £ £ Pi o o o o Ci 3 o 3 PJ s 3 61 S"i fi] Co Co Co Ci Ci 3 - 3 -Pi Ci 3 -Pi o -:. J . J . 3 s S' 3 N ? a 3" <*3 o 3 O Pi 3 5 Ci Ci O o 3 -rs Z w O N G O H r is w - H > O to to Q ^ 0 O m « i_ -v 01 01 tri ^ o X X r > X p CD O o T3 M C 1 I "A W x _ c:- co > ^ > X n H o O l N O o ~-J N O O N P P P ft 3 O CD CO P 5' 3 C c V p p co n. 00 4^ to O l O N O N O I ~ J O i O N N O O N to 01 o l r ft P o A- CD o cc 5' p c CT" O i CD X o o X to o O l 4i. ~ J 00 to N O O O O l U ) O l to O i 00 cr CD P C L CD ^ c a B . c. c. N < 3 — ^ -o 3 — 5" > CD P > H p Cf" Typ i fac AD pore CD 0 *? a "-1 0 c r 3 00 0 0 ~ , CD 3 CO 3 O •-t era CD 0 p CD CO 0 5' 5' >< 3 0Q m 3 ' 09 1 a> 3 to CD N O 00 to to N O N O to 00 _I O N to N O to O N >—' - J 4*. i—. N O O l O l N O 0 0 to O l ~-4 1—1 00 N O u> N O to 0 O N O O O N O N O O 0 O N O N O l 4*. 0O 00 O O l - O O l N O 1—» - J O N 1—. to 0 0 O 4^ to O N O N O O l to - O O l O0 O l l—* »—» - 0 to u> l>i 00 N O O l N O O l ~J N O 4*. O l O N 00 »—» ^4 4^ O0 0 U J u> 00 O i N O 00 --4 0 U i O l 4 -^ U ) O i 4 .^ ~ J 4i- 4 .^ 0 N O 00 J O O N to '* N O N O O l 0 to N O O0 )_ to ~ J to 1—' N O 00 4^ O l N O 0 t ' 00 O N O l 0 ~ J N O O l O l O 00 - 4 00 4*. N O O N N O 4i . to O O l 00 O - 0 N O 0 N O O O - 0 U ) N O to O N O l -4 XA o »-*• 1—». o 0 o Cu p a R 5 O 3 tr CD < O Cu a c ^ p CD 3.5.2 BLAST Analysis and Identification of Putative Protein Homologs A B L A S T search was performed on the listed pathogencity factors against the C. elegans genome to identify possible protein homologs between bacterial pathogens and C. elegans. The criteria for notable homologs consisted of amino acid similarity greater than 30 % and an e-value less than e"20. A selection of the list of the top homolog hits between C. elegans and pathogens is displayed in Table 8. Some of these high hits include alginate regulatory protein, internalin, tRNA-ribosyltransferase, and ATP binding cassette (ABC) transporters; however, the overall majority of VGS database entries did not yield high sequence homology hits with C. elegans proteins. 84 £8 ' 3' ST 0Q o 2 5" cr o x Q. > cr o 3 < o O cr o 3 £3 W 73 < CO > H T3 3 Q. 3' HQ o O 3 T3 O 3 ft 3 EL CTQ 5' ?2 ft <JQ O o 2 5' £L 5" > > — cr 5' 5" OQ -a - i o •0 | (era : ft ^1 U i U i U l N O O N U i U l U i N O N O N O U i O N O OO o o U l U l O N N O O U i o U J K) N O 0 0 —• 0 0 o o K ) o U i oo O N U J ^1 ^1 0 0 0 0 U J oo OO ^1 N O N O •— U J O N K > O TO o TO TO s c^  t 3 3 TO K 3 O 3 S' ro -3 o TO o TO TO s 13 3 TO S 3 o 3 S' TO t O TO O TO TO S 3 TO S 3 o 3 S' TO 3 TO 3 TO TO S §• 3 o 3 TO 3 ' p TO S §" 3 © 3 8 o TO 3 ' O TO* O 3 O TO 3 TO 3 -TO >t TO' 3 -S' TO O 8) o era re CO o to oo O N o U l o N 7< oo N O O N O O a N 8 oo u i > 7^ H — o o NO O > K> U l I— N O O o ^ 3 U J u> U J o — U J o U J U J 4> o U J U J U J 0 0 — — U J 4^ era o o o © o rt o o j> — — o o ft o o o o U J b b o o o o o o o o o o M l < 69 C H A P T E R IV 4.0 DISCUSSION 4.1 Re-Examining C. elegans as a Host Model The utilization of C. elegans as a host model for infection has been recently addressed by various research groups. Hodgkin et al. (2001) have successfully demonstrated an infection between M. nematophilum and C. elegans which results in a specific and identifiable phenotype (i.e., the Dar phenotype). Other research groups such as those of Ausubel, Manoil, Jeddeloh, Ewbank, and their colleagues, have reported infection within C. elegans using a variety of bacterial pathogens (e.g., P. aeruginosa, S. typhimurium, Burkholderia spp., and E. faecalis), in which infection was characterized by elevated worm fatality rates and distended gastrointestinal tracts (Darby et al., 1999; Tan et al, 1999a; Aballay et al., 2000; Labrousse et al., 2000; Garsin et al., 2001; O'Quinn et al., 2001). However, due to the lack of a definitive infection phenotype as described by Hodgkins et al. (2001) and the admittance by several groups that observed elevated worm death from exposure to P. aeruginosa, B. thuringiensis, and Burkholderia spp. is the result of toxicity (Darby et al., 1999; Mahajan-Miklos et al, 1999; Marroquin et al., 2000; Gallagher and Manoil, 2001; O'Quinn et al., 2001), the interpretation of an established infection in C. elegans using mammalian pathogens remains ambiguous. To investigate the possibility of characterizing an infection in C. elegans, this thesis examined the effects of culturing C. elegans on bacterial pathogens such as S. typhimurium, Y. enterocolitica, Y. pseudotuberculosis, and L. monocytogenes. 86 Initial assays were designed following the protocol outlined in the paper by Tan et al. (1999a) in which C. elegans was introduced to a P. aeruginosa bacterial lawn grown on N G or PGS agar plates at 25°C. For this project, C. elegans was introduced to Y. enterocolitica and S. typhimurium bacterial lawns grown o n N G agar plates and scored for worm death. In all initial attempted assays, no elevated worm death, worm infertility, or visible mutant phenotype was observed after 120 h from the initial exposure to pathogenic bacteria compared to worms feeding on E. coli OP50. The results differ from the results of Tan et al. (1999a) who reported increased rates of worm death within 4 - 72 h using P. aeruginosa. The success of the assay established by Tan et al. (1999a) was limited to a specific strain of P. aeruginosa, namely PA14. Other strains utilized by Tan et al. (1999a) such as P A K and P A O l , did not yield significant worm fatalities. The clinical isolate P. aeruginosa PA 14 strain was originally isolated from a plant infection and was later found to be virulent in mice (Rahme et al., 1995; Mahajan-Miklos et al, 1999). Thus the success of the assay observed by Tan et al (1999a) may be due to the broad host range of this specialized strain of P. aeruginosa. The successful establishment of the C. elegans - P. aeruginosa pathogenesis model may be an unique circumstance since P. aeruginosa is primarily a plant pathogen and an opportunistic pathogen in human hosts. The broader host range of P. aeruginosa compared to other enteric pathogens such as Y. enterocolitica and S. typhimurium may explain the sole success using P. aeruginosa in assays with C. elegans. On the other hand, despite a prolonged exposure time ( 5 - 8 days), a series of successful infection assays were reported using C. elegans with other Gram-negative (i.e., S. 87 typhimurium and S. enterica) (Aballay et al, 2000; Labrousse et al, 2000; Aballay et al, 2001) and Gram-positive (i.e., E. faecalis, Streptococcus pneumonia, and Staphylococcus aureus) (Garsin et al, 2001) human pathogens, suggesting that C. elegans could be infected with enteric human pathogens. In addition, Darby et al. (1999) later demonstrated that P. aeruginosa P A O l grown under different conditions caused lethal paralysis of C. elegans after 6 h of exposure. The P. aeruginosa culture methods utilized by Darby et al. (1999) were different from those utilized by Tan et al (1999a), which suggested the importance of bacterial culture media and growth conditions to establish a successful assay with C. elegans. 4.2 General Assessment of Mammalian Infection Environment Subsequent directions of this project investigated several possible factors that would contribute to the lack of bacterial infection within C. elegans. Since mammalian pathogens were being utilized in this investigation, the significance of proper culture media and incubation temperature for mammalian pathogens was investigated. 4.2.1 Relevance of Nematode and Bacteria Media A first consideration was the nutritional content of bacterial and nematode culture media. Tan et al. (1999a) observed different worm death rates using different nematode media. For example, worm death was observed between 48 - 60 h after initial infection when bacterial strains were grown on N G plates. However, when PGS agar plates (i.e., 1 % Bacto-Peptone; 1 % NaCl; 1 % glucose; 0.15 M sorbitol; 1.7 % Bacto-Agar) were utilized, worm death was observed in 4 - 10 h after initial 88 exposure to the pathogen. This so-called "fast-killing" observation was also reported by Mahajan-Miklos et al (1999) when PGS was utilized for the C. elegans - P. aeruginosa infection assay. The elevated fatality rates were attributed to added sorbitol within the culture media which produces high osmolality conditions within the growth environment, causing increased diffusion rates of toxins from P. aeruginosa (Mahajan-Miklos et al, 1999). The fact that worm paralysis and death was discovered to be mediated by excreted toxins from P. aeruginosa (Darby et al., 1999; Mahajan-Miklos et al, 1999; Gallagher and Manoil, 2001) brings about debate whether C. elegans death could be accurately labeled as an infection. Essential nutrients are important for virulent gene expression in pathogens and the importance of appropriate culture medium has been reviewed by Tan et al. (2000). A n increase in worm death was observed when P. aeruginosa P A O l was grown in BHI media compared growth in LB media (Darby et al, 1999). Investigations of infection models using C. elegans and Gram-positive human pathogens using BHI media have also been reported (Garsin et al, 2001). Therefore, this study tested BHI broth by substituting it for L B as overnight culture media for Y. enterocolitica and S. typhimurium strains. As N G M agar plates are optimized for C. elegans growth, additional directions were undertaken to observe C. elegans behaviour on BHI agar plates in order to determine the effect of BHI media on worm growth and mortality. C. elegans fed on N G M plates at 25°C displayed a 7.1 % (+ 1.0 %) worm fatality after 72 h of incubation. When exposed to BHI media, the C. elegans fatality was observed to be five times higher (38.6 %, ± 1.0 % after 72 h) than worms on N G M 89 control media. Moreover, when cultivating C. elegans on BHI agar plates, Garsin et al. (2001) also noted a detrimental effect on the C. elegans life-span which suggests that infection assays using C. elegans on BHI agar plates may not be suitable. 4.2.2 Relevance of the Incubation Temperature A n additional factor in establishing an infection model between C. elegans and a pathogen was the issue of incubation temperature. The optimal growth temperature for C. elegans is 20°C and the maximum temperature in which C. elegans remains viable is 25°C (Lewis and Fleming, 1995). Temperatures exceeding 25°C result in worm infertility and a shortened life-span. However, virulent gene expression in enteric pathogens is regulated by temperature, with the optimal temperature being 37°C. For example, in Yersinia spp., the virulent genes yadA and psaA, as well as the yop operons, are up-regulated at 37°C and down-regulated at temperatures below 26°C (Straley and Perry 1995). A possible explanation of unsuccessful infection of C. elegans with an enteric bacterium could be due to the lack of virulent gene expression during room temperature incubation (i.e., ~ 22°C) or the lack of essential environment cues that are involved with the initial stages of pathogenesis (i.e., proper ion concentration, pH, and/ or nutrient content). To address the problems of culture media and temperature in an infection assay using mammalian enteric pathogens, it would be beneficial to utilize a Caenorhabditis strain that could grow on BHI media and survive at temperatures close to 37°C, the temperature of a mammalian infection environment. 90 4.2.3 Utilization of Thermotolerant Caenorhabditis Strains The search for Caenorhabditis strains that were capable of remaining viable and fertile at higher temperatures than 25°C resulted in the acquisition of two Caenorhabditis species, namely a C. elegans daf-2 mutant and the wild-type C. briggsae, var. Gujarati G16. The mutant C. elegans daf-2 has been reported to survive at higher temperatures than 25°C (Lithgow et al, 1995; Gems et al, 1998) and another group reported that wild-type C. briggsae G16 strain remains viable at 30°C (Yarwood and Hansen, 1969). Thermotolerance testing was performed on the C. elegans daf-2 mutant and C. briggsae G16 strains to evaluate the maximum temperature each organism could survive at and remain fertile. From this experiment it was determined that C. briggsae G l 6 exhibited the highest thermotolerance, remaining viable and semi-fertile at 30°C for 72 h. In addition to thermotolerance, C. briggsae G16 also grew better on bacterial BHI media streaked with E. coli OP50 compared to C. elegans. After 72 h at 25°C, 9.8 % (± 0.2 %) and 9.1 % (± 1.3 %) of C. briggsae died on N G M and BHI agar plates, respectively. However, after 72 h of growth at 30°C, the fatality rates of C. briggsae increased to 10.0 % (± 1.4 %) when grown on N G M agar plates and 36.4 % (± 1.8 %) when cultivated on BHI agar media. Despite the sharp increase of C. briggsae death on BHI agar media at 30°C, the C. briggsae fatality rate is considerably less than C. elegans N2 incubated 30°C on BHI solid media (61.8 %, + 3.5% after 72 h). From the results, the C. briggsae G16 is a wild-type strain capable of remaining alive while growing on BHI solid media at 30°C. Therefore, C. briggsae G16 was selected for testing as a host model that could be potentially 91 infected with mammalian enteric pathogens in conditions close to the mammalian infection environment. 4.3 C. briggsae as a Host Model for Mammalian Enteric Pathogens Subsequent assays were performed at 30°C and involved placing C. briggsae G16 on BHI culture plates spread with various laboratory wild-type and mutant strains of P. aeruginosa, S. typhimurium, L. monoctyogenes, and Y. enterocolitica. In addition, several clinical isolates of L. monoctyogenes, Y. enterocolitica, and Y. pseudotuberculosis were obtained and tested. As a control, C. briggsae was placed on BHI agar plates spread with E. coli OP50. In total, 27 different bacterial strains were assayed. In each case, no significant worm death was observed after 96 h (4 days) of exposure. Of the 27 bacterial pathogens assayed in this investigation with C. briggsae only two bacterial strains, P. aeruginosa P A K and S. typhimurium SL1344, have been utilized by other groups investigating C. elegans as a host model. After 3 days at 25°C, Tan et al. (1999a) reported no sign of elevated worm death using the P. aeruginosa P A K strain with C. elegans. Published data regarding infection assays using C. elegans grown on S. typhimurium SL1344 at 25°C reported elevated worm death only when C. elegans was exposed to S. typhimurium for more than 6 days (Aballay et al., 2000; Aballay and Ausubel, 2001). Therefore, comparing results only within a 4 day exposure period, the results of the assays using C. briggsae grown on P. aeruginosa P A K and S. typhimurium SL1344 are similar to the published data of assays using C. elegans, as no elevated worm death was reported during a 4-day time frame. These results demonstrated the rarity of finding a 92 detrimental effect of growing C. briggsae on mammalian bacterial pathogens, suggesting the need for additional investigations to determine specific assay conditions in establishing an infection model. 4.4 C. briggsae - Y. enterocolitica Infection Assay in a Low C a 2 + Environment The pathogenic process of Yersinia is highly dependent on the expression of the Yersinia virulence plasmid, p Y V , which encodes most of the Yersinia virulence factors (i.e, Yops, Yscs, and Syns). The p Y V has been found to be tightly regulated by the L C R system, in which m M changes in C a 2 + levels within the surrounding environment can alter the transcription levels of Yersinia virulence proteins (Straley, 1991). The observed L C R mechanism is that in the absence of C a 2 + ions, virulent Yersinia strains would actively secrete Yops, Yscs and Sycs in large quantities (Straley and Bowmer, 1986; Cornelis etal.; 1989; Price etal., 1991; Straley, 1991; Koornhof et al., 1999). It is the understanding that such secretion behaviour of virulence proteins would enable Yersinia to invade a eukaryotic host cell. Experimentally, in Yersinia spp., the L C R can be activated in vitro by the presence of E G T A which chelates C a 2 + ions within the culture medium grown at 37°C (Cornelis, 1998). As L C R gene products sense external Ca levels within the medium, it is possible that L C R may be also involved in sensing the proximity of a host cell and/or the host environment to initiate an infection, such that low levels of C a 2 + ions may be the environmental cue of a neighbouring host cell. Since N G M and BHI mediums are rich in calcium and the Caenorhabditis intestinal lumen is not as differentiated as the 93 mammalian gastrointestinal tracts, perhaps the failure to observe infection of Yersinia spp. in C. briggsae may be explained by the lack of proper environmental cues (i.e., low calcium levels) within the lumen of C. briggsae. Therefore, new approaches were undertaken to design C. briggsae - Y. enterocolitica infection assays involving the usage of E G T A within the bacterial culture medium. The first attempted infection assay using E G T A involved two clinical Y. enterocolitica 0:3 isolates (1613 and 1564) and a Y. enterocolitica YopE mutant. Within 48 h of exposure to Y. enterocolitica strains, the average fatality rates were four times higher than the control. Assays utilizing Y. enterocolitica serotype 0:8 strains showed similar elevated worm death patterns as previously observed using Y. enterocolitica 0:3 strains, indicating that the serotype of Y. enterocolitica strains has no direct effect on the incidence of observed worm death. In addition to elevated worm death, the observed brood size of C. briggsae exposed to Y. enterocolitica strains was 46 % lower compared to C. briggsae feeding on E. coli OP50 and survival of the Fi generation decreased by three-fold. The decrease in brood size and progeny death was also reported in C. elegans infection assays using P. aeruginosa PA14 (Darby et al., 1999; Tan et al., 1999a), S. typhimurium SL1344 (Aballay et al., 2000), and E. faecalis strains (Garsin etal., 2001). Additional observations including an egg laying defect observed in C. elegans exposed to S. typhimurium, in which worms became laden with eggs and progeny hatched internally (Aballay et al, 2000); however, in the infection assay between C. briggsae and Y. enterocolitica, no egg laying defect was observed. 94 The data collected from the experiment wherein the E G T A concentration was varied within the media suggested that the optimal concentration of E G T A for infection assays is 5 m M and E G T A concentrations exceeding 5 m M may exert a toxic effect on C. briggsae. Within the literature, there have been no direct experiments performed on C. elegans regarding the nutritional importance of calcium. However, several groups have indicated numerous diverse roles that calcium plays within C. elegans in the nervous system (e.g., function of olfactory neurons, isothermal tracking, and axonal branching) (Troemel etal., 1999; Gomez etal., 2001; Wang and Wadsworth, 2002), fertilization (Samuel et al, 2001), and muscle contraction (e.g., pharyngeal pumping) (Kerr et al, 2000). Within in this study, it was noted that the rate of pharyngeal pumping and locomotion decreased during the course of the assay, which may be explained by the lack of calcium in the media, resulting in the decreased stimulation of muscle contractions. 4.4.1 The Role of the Yersinia Virulence Plasmid The decreased survival of C. briggsae observed using Y. enterocolitica strains suggested the possibility that Y. enterocolitica virulence determinants attributed to the killing of C. briggsae; however, the same detrimental effects were also observed when C. briggsae was grown on the Y. enterocolitica YopE mutant and the avirulent W A - C strain. Y. enterocolitica strains harbouring ayopE mutation are unable to initiate an infection as YopE is known to be essential virulence cytotoxin which is injected into the host cell during the initial stage of infection (Rosqvist et al. 1991; Rosqvist et al, 1994). The Y. enterocolitica WA -C strain does not harbour the Yersinia virulence plasmid and therefore lacks the yop, syc, and ysc genes that 95 express Yersinia virulence determinants. However, C. briggsae grown on both attenuated Y. enterocolitica strains resulted in similar worm death as C. briggsae grown on virulent Y. enterocolitica strains. Therefore, the detrimental effects of growing C. briggsae on Y. enterocolitica strains do not correlate with the expression and active secretion of virulence factors by the L C R in Yersinia spp. One possible explanation of the observed elevated worm death, other than a supposed infection, was that the worms were malnourished or starving during exposure to Y. enterocolitica strains. When this possibility was tested, it was found that within 12 h without a food source, 85.7 % (± 14.3 %) of the worms died showing that a lack of food source dramatically shortens the worm life-span. This data demonstrated that C. briggsae death was not due to starvation; however, the possibility of death due to malnutrition remained unresolved. The issue that the life-span reduction of worms grown on pathogenic bacteria could be due to malnutrition was recently assessed by Couillault and Ewbank (2002). The group measured worm survival when C. elegans was grown on various heat-killed bacterial species and showed that worms grown on most heat-killed pathogens (e.g., Agrobacterium tumefaciens, Aeromonas hyrdophila, Erwinia carotovora, and Photorhabdus luminescent) displayed similar survival rates to worms grown on E. coli OP50, suggesting that worm death was independent to dietary deficiency (Couillault and Ewbank, 2002). The only exception to this observation was worms grown on heat-killed Xenorhabdus nematophila, which resulted in reduced life-span compared to C. elegans grown on viable X. nematophila. The authours suggest that X. nematophila possibly contained a heat-resistant product with hemolytic activity which was had a 96 deleterious effect on C. elegans (Couillault and Ewbank, 2002). Evidence of a toxin-mediated killing has already been presented by several groups investigating interactions between C. elegans and various pathogens such as, P. aeruginosa (Darby et al., 1999; Mahajan-Miklos et al. 1999; Gallagher and Manoil, 2001), B. thuringiensis (Marroquin et al., 2000), and Burkholderia spp. (O'Quinn et al., 2001). In addition, Garsin et al. (2001) also noted that worms grown on E. coli OP50 shorten the worm life-span when using BHI media. The group suggested that rich bacterial media induces expression of E. coli factors which are lethal for C. elegans. The possibility that bacterial products that are not identified as mammalian virulence determinants could be lethal the worm suggests that there may be similar lethal products within Y. enterocolitica that are detrimental to C. briggsae. 4.5 Visualization of Y. enterocolitica in C. briggsae To determine whether Y. enterocolitica was being ingested and/or retained within C. briggsae, GFP-expressing E. coli OP50 and Y. enterocolitica were utilized. For E. coli, green fluorescence was restricted to the anterior region of the intestinal lumen. In comparison, Y. enterocolitica-QYY proliferation was observed throughout the intestinal lumen in the worm. Not all worms growing on Y. enterocolitica as a food source showed Yersinia-GFF strains within the gastrointestinal tract after 24 h; however, at the 48 h interval the majority of worms displayed a fully distended gut. These results demonstrated that C. briggsae ingest but may not digest Y. enterocolitica within the gastrointestinal tract Since C. briggsae digested E. coli, expression of the GFP gene decreased resulting in diminished fluorescence. Only 97 small amounts of undigested E. coli were seen posterior to the pharynx. This GFP data shows that some E. coli and Y. enterocolitica strains escape the mechanical digestion from the grinder in the worm's pharynx and enter the gastrointestinal tract of C. briggsae undigested. However, GFP data also shows that the E. coli strains are eventually digested within the gastrointestinal tract (loss of fluorescence), while the Y. enterocolitica strains are not properly digested as the GFP expressing Y. enterocolitica remains visible within the intestine of C. briggsae. Several published papers have used bacterial GFP-expressing strains to visualize bacteria within C. elegans. Tan et al. (1999a) showed that C. elegans grown on the P. aeruginosa PA14-GFP strain resulted in a fluorescent distended lumen after 36 h of exposure. In contrast, C. elegans grown on an E. coli-GFF DH5a strain or a P. aeruginosa-GFF GacA mutant showed no detectable green fluorescence within the lumen (Tan et al, 1999a). Other groups demonstrated the same distended gut phenotype within the worm when utilizing GFP-expressing S. typhimurium strains in infection assays with C. elegans (Aballay et al, 2000; Labrousse et al, 2000). In each case the authours attribute the distended gastrointestinal tracts as evidence of a bacterial infection. Similarly, in this study, obtained GFP data from the Y. enterocolitica-GFF strain shows a fluorescent distended gut within C. briggsae; however, whether this demonstrates infection remains unresolved. Recently Darby et al (2002) performed GFP studies with Yersinia spp. and demonstrated a biofilm formation around the mouth of C. elegans after exposure to Y. pestis and Y. pseudotuberculosis. In their paper the authours demonstrate that GFP-expressing Y. pseudotuberculosis strain form this biofilm, resulting in the blockage of 98 food intake and ultimately lowers nematode survival. In this thesis, a related Yersinia species, Y. enterocolitica, was ingested by C. briggsae and no prominent biofilms around the mouth of C. briggsae were observed. Perhaps the hmsHFRS-dependent extracellular biofilms are unique to Y. pseudotuberculosis and Y. pestis or the different assay conditions described in the paper explain the biofilms reported by Darby et al. (2002). 4.5.1 Calcium Role in the Retention of Y. enterocolitica in C. briggsae The availability of calcium ions in the culture media had an effect on the viability of Y. enterocolitica strains within C. briggsae. In the absence of EGTA, the observed pattern of green fluorescence within C. briggsae exposed to Y. enterocolitica-GF? strains displayed a similar pattern as worms feeding E. coli OP50-GFP in gut (i.e., illumination localized to anterior regions of the pharynx). However, in the presence of EGTA, distended lumens are observed in C. briggsae grown on Y. enterocolitica-GF? strains. This suggests that calcium ion levels may be involved with proper digestion of bacteria or possibly provide a defense mechanism against exposure to foreign bacterial pathogens. 4.6 Persistence of Y. enterocolitica has a Deleterious Effect on C. briggsae C. briggsae can overcome a shortened exposure to Y. enterocolitica strains as the fatality rates of C. briggsae leveled off once the worms were transferred to a nutritional food source after 24 h (i.e., E. coli OP50). These results indicated that the deleterious effect of Y. enterocolitica in C. briggsae can be slowed, i f not reversed, when proper dietary source is presented. Shifting experiments were also performed 9 9 in the C. elegans - P. aeruginosa infection assay by Tan et al. (1999a) in which C. elegans was shown to evade possible infection by P. aeruginosa PA14 strain if transferred to an E. coli OP50 food source within 18 h of incubation. However, the availability of E. coli did not clear Y. enterocolitica from the intestinal tract of C. briggsae. GFP visualization data showed Y. enterocolitica being retained within C. briggsae after growing for 24 h on E. coli OP50. The results show that C. briggsae is unable to void Y. enterocolitica from its gastrointestinal tract. The possibility that worms were constipated in the growth conditions was taken into consideration. However, the GFP data shows that C. briggsae grown on E. coli OP50 were able to properly digest and void bacteria from their gastrointestinal tract. The exposure of C. briggsae to a bacterial lawn containing both Y. enterocolitica and E. coli strains resulted in an additive effect on the incidence of worm death as the on-set and extent of worm death was observed at higher rates than exposure to Y. enterocolitica strains alone. When C. briggsae is fed E. coli OP50 and Y. enterocolitica at same time, an increased amount of viable Y. enterocolitica may to enter the gut leading to faster proliferation within the intestinal tract. In addition, the results suggest that both Y. enterocolitica and E. coli grown on BHI media are independently harmful to C. briggsae. A n investigation was also undertaken to examine worm fatality rates exposed other Gram-negative enteric pathogens grown on BHI E G T A 5 m M media. C. briggsae exposed to S. typhimurium displayed the same trend of worm fatality and distended gastrointestinal tract as C. briggsae exposed to Y. enterocolitica, demonstrating that the lethal effect on C. briggsae was not limited to Yersinia species. 100 4.7 Possible Mechanisms of C. briggsae Killing 4.7.1 The Possible Protective Role of Calcium The connection of calcium could also suggest that calcium is involved with worm immunity against invading bacteria. Studies focusing on the role of dietary calcium in mice demonstrate that dietary calcium protects against S. typhimurium infection (Bovee-Oudenhoven and Van der Meer, 1997; Bovee-Oudenhoven et al, 1997). The authours suggest that calcium supplements the proliferation of lactobacilli which in return help protect the gastrointestinal lumen from intruding bacterial pathogens such as S. typhimurium (Bovee-Oudenhoven et al, 1997; Bovee-Oudenhoven et al, 1999). However, it has been undocumented whether Caenorhabditis possess intestinal lactobacilli or other microflora that could help protect against invading bacterial pathogens. Further evidence to suggest a protective effect of calcium was demonstrated in studies with Burkholderia spp. Loss of function the egl-19(n582) and unc-43(e251) C. elegans mutants growing on B. pseuodomallei and B. thailandensis resulted in decreased worm life-span (O'Quinn et al, 2001). egl-19 and unc-43 genes encode subunits for the L-type C a 2 + channel in C. elegans that is essential for the calcium signal-transduction pathway involving calcium- and calmodulin-dependent protein kinase II. The authours suggest that the worm-killing mechanism of Burkholderia spp. was attributed to the disruption of normal calcium-signal transduction pathways within the worms (O'Quinn et al, 2001). However, Ausubel and colleagues (Kim et al, 2002) have recently published results regarding a genetic screen of C. elegans mutants with enhanced susceptibility to killing by bacterial nathogens such as P. 101 aeruginosa (i.e., the Esp phenotype). Their data suggest that M A P kinases may be involved with innate immune response within C. elegans. Among the mutants tested, two different unc-43 null mutants, nll85 and el55 failed to show the Esp phenotype. 4.7.2 Implications of the V G S Database Although it was determined within this project that death of C. briggsae grown on Y. enterocolitica strains is not directly caused by virulence determinants, the search of commonalities in biological processes between host and pathogen can provide valuable insights to the process of pathogenesis. Not all of the components that determine the onset and/or propagation of a bacterial infection have been properly identified, making these missing links the holy grails for researchers of pathogenesis. With the accumulation of complete sequenced genomes and identified proteins, it is reasonable to take a step back to compile and compare the data for analysis. The development of the V G S database provides such a tool by assembling identified virulence determinants to allow quick comparative analysis to identify host proteins that may have not been considered to play a protective or detrimental role in worm defense against invading microbes. Already comparative data from the VGS database suggests that pathogens may utilize common cellular mechanisms such as synthetase carboxylase and tRNA-ribosyltransferases in the worm for biological routes of pathogenesis. 4.8 Conclusions This thesis has demonstrated that C. briggsae has potential as a host model for the study of mammalian pathogenesis mechanisms. 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