[
C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting,
2008]
Vibrio cholerae (VC), the causative agent of cholera, is responsible for devastating epidemics across the world. The major virulence factor underlying the pathogenesis of cholera is cholera toxin (CT). However, CT negative VC non-O1 and non-O139 strains and CT deletion vaccine mutant strains are still able to cause severe diarrheal disease through mechanisms that are currently unclear. Additional virulence factors such as zonula occludens toxin (zot), accessory cholerae toxin (ace), and hemolysin (hlyA), are implicated in cholera pathogenesis. VC causes lethal infection in the nematode C. elegans via a CT-independent process (1, 2), providing an excellent model to determine the roles of the other virulence factors in pathogenesis. We fed CVD110, a VC vaccine strain that is deficient in several virulence factors (3), to the worms, and observed an attenuated killing. CVD110 is deficient in zonula occludens toxin (zot), accessory cholerae toxin (ace), and hemolysin (hlyA) as well as the CT genes ctxA and ctxB. These data suggest that the participation of one or more of these factors was/were responsible for the lethality in C. elegans. To test the hypothesis that hlyA was responsible for lethality in the C. elegans model, the effect of strain CVD109, which has an intact hlyA and otherwise genetically identical with CVD110, was evaluated. We found that the presence of intact hlyA locus was sufficient to promote same level of killing as the wild type VC. Furthermore, worms fed with a VC strain carrying a single locus deletion of hlyA gene, found to be attenuated in C. elegans lethality, a result which is similar to that observed for CVD110. Together, our data strongly suggest that Vibrio cholerae hly A is responsible for killing of C. elegans. hemolysin A is a pore forming exotoxin whose role in VC pathogenesis is not fully understood. Further studies in C. elegans host model will help us understand the contribution of hemolysin virulence in VC pathogenesis.
[
International Worm Meeting,
2007]
Vibrio species are responsible for foodborne illnesses around the world, particularly in underdeveloped countries. Recent studies have shown that the human pathogens Vibrio cholerae (1) and Vibrio vulnificus (2) cause lethal infections in C. elegans. Using V. cholerae wild type strain A1552, we found that, in addition to the induction of the slow killing in adult worms, A1552 also killed larvae and caused escape behavior when killing assays were performed on brain heart infusion (BHI) agar plates. V. cholerae: OP50 mixed inoculation (1:10000) was as effective as the V. cholerae single inoculation in killing adults and larvae, indicating that the infectious dose of V. cholerae is very low. Furthermore, L4 stage worms which were exposed to a GFP-tagged V. cholerae strain for one hour and subsequently fed with OP50, exhibited gut colonization of V. cholerae which resulted in worm death similar to that observed in the continuous feeding experiments. To determine whether C. elegans prefers OP50 over V. choleare, 20 <font face=symbol>m</font>l of each bacterial culture were seeded on opposite sides of BHI plates and worms were placed at the midline. N2 adults were equally distributed to the OP50 and V. cholerae sides of the plate which suggests that they do not show a preference for OP50. However, worms on the OP50 side of the plate were located on the bacterial lawn while worms on the V. cholerae side tended to wander about and did not show a preference for the bacterial lawn. Once the OP50 lawn became contaminated with V. cholerae, which was carried through worms, all the worms on the plate adopted ‘wandering about behavior. These observations suggest that C. elegans may sense V. cholerae as a harmful agent and possibly avoids it upon exposure. Alternatively, V. cholerae may have destroyed sensory processes in worms resulting in loss of their ability to locate the food. Other clinical isolates of V. cholerae are being tested in killing and avoidance assays. A V. cholerae transposon insertion library will also be screened to identify genes required for the observed phenotypes. These studies will be extended to other pathogenic Vibrio species. (1) Vaitkevicius K. et al. PNAS, 103 (2006) 9280-9285 (2) Dhakal BK et al. Biochemical and Biophysical Research Communications, 346 (2006) 751-757.