| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 92 ===
Vibrio vulnificus, a gram-negative halophilic marine bacterium, is an opportunistic human pathogen. It causes severe wound infection and septicemia, particularly in those with underlying diseases. V. vulnificus produce a number of potential virulence factors including capsular polysaccharide, siderophores, protease, cytolysin and phospholipase. Our previous study showed that disruption of the genes encoding protease, cytolysin and phospholipase did not influence the virulence in mice or cytotoxicity to HEp-2 cells. Hence we concluded that these three factors are not the major virulence factors in V. vulnificus. However, a non-cytotoxic mutant, NY303, isolated fortuitously from a phospholipase-cytolysin double mutant was shown to be much less virulent in mice. This data suggested that cytotoxicity may be an important virulence factors. We suspected that NY303 might have lost the activity of an unidentified cytotoxin. In this study, we tried to identify the proteins that are lost in NY303 by proteomic analysis. The total soluble proteins, outer membrane proteins, secreted extracellular proteins and periplasmic proteins of NY303 and its isogenic, cytotoxic strain, NY303-2, were extracted and separated by one-dimensional (SDS-PAGE) or two-dimensional electrophoresis (2-DE). When we separated the periplasmic and outer membrane proteins by SDS-PAGE, one band about 35 kDa was found lost in NY303 in each fraction. These bands were subjected to LCQ/MS for protein identities and the transcriptions of genes encoding these proteins were examined by RNA slot blot hybridization. Only with VV2338, the open-reading-frame of one of the identified proteins, the transcript was more abundant in NY303-2 than NY303. However, VV2338 in NY303 can be amplified without any deletion, indicating that the lost of VV2338 protein in NY303 was not caused by a deletion in the gene. Meanwhile, some protein spots differentially expressed in NY303-2 and NY303 were identified on 2-DE gels of the periplasmic and extracellular fraction, suggesting that the lost of cytotoxicity in NY303 might be due to the dysfunction of a regulatory factor.
On the other hand, TolC and its homologues are outer membrane proteins essential for the transport of a variety of molecules, including the antibiotics and toxins, across the cell envelope. In our previous study, we found that a TolC-deficient V. vulnificus mutant, MW021, was more sensitive to bile and erythromycin, non-cytotoxic to HEp-2 cells, and much less virulent in mice compared to the parental strain, YJ016. Since the disruption of tolC did not influence the secretion of protease and cytolysin, we suspected that the activity or transport of an unknown cytotoxin was affected by TolC dysfunction. To confirm that the phenotypes we observed were associated with the disruption of tolC, we performed a complementary experiment. We found it’s very difficult to clone the wild-type V. vulnificus tolC gene in Escherichia coli with a high copy number vector, and we always obtained at least one point mutation in the coding sequence of cloned tolC. These suggested that overexpression of V. vulnificus tolC might be harmful for E. coli. When we used the low copy number vector, partial complementation with delayed growth in the 0.02% bile-containing LB medium was observed in one strain, YC019, which contained one point mutation 19-bp upstream of the cloned tolC open reading frame and one silent mutation in the coding sequence. We plated the bacteria grown in the bile-containing LB medium on TCBS (Thiosulfate-Citrate-Bile salt-Sucrose) agar and screened for those that grew as well as YJ016 in bile-containing LB medium. One strain, YC025, was obtained and its phenotypes, including cytotoxicity to HEp-2 cells and susceptibility to erythromycin were similar to YJ016. We found that the tolC gene on the plasmid contained in YC025 had one additional silent mutation and one amino acid deletion. We suspected that the deletion of the amino acid might have altered the TolC structure that resulted in restoration of the phenotypes. Whether TolC has an effect on the activity of an unknown cytotoxin or RTX toxin awaits further investigation.
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