| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 101 === Vibrio vulnificus, a Gram-negative, curved-bacillus, may cause severe infectious diseases in both the human and eels. Only a fraction of V. vulnificus can infect the eels, and they are classified as biotype (BT) 2. We have previously demonstrated that the ability of BT2 V. vulnificus strains to infect eels is associated with a BT2 specific virulence plasmid. Further, by characterization of mutants with large sectional deletion in the virulence plasmid, pR99, of BT2 strain CECT4999, it was suggested that in this plasmid only vep07, vep20 and vep71, are required for bacterial survival in eel serum. However, the ability of pR99-cured strain complemented with these three genes to survive in eel serum was not observed. The aim of this study is to determine whether vep07, vep20 and vep71 together are sufficient to convey resistance to eel serum. First, I determined the importance of vep07, vep20 and vep71 for survival in eel serum by deleting each gene individually from pKT391, and confirmed that vep07 and vep20 are essential while vep71 is important only when the bacteria were in stationary, but not exponential phase. Then, I inserted a toxin-antitoxin module into a broad-host range vector, pBBR1MCS4, which is compatible with pIT009, to increase its stability, and used the resulting plasmid in combination with pIT009 to establish a double vector system. With this system, I transferred vep07, vep20 and vep71 to V. vulnificus strains to determine the importance of these genes for bacterial survival in eel serum. The results showed that these three genes together are sufficient to restore the survival of pR99-cured BT2 strain. However, these three genes together or vep71 alone in the BT1 strain YJ016 conferred only 1/3 of the level in BT2 for survival in eel serum. By real-time reverse transcription PCR and Western blotting, it was shown that both vep07 and vep20 were transcribed and Vep07 was expressed in YJ016. These data suggest that, among these three genes, only vep71 conveyed partial survival of YJ016 in eel serum. Therefore, it is likely that some other BT2-specific chromosomal genes are also involved in resistance to eel serum, thus leading to a better survival of BT2 than BT1 strains in eel serum.
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