Signature-tagged mutagenesis of Vibriovulnificus

[摘要] References(35)Vibrio vulnificus is the causative agent of primary septicemia, woundinfection and gastroenteritis in immunocompromised people. In this study, signature-taggedmutagenesis (STM) was applied to identify the virulence genes of V.vulnificus. Using STM, 6,480 mutants in total were constructed and divided into81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT poolwas intraperitoneally injected into iron-overloaded mice, and in vivosurviving mutants were collected from blood samples from the heart (OUTPUT pools). Fromthe genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against thetagged region were prepared and used for dot hybridization. Thirty tentatively attenuatedmutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes,were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000.Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase,UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidinecyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunizedwith an attenuated mutant strain into which IMP dehydrogenase had been inserted with atransposon, they were protected against V. vulnificus infection. In thisstudy, we demonstrated that the STM method can be used to search for the virulence genesof V. vulnificus.

[学科分类] 兽医学

[关键词] foodborne disease;signature-tagged mutagenesis;vaccine;Vibrio vulnificus;virulence