Signature-tagged mutagenesis of Vibrio
vulnificus |
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| Authors: | Mai YAMAMOTO Takashige KASHIMOTO Ping TONG Jianbo XIAO Michiko SUGIYAMA Miyuki INOUE Rie MATSUNAGA Kohei HOSOHARA Kazue NAKATA Kenji YOKOTA Keiji OGUMA Koichiro YAMAMOTO |
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| Institution: | 1)Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama 719–1197, Japan;2)School of Veterinary Medicine, Kitasato University, Towada, Aomori 034–8628, Japan;3)Graduate School of Health Sciences, Okayama University, Okayama 700–8558, Japan;4)Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama 700–8558, Japan |
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| Abstract: | Vibrio vulnificus is the causative agent of primary septicemia, wound
infection and gastroenteritis in immunocompromised people. In this study, signature-tagged
mutagenesis (STM) was applied to identify the virulence genes of V.
vulnificus. Using STM, 6,480 mutants in total were constructed and divided into
81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool
was intraperitoneally injected into iron-overloaded mice, and in vivo
surviving mutants were collected from blood samples from the heart (OUTPUT pools). From
the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the
tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated
mutants, 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 than
1/100; 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, phosphoribosylformylglycinamidine
cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized
with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a
transposon, they were protected against V. vulnificus infection. In this
study, we demonstrated that the STM method can be used to search for the virulence genes
of V. vulnificus. |
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| Keywords: | foodborne disease signature-tagged mutagenesis vaccine Vibrio vulnificus virulence |
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