UM
Signature-tagged mutagenesis of vibrio vulnificus
Yamamoto M.3; Kashimoto T.4; Tong P.3; Xiao J.3; Sugiyama M.3; Inoue M.3; Matsunaga R.3; Hosohara K.3; Nakata K.3; Yokota K.1; Oguma K.1; Yamamoto K.3
2015
Source PublicationJournal of Veterinary Medical Science
ISSN13477439 09167250
Volume77Issue:7Pages:823-828
AbstractVibrio 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.
KeywordFoodborne disease Signature-tagged mutagenesis Vaccine Vibrio vulnificus Virulence
DOI10.1292/jvms.14-0655
URLView the original
Language英語
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被引频次[WOS]:4   [WOS记录]     [WOS相关记录]
Document TypeJournal article
专题University of Macau
Affiliation1.Okayama University
2.Macau Univ. Sci. Technol
3.Okayama Prefectural University
4.Kitasato University
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GB/T 7714
Yamamoto M.,Kashimoto T.,Tong P.,et al. Signature-tagged mutagenesis of vibrio vulnificus[J]. Journal of Veterinary Medical Science,2015,77(7):823-828.
APA Yamamoto M..,Kashimoto T..,Tong P..,Xiao J..,Sugiyama M..,...&Yamamoto K..(2015).Signature-tagged mutagenesis of vibrio vulnificus.Journal of Veterinary Medical Science,77(7),823-828.
MLA Yamamoto M.,et al."Signature-tagged mutagenesis of vibrio vulnificus".Journal of Veterinary Medical Science 77.7(2015):823-828.
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