Signature-tagged mutagenesis of Vibrio vulnificus.
Signature-tagged mutagenesis of Vibrio vulnificus.
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DOI：
10.1292/jvms.14-0655
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发表时间：
2015-07
期刊：
影响因子：
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通讯作者：
Yamamoto K
中科院分区：
文献类型：
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作者：
Yamamoto M;Kashimoto T;Tong P;Xiao J;Sugiyama M;Inoue M;Matsunaga R;Hosohara K;Nakata K;Yokota K;Oguma K;Yamamoto K
创伤弧菌是免疫功能低下人群中原发性败血症、伤口感染和胃肠炎的病原体。本研究应用特征标记突变技术（STM）对创伤弧菌的毒力基因进行了鉴定。使用STM，总共构建了6，480个突变体，并将其分成81组（输入池）;组中的每个突变体被分配了不同的标签。将每个输入池腹膜内注射到铁超载小鼠中，并从心脏的血液样品中收集体内存活的突变体（输出池）。从混合的输入或输出池的基因组DNA中，制备针对标记区域的地高辛标记的DNA探针，并用于斑点杂交。阴性选择了30个暂时减毒的突变体，这些突变体与输入探针明显杂交，但与输出探针几乎没有杂交。30个突变体中有11个的致死剂量降低到1/100以上;其中2个的致死剂量降低到1/100，000。转座子插入的基因包括IMP脱氢酶、UDP-N-乙酰葡萄糖胺-2-差向异构酶、葡萄糖激酶、磷酸核糖甲酰甘氨脒环连接酶、苹果酸Na（+）同向转运体和假想蛋白。当小鼠用IMP脱氢酶插入转座子的减毒突变株免疫时，它们被保护免受创伤弧菌感染。本研究证明STM方法可以用于创伤弧菌毒力基因的研究。
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.