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Analysis of the regulation mechanisms for the expression of virulence factors in gram-positive cocci.

革兰阳性球菌毒力因子表达调控机制分析。

基本信息

批准号：
14370090
负责人：
OHTA Michio
金额：
$ 7.17万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

We analyzed the mechanisms of the expression of exoproteins such as toxins and enzymes in Staphylococcus aureus and Streptococcus pyogenes. We performed comprehensive analyses of exoproteins of these bacteria by two dimensional gel electrophoresis (2-DE) and LC/MS/MS and constructed 2-DE maps. We detected putative genes that affect the expression of exoproteins in genome databases and constructed knockout mutants of these genes and analyzed the functions of these genes. The results were as follows.Molecular epidemiological study for 23 exotoxin genes revealed that most Japanese MRSA strains were TSST-1,SEC3 and egc locus-positive, suggesting that these MRSA are very clonal. The differences of the expressed amount of each toxin were analyzed by 2-DE. We found that the expression of TSST-1 was very high in the strains that caused neonatal toxic shock-like exanthematous disease. The production of TSST-1 was suppressed by glucose-mediated catabolite repression. The difference of agr locus … More was not observed in theses trains.We found that the expression of Sic protein was regulated by mga, a global regulator of S.pyogenes, since the production of Sic was reduced in an mga-knockout mutant, and that other several exoproteins were also regulated by mga.It is very likely that the specific secretion machineries are involved in the expression of exoproteins in S.aureus and S.pyogenes, respectively. We found a tat-like gene similar to tat gene of Escherichia coli in the genome of S.aureus. We constructed tat-knockout mutants and found that the expression of several exoproteins were reduced in these mutants, suggesting that this tat-like gene is functional in S.aureus. Interestingly, fat-like gene was found in S.intermedius but not in other coagulase-negative staphylococci. These results suggested that Tat was involved in the virulence of staphylococci.We detected four signal peptidase I (SPaseI)-like genes in the genome of S.pyogenes and constructed knockout mutants of sipC and spi genes. A sipC mutant reduced the expression of 54 exoproteins and a spi mutant reduced the expression of 36 exoproteins. We found preferential expression of some exoproteins in each mutant. Less

我们分析了金黄色葡萄球菌和化脓性链球菌中毒素和酶等外蛋白的表达机制。采用双向凝胶电泳（2-DE）和LC/MS/MS对这些细菌的胞外蛋白进行了综合分析，并构建了2-DE图谱。我们在基因组数据库中检测了影响外蛋白表达的推定基因，构建了这些基因的敲除突变体，并分析了这些基因的功能。结果如下：对23种外毒素基因的分子流行病学研究显示，大多数日本MRSA菌株均为TSST-1、SEC 3和egc基因座阳性，表明这些MRSA的克隆性很强。通过双向电泳分析各毒素表达量的差异。我们发现TSST-1在引起新生儿中毒性休克样外生性疾病的菌株中表达非常高。TSST-1的产生受到葡萄糖介导的分解代谢产物阻遏的抑制。agr位点的差异 ...更多信息我们发现，Sic蛋白的表达受化脓性链球菌的全局调节因子mga的调控，在mga基因敲除的突变株中，Sic蛋白的表达减少，其他几种胞外蛋白的表达也受mga的调控，这很可能分别涉及金黄色葡萄球菌和化脓性链球菌胞外蛋白表达的特异性分泌机制。我们在金黄色葡萄球菌基因组中发现了一个与大肠杆菌达特基因相似的tat样基因。我们构建了Tat敲除突变体，发现在这些突变体中几种外蛋白的表达减少，表明这种Tat样基因在金黄色葡萄球菌中是有功能的。有趣的是，脂肪样基因在中间型葡萄球菌中发现，而在其他凝固酶阴性葡萄球菌中未发现。我们在化脓性链球菌基因组中检测到4个信号肽酶I（SPaseI）样基因，并构建了sipC和spi基因的敲除突变体。sipC突变体减少了54个外蛋白的表达，spi突变体减少了36个外蛋白的表达。我们发现在每个突变体中的一些外蛋白的优先表达。少