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

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。我们通过二维凝胶电泳（2-DE）和LC/MS/MS对这些细菌的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可能影响外蛋白表达的基因，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：23个外毒素基因的分子流行病学研究显示，日本MRSA菌株多数为tst -1、SEC3和egc位点阳性，表明这些MRSA具有很强的克隆性。用2-DE法分析各毒素表达量的差异。我们发现在引起新生儿中毒性休克样疹性疾病的菌株中，TSST-1的表达非常高。葡萄糖介导的分解代谢抑制抑制了TSST-1的产生。在这些系中，agr位点的差异没有观察到更多。我们发现Sic蛋白的表达受化脓性s.p ogenes的全局调控因子mga的调控，因为在mga敲除突变体中Sic的产生减少，并且其他几种外蛋白也受mga的调控。金黄色葡萄球菌和化脓性葡萄球菌的外蛋白表达很可能与特定的分泌机制有关。我们在金黄色葡萄球菌的基因组中发现了一个类似于大肠杆菌的基因。我们构建了敲除突变体，发现这些突变体中几种外蛋白的表达减少，这表明这种类似于塔的基因在金黄色葡萄球菌中起作用。有趣的是，脂肪样基因在中间葡萄球菌中发现，而在其他凝固酶阴性葡萄球菌中未发现。这些结果表明Tat参与了葡萄球菌的毒力。在化脓链球菌基因组中检测到4个信号肽酶I （SPaseI）样基因，构建sipC和spi基因敲除突变体。sipC突变体降低了54种外蛋白的表达，spi突变体降低了36种外蛋白的表达。我们发现一些外蛋白在每个突变体中优先表达。少

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