Investigation of antibiotic resistance by total analysis of cell wall biosynthesis in methicillin-resistant

用差异显示法检测了甲氧西林/万古霉素耐药金黄色葡萄球菌和敏感金黄色葡萄球菌中参与细胞壁生物合成的30个因子的表达水平，发现它们之间没有差异。由于我们先前发现万古霉素中间体金黄色葡萄球菌对莫诺霉素表现出抗药性，因此我们重点研究了两种单功能转糖基酶：sgtA和sgtB。我们构建了它们的敲除突变体和过度表达SGTA或SGT B的突变体。两株过表达突变株对莫诺霉素和万古霉素的敏感性均降低。然后，我们从5株临床分离的金黄色葡萄球菌中分离出对莫诺霉素耐药的突变株，发现所有突变株对万古霉素的敏感性降低，对甲氧西林的敏感性增加。然而，这些突变体并没有改变SgtA和SgtB的表达。提示SGT A和SGT B与万古霉素的易感性有关，但也与其他因素有关。然后，我们分离到了两个Tn551插入突变体，它们增加了对莫诺霉素的敏感性。两个突变株对万古霉素的敏感性均降低。我们鉴定了Tn551插入的基因lysC和FMTC。LysC参与了赖氨酸的生物合成，FMTC参与了细菌膜上赖氨酰磷脂酰甘油的合成。这些都是影响金黄色葡萄球菌对万古霉素敏感性的新因素。

We investigated the expression levels of 30 factors involved in cell wall biosynthesis by differential display method in methicillin/vancomycin-resistant and -sensitive Staphylococcus aureus, and found no difference between them. Since we previously found that vancomycin-intermediate S.aureus showed the moenomycin-resistance, we focused on two monofunctional transglycosylases, sgtA and sgtB. We constructed their knockout mutants and the mutants overexpressing SgtA or SgtB. Both overexpressed mutants showed the decreased susceptibility to moenomycin and vancomycin. Then, we isolated the moenomycin-resistant mutants from 5 clinical S.aureus strains, and found that all mutants showed decreased susceptibility to vancomycin and increased susceptibility to methicillin. However, these mutants did not alter the expression of SgtA and SgtB. These results indicate that SgtA and SgtB are associated with the susceptibility to vancomycin, but other factors are also involved. Then, we isolated two Tn551-insertional mutants, which increased the moenomycin-susceptibility. Both mutants also showed the decreased susceptibility to vancomycin. We identified the genes, lysC and fmtC, in which Tn551 inserted. lysC is involved in lysine biosynthesis, and fmtC is involved in the synthesis of lysyl-phosphatidylglycerol in bacterial membrane. These are novel factors affecting the vanocmycin-susceptibility in S.aureus