Interaction of sugar metabolism with antibiotic susceptibility in Staphylococcus aureus

金黄色葡萄球菌糖代谢与抗生素敏感性的相互作用

• 批准号: 17590392
• 负责人: KOMATSUZAWA Hitoshi
• 金额: $ 2.3万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590392/
• 关键词: Staphylococcus aureus; antibiotic resistance; sugar metabolism; Microarray; ribozyme

We investigated the interaction of sugar metabolism with the susceptibility to vancomycin and methicillin in Staphylococcus aureus, and had several findings described below.1) Construction of PTS mutants and characterization of these mutants : We found possible 17 PTS systems such as specific PTS for glucose and for fructose, so we constructed 17 PTS mutants. We measured the susceptibility to antibiotis, and found no significant difference between the mutant and the parent.2) Construction of ccpA-mutants and its characterization: We constructed the mutant of ccpA, which is involved in global regulation of glucose metabolism. CcpA-mutant reduced the resistance level to methicillin. From the microarray analysis, we found the genes regulated by CcpA.3) Analysis of GlmS-ribozyme: To know the critical regions for ribozyme activity in glmS-mRNA, we constructed the various length of DNA fragment containing promoter region, and cloned these fragments in one vector for XylE-reporter analysis. As a result, the region responsible for ribozyme activity is about 300 by region upstream of glmS-orf. To evaluate the ribozyme activity, we set up the method using real-Time PCR, and found that glmS-mRNA was cleaved in specific sites by additon of N-acetylglucosamine, and also digested glmS-mRNA itself.4) Microarray analysis of the expression profile by addition of various kinds of sugars: We analyzed the expression of all genes in S. aureus by addition of various kinds of sugars, and found specific PTS for each sugar. Also, we evaluated the specific genes affected by each sugar.

我们研究了金黄色葡萄球菌中糖代谢与万古霉素和甲氧西林敏感性之间的相互作用，并得到了如下所述的几个发现。 1) PTS 突变体的构建和这些突变体的表征：我们发现了可能的 17 个 PTS 系统，例如葡萄糖和甲氧西林的特异性 PTS。对于果糖，因此我们构建了 17 个 PTS 突变体。我们测量了对抗生素的敏感性，发现突变体与亲本之间没有显着差异。2）ccpA突变体的构建及其表征：我们构建了参与葡萄糖代谢全局调节的ccpA突变体。 CcpA 突变体降低了对甲氧西林的耐药水平。通过微阵列分析，我们找到了受CcpA调控的基因。3）GlmS-核酶分析：为了了解glmS-mRNA中核酶活性的关键区域，我们构建了包含启动子区域的各种长度的DNA片段，并克隆了这些片段在一个载体中进行 XylE 报告基因分析。结果，负责核酶活性的区域是glmS-orf上游约300个区域。为了评估核酶活性，我们建立了实时PCR方法，发现glmS-mRNA通过添加N-乙酰氨基葡萄糖在特定位点被切割，并且也消化了glmS-mRNA本身。4)表达的微阵列分析添加各种糖的谱：我们通过添加各种糖分析了金黄色葡萄球菌中所有基因的表达，并发现了每种糖的特定PTS。此外，我们还评估了每种糖影响的特定基因。