Insertional inactivation of branched-chain α-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses
Insertional inactivation of branched-chain α-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses
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DOI：
10.1128/aem.00882-08
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发表时间：
2008-10-01
影响因子：
4.4
通讯作者：
Wilkinson, Brian J.
中科院分区：
文献类型：
--
作者：
Singh, Vineet K.;Hattangady, Dipti S.;Wilkinson, Brian J.
金黄色葡萄球菌是一种主要的社区和医院病原体。它能够承受多种应激条件并迅速对抗生素产生耐药性，使葡萄球菌感染的控制复杂化。适应较低的温度是细菌物种在宿主外生存的关键。支链α-酮酸脱氢酶（BKD）是一种酶复合物，催化支链脂肪酸（BCFA）生产的早期阶段。在这项研究中，BKD被灭活，导致S.金黄色。BKD失活突变体的生长比野生型S.金黄色葡萄球菌随着温度的降低，突变体不能在12摄氏度下生长。突变体的生长显着刺激包括2-甲基丁酸在生长培养基中在所有温度下测试。2-丁酸甲酯是奇数反异构脂肪酸的前体，并绕过BKD。有趣的是，野生型S.金黄色葡萄球菌也通过在培养基中包括2-甲基丁酸酯而被刺激，尤其是在较低温度下。通过在培养基中加入2-甲基丁酸盐，BKD失活突变体的anteiso脂肪酸含量得以恢复。荧光偏振测量表明，BKD失活突变体的细胞膜比野生型S.金黄色。与该结果一致，突变体显示出降低的甲苯耐受性，其可以通过在培养基中包含2-甲基丁酸酯来增加。BKD失活突变体更容易受到碱性pH和氧化应激条件的影响。在S.金黄色葡萄球菌还导致突变体对真核细胞的粘附减少及其在小鼠宿主中的存活减少。此外，该突变体为研究膜流动性在S.金黄色葡萄球菌与抗菌物质。
Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.