Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis
Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis
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DOI：
10.1128/aac.01819-10
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发表时间：
2011-09-01
影响因子：
4.9
通讯作者：
Helmann, John D.
中科院分区：
文献类型：
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作者：
Hachmann, Anna-Barbara;Sevim, Elif;Helmann, John D.
达托霉素（DAP）是一种环脂肽，以Ca2（+）依赖性的方式破坏革兰氏阳性细菌细胞膜的功能完整性。在这里，我们介绍了进化的Dap抗性分离物Dap(R)1的遗传、基因组和表型分析，Dap(R)1来自模式细菌枯草芽孢杆菌168。随着Dap浓度的增加，通过连续传代得到Dap(R)1，其抗性是亲本菌株的30倍，并对万古霉素、莫诺霉素和杆菌肽具有交叉抗性。Dap(R)1的特征是异常的间隔位置，细胞极点的肽聚糖增厚，转录组和蛋白质组水平的多向性改变。Dap(R)1的基因组测序发现44个点突变，其中31个改变了蛋白序列。对DAP的抗性比野生型高20倍的中间分离物只有三个点突变：影响细胞形状调节基因mreB、严格反应基因relA和磷脂酰甘油合成酶基因pgsA的突变。遗传重建研究表明，pgsA（A64V）等位基因是DAP抗性的主要原因。用野生型pgsA替换等位基因后，DAP敏感性恢复到野生型水平。进化菌株中额外的点突变可能进一步促进DAP抗性，用于补偿膜组成改变的有害影响，或代表中性变化。这些结果表明了一种抗性机制，通过这种机制，磷脂酰甘油水平的降低减少了膜的净负电荷，从而减弱了与带正电荷的Ca2(+)-DAP复合物的相互作用。
Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2(+)-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap(R)1, from the model bacterium Bacillus subtilis 168. Dap(R)1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap(R)1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap(R)1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB, the stringent response gene relA, and the phosphatidylglycerol synthase gene pgsA. Genetic reconstruction studies indicated that the pgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca2(+)-DAP complex.