Division of Infectious Diseases, Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, USA.
Antimicrob Agents Chemother. 2012 Jan;56(1):92-102. doi: 10.1128/AAC.00432-10. Epub 2011 Oct 10.
Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains.
达托霉素（DAP）是一种新型的环脂肽类抗生素，对耐甲氧西林金黄色葡萄球菌（MRSA）感染具有高度活性。其作用机制涉及以 Ca 依赖性方式破坏细菌膜的功能完整性。在本工作中，我们研究了一组从接受 DAP 治疗的金黄色葡萄球菌感染患者中分离得到的同源性 MRSA 临床株中 DAP 耐药的分子基础。在 DAP 耐药（DR）株中发现 mprF 基因的不同点突变。通过在 DAP 敏感（DS）株中灭活和转互补全长野生型或突变 mprF，研究了 DR 株中的 mprF L826F 突变，结果表明它们与 DR 表型在机制上相关。然而，我们的数据表明 mprF 不是决定 DAP 耐药的唯一因素。差异基因表达分析显示双组分调控系统 vraSR 的上调。vraSR 的失活导致 DAP 敏感性增加，而 vraSR 突变株的互补则使 DAP 耐药性恢复到与相应 DR 野生型菌株观察到的水平相当。电子显微镜分析显示 DR CB5012 的细胞壁比 DS CB5011 更厚，这种效应与 vraSR 和 mprF 突变对细胞壁的影响有关。此外，DS 株中 vraSR 的过表达导致 DAP 耐药性增加和 oxacillin 耐药性降低，类似于 DR 株的表型。这些结果支持了这样一种观点，即在 mprF 突变之外，vraSR 也有助于本研究组临床株中的 DAP 耐药性。
