摘要

达托霉素是一种钙依赖性脂肽类抗生素，其完整作用方式尚不完全清楚，已成为治疗耐甲氧西林金黄色葡萄球菌（MRSA）感染的标准治疗剂。达托霉素抗性（DAP-R）金黄色葡萄球菌突变体在治疗期间出现，其特征在于分离物，其在大多数情况下在mprF基因中具有点突变。 MprF是双功能细菌抗性蛋白，其合成带正电荷的脂质赖氨酰 - 磷脂酰甘油（LysPG）并随后将其从内膜小叶移位至外膜小叶。该过程导致阳性金黄色葡萄球菌表面电荷增加并降低对阳离子抗微生物肽和阳离子抗生素的敏感性。我们在定义的遗传背景中对DAP-R金黄色葡萄球菌菌株中最常报道的MprF突变进行了表征，发现只有某些突变，包括经常报道的T345A单核苷酸多态性（SNP），才能重现地引起达托霉素抗性。令人惊讶的是，T345A没有改变LysPG合成，LysPG易位或金黄色葡萄球菌细胞表面电荷。 MprF介导的DAP-R依赖于功能性翻转酶结构域，并且限于达托霉素和相关的环状脂肽抗生素，friulimicin B，表明这些突变调节与这两种抗生素的特异性相互作用。值得注意的是，T345A突变导致MprF的分子内结构域相互作用减弱，表明达托霉素和friulimicin抗性突变可能改变MprF翻转酶的底物范围，从而直接转移这些脂肽抗生素或其他膜组分，从而在这些活性中起关键作用。抗菌剂。我们的研究指出金黄色葡萄球菌用于抵抗钙依赖性脂肽抗生素的新机制，并增加了我们对细菌磷脂翻转酶MprF的理解。重要性自从达托霉素被引入临床以来，已经报道了达托霉素抗性分离株。在大多数情况下，抗性分离株在MprF中具有点突变，其产生并翻转带正电荷的磷脂LysPG。这导致假设抗性机制依赖于LysPG的过量产生，因为增加的LysPG产生可导致带正电荷的抗微生物化合物（包括达托霉素）的静电排斥增加。在这里我们显示抗性机制是高度特异性的并且依赖于涉及功能性MprF翻转酶的不同过程，表明赋予抗性的突变可以使翻转酶适应达托霉素或对其活性至关重要的未知组分。我们的报告提供了对主要抗生素耐药机制的新观点。

Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF.IMPORTANCE Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic.

https://www.ncbi.nlm.nih.gov/pubmed/30563904