Thioridazine Induces Major Changes in Global Gene Expression and Cell Wall Composition in Methicillin-Resistant Staphylococcus aureus USA300

Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics.

众所周知，抗精神病药物硫利达嗪（thioridazine, TDZ）的亚抑菌浓度可增强β-内酰胺类抗生素对耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）的杀菌活性，但TDZ与β-内酰胺类药物协同作用的具体分子机制尚未完全阐明。本研究探讨了亚抑菌浓度TDZ对MRSA USA300的抗菌耐药性、全局转录组及细胞壁组成的影响。结果显示，TDZ可使细菌对多种靶向肽聚糖（peptidoglycan, PGN）合成后期阶段的抗菌药物敏感。此外，微阵列分析表明，TDZ可调控编码膜蛋白、表面蛋白、转运体以及氨基酸生物合成相关酶类的基因表达。值得注意的是，TDZ处理后的转录谱与金黄色葡萄球菌对已知细胞壁合成抑制剂的转录应答高度相似，提示TDZ可在青霉素结合蛋白（penicillin-binding proteins, PBPs）介导的转肽作用之前的阶段干扰肽聚糖的生物合成。为验证这一假说，我们观察到在TDZ存在下培养的USA300菌株的胞壁肽谱发生了显著变化，表明TDZ可干扰五甘氨酸侧链的形成。令人惊喜的是，向培养基中添加甘氨酸可逆转TDZ对胞壁肽谱的影响。此外，外源性甘氨酸可适度缓解TDZ诱导的β-内酰胺类药物敏感性增强现象。我们推测，TDZ暴露会导致细胞内包括甘氨酸在内的氨基酸匮乏，而甘氨酸是合成带有五甘氨酸侧链的正常肽聚糖前体所必需的物质，该前体正是金黄色葡萄球菌PBPs的正确底物。综上，本研究证实TDZ对金黄色葡萄球菌的细胞壁生物合成通路具有显著影响，并为MRSA如何被增敏至β-内酰胺类抗生素提供了新的见解。