Contribution of altered cell envelope metabolism to resistance to cell envelope-targeting antimicrobials in MRSA

耐甲氧西林金黄色葡萄球菌(MRSA)引起的侵袭性感染较差 由于对一线药物万古霉素(糖肽；GP)的敏感性降低而恶化的结果，以及 达托霉素(脂肽；Lp)。长效脂糖肽(LGP)dalbavancin是一种替代药物，可以 每周给药或单次给药，这可以促进出院和降低成本。然而，我们已经表明， 其较长的半衰期可能会增加其抗性选择潜力，并可对万古霉素进行交叉抗性选择 还有达托霉素。因此，迫切需要了解细胞间交叉耐药的机制(S 在耐甲氧西林金黄色葡萄球菌中使用包膜靶向药物，并研究减轻或克服这种耐药性的策略。我们的 前一批资助期间的工作发现，75%的GP/LP/LGP不敏感分离株来自体外PK/PD 模拟Dalbavancin暴露的模型获得与基本双组分调节相关的突变 系统WalKR。此外，我们最近发表了一个病例，其中达巴文治疗被选为 1例心内膜炎患者通过Walk突变产生GP/LP/LGP耐药MRSA。这些最新的发现导致了 本次更新的目标：阐明walKR突变导致GP/LP/LGP的多种机制 交叉耐药并揭示β-内酰胺类和其他代谢调节剂如何与WalKR调节的相互作用 代谢网络与GP/LP/LGP协同作用，防止耐药性。我们假设walKR突变 Gp/lp/lgp交叉耐药表型通过细胞被膜和核苷酸的调节 代谢，代谢调节剂可以使这些菌株对GP/LP/LGP重新敏感，或通过以下方式防止耐药性 进一步改变细胞被膜或核苷酸代谢。在目标1中，我们将衡量Reduced WalKR在耐甲氧西林金黄色葡萄球菌交叉耐药表型中的作用 和蛋白质组学方法，结合敏感性测试和定量生物物理评估 单元格信封属性。在AIM 2中，我们将检验以下假设：β-内酰胺类药物和其他代谢 调节剂可以使步行敲除菌株对GP/LP/LGP重新敏感。我们将研究以下各项的协同效应 细胞壁抑制剂，β-内酰胺类和磷霉素，脂类合成抑制剂，以及抗叶酸药物， 甲氧苄啶/磺胺甲恶唑，它将告知对walKR突变至关重要的代谢途径- 引发了抵抗。在AIM 3中，我们将评估β-内酰胺类和其他代谢调节剂的潜力 用串联传代和PK/PD模型体外阻断达巴文星对GP/LP/LGP耐药的选择。 这项工作很有意义，因为达班星的暴露很容易选择万古霉素和达托霉素耐药 预防耐甲氧西林金黄色葡萄球菌耐药和/或使耐甲氧西林金黄色葡萄球菌对gp/lg/lgp重新敏感的策略对保存 这些药物，特别是在目前达巴文使用量增加的情况下。这项建议的创新之处 包括利用代谢表型来通知和调节耐药性表型以及应用 高通量脂质组学和代谢组学，以表征耐药性和反应。

Invasive infections due to the “superbug” methicillin-resistant Staphylococcus aureus (MRSA) have poor outcomes that are worsened by reduced susceptibility to first-line agents, vancomycin (glycopeptide; GP), and daptomycin (lipopeptide; LP). The long-acting lipoglycopeptide (LGP), dalbavancin, is an alternative that can be given weekly or as a single dose, which can facilitate discharge and reduce costs. However, we have shown that its long half-life may increase its resistance selection potential and can select for cross-resistance to vancomycin and daptomycin. Thus, there is a critical need to understand the mechanism(s) of cross-resistance among cell envelope-targeting drugs in MRSA and to investigate strategies to mitigate or overcome such resistance. Our work from the previous grant periods found that 75% of GP/LP/LGP non-susceptible isolates from in vitro PK/PD models simulating dalbavancin exposures acquired mutations related to the essential two-component regulatory system walKR. Furthermore, we recently published a case in which dalbavancin treatment selected for GP/LP/LGP-resistant MRSA in a patient with endocarditis, via a walK mutation. These recent findings led to the goal for this renewal: to elucidate the multiple mechanisms through which walKR mutations lead to GP/LP/LGP cross-resistance and reveal how beta-lactams and other metabolic modulators interact with WalKR-regulated metabolic networks to synergize with GP/LP/LGP and prevent resistance. We hypothesize that walKR mutations underlie GP/LP/LGP cross-resistance phenotypes through modulation of both cell envelope and nucleotide metabolism, and metabolic modulators can re-sensitize these strains to GP/LP/LGP or prevent resistance by further altering cell envelope or nucleotide metabolism. In AIM 1, we will measure the contribution of reduced WalKR function to cross-resistance phenotypes in MRSA using genetic, lipidomic, metabolomic, transcriptomic, and proteomic approaches in combination with susceptibility testing and quantitative biophysical assessment of the cell envelope properties. In AIM 2, we will test the hypothesis that beta-lactams and other metabolic modulators can re-sensitize walK-knockdown strains to GP/LP/LGP. We will examine the synergistic effects of cell wall inhibitors beta-lactams and fosfomycin, lipid synthesis inhibitors, and anti-folate drugs, trimethoprim/sulfamethoxazole, which will inform metabolic pathways that are important for walKR mutation- caused resistance. In AIM 3, we will evaluate the potential of beta-lactams and other metabolic modulators to prevent the selection of GP/LP/LGP resistance by dalbavancin in vitro using serial passage and PK/PD models. This work is significant because dalbavancin exposures readily select for vancomycin and daptomycin-resistant S. aureus and a strategy to prevent resistance and/or re-sensitize MRSA to GP/LG/LGP is critical to preserve these drugs, especially in the current context of increasing dalbavancin use. The innovation of this proposal includes leveraging metabolic phenotypes to inform and modulate resistance phenotypes and the application of high-throughput lipidomics and metabolomics to characterize drug resistance and responses.