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

由于"超级细菌"耐甲氧西林金黄色葡萄球菌（MRSA）的侵袭性感染， 对一线药物万古霉素（糖肽类; GP）敏感性降低导致结局恶化，以及 达托霉素（脂肽; LP）。长效脂糖肽（LGP）达巴万星是一种替代品， 每周一次或单次给药，这可以促进出院并降低成本。然而，我们已经证明， 其较长半衰期可增加其抗性选择潜力，并可选择对万古霉素的交叉抗性 和达托霉素。因此，迫切需要了解细胞间交叉抗性的机制 靶向MRSA的药物，并研究减轻或克服这种耐药性的策略。我们 从以前的资助期的工作发现，75%的GP/LP/LGP非敏感分离株，从体外PK/PD 模拟达巴霉素暴露的模型获得了与必需的双组分调控相关的突变， 系统walkKR。此外，我们最近发表了一个病例，其中达巴霉素治疗选择了 1例心内膜炎患者通过WalK突变发生GP/LP/LGP耐药MRSA最近的这些发现导致了 更新的目标：阐明walKR突变导致GP/LP/LGP的多种机制 交叉耐药性，并揭示β-内酰胺和其他代谢调节剂如何与WalKR调节的 代谢网络与GP/LP/LGP协同作用并防止耐药性。我们假设walKR突变 通过调节细胞包膜和核苷酸， 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途径防止耐药性： 进一步改变细胞包膜或核苷酸代谢。在AIM 1中，我们将测量减少的 WalKR在MRSA中使用遗传、脂质组学、代谢组学、转录组学 和蛋白质组学方法结合敏感性测试和定量生物物理评估， 细胞包络特性。在AIM 2中，我们将检验β-内酰胺和其他代谢产物 调节剂可以使WalK敲低菌株对GP/LP/LGP再敏感。我们将研究的协同效应， 细胞壁抑制剂β-内酰胺和磷霉素，脂质合成抑制剂，和抗叶酸药物， 甲氧苄啶/磺胺甲恶唑，这将告知对walKR突变重要的代谢途径- 引起了抵抗。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， 在体外使用连续传代和PK/PD模型防止达巴霉素选择GP/LP/LGP抗性。 这项工作是重要的，因为达巴霉素暴露容易选择万古霉素和达托霉素耐药 S.金黄色葡萄球菌和战略，以防止耐药性和/或重新敏感MRSA GP/LG/LGP是至关重要的，以保持 这些药物，特别是在目前越来越多的达巴万星使用的情况下。本提案的创新之处在于 包括利用代谢表型来告知和调节抗性表型， 高通量脂质组学和代谢组学来表征耐药性和反应。

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.