Molecular Mechanisms Of Daptomycin Resistance In Enterococci

描述（由申请人提供）：抗生素耐药性细菌的出现是21世纪影响人类的最具挑战性的公共卫生问题之一。在这些细菌中，耐万古霉素肠球菌（VRE）是美国医院中最难治疗的微生物之一。目前只有两种抗菌化合物被FDA批准用于治疗VRE感染;即利奈唑胺和奎奴普汀-达福普汀（Q/D）。然而，这两种药物对VRE的使用受到严重感染的次优治疗结局、副作用的频繁发生以及利奈唑胺和Q/D耐药VRE分离株的出现和广泛传播的阻碍。达托霉素（DAP）是一种脂肽抗生素，其杀伤机制涉及以钙依赖性方式与细菌细胞膜（CM）相互作用。DAP是目前唯一可用的具有抗VRE活性的杀菌抗生素。虽然DAP没有FDA批准的用于治疗VRE感染的适应症，但由于缺乏更好的替代方案来治疗感染VRE的患者，临床医生经常被迫使用DAP，这些患者通常病情严重，免疫系统受到严重损害。在VRE治疗期间，DAP的标签外使用在几种情况下导致了DAP耐药（DAP-R）的发生，从而进一步恶化了临床情况。本基金申请的长期目标是了解在VRE治疗期间导致DAP-R发展的分子事件，以便能够i）设计改进的治疗策略以防止DAP-R的出现，以及ii）确定未来抗微生物开发的新的潜在靶标，目的是保护DAP对抗VRE的功效。根据对DAP敏感和DAP耐药的粪肠球菌（VREfs）和大肠杆菌（E.在屎肠球菌（VREfm）中，我们已经鉴定了两个极有可能参与DAP-R的发展的基因：i）在VREfs和VREfm中编码心磷脂合酶的基因（cls），其参与细胞膜稳态，和ii）liaF基因的VREfs同源物，其是参与细菌细胞被膜对抗微生物剂和抗微生物肽的应答的三组分基因系统的一部分。因此，我们的目标是a）调查上述基因中突变的贡献（VREfs中的cls和VREfs中的liaF）对DAP耐药性的影响，和B）通过测试递增剂量的DAP和DAP与i）氨苄青霉素的组合疗法的效果，评估优化DAP用于VRE的使用的策略（对于VREfs），和ii）替加环素或利福平（对于VREfm），使用感染性心内膜炎的鼠模型预防DAP-R的出现。我们预计，这些研究将有助于更深入地了解CM磷脂稳态和细胞包膜调节在抗生素耐药性和抗菌肽作用的发展中的作用，并肯定会促进DAP作为一种有用的抗生素在未来治疗VRE感染的保护。

DESCRIPTION (provided by applicant): The emergence of antibiotic resistant bacteria is one of the most challenging public health problems affecting humankind in the 21st century. Among these bacteria, vancomycin-resistant enterococci (VRE) are one of the most difficult organisms to treat in hospitals across the US. Only two antimicrobial compounds are currently FDA-approved for the treatment of VRE infections; namely, linezolid and quinupristin-dalfopristin (Q/D). However, the use of these two agents against VRE has been hampered by suboptimal therapeutic outcomes in severe infections, frequent occurrence of side effects and the emergence and widespread dissemination of linezolid- and Q/D-resistant VRE isolates. Daptomycin (DAP) is a lipopeptide antibiotic whose mechanism of killing involves the interaction with the bacterial cell membrane (CM) in a calcium-dependent manner. DAP is the only bactericidal antibiotic currently available with activity against VRE. Although DAP does not have an FDA-approved indication for the treatment of VRE infections, clinicians are often pushed to use DAP due to the lack of better alternatives to treat patients infected with VRE who are often severely ill and with important compromise of the immune system. The off-label use of DAP during VRE therapy has led in several instances to the development of DAP resistance (DAP-R), thus, worsening the clinical scenario even further. Our long- term goal for this grant application is to understand the molecular events that lead to the development of DAP- R during VRE therapy to be able to i) design improved therapeutic strategies to prevent the emergence of DAP-R, and ii) identify new potential targets for antimicrobial development in the future with the aim of protecting the efficacy of DAP against VRE. Based on the information gathered from the comparative whole- genome, CM and cell envelope ultrastructural analysis of VRE clinical strain pairs of DAP-susceptible and DAP-resistant Enterococcus faecalis (VREfs) and E. faecium (VREfm), we have identified two genes that are highly likely to be involved in the development of DAP-R: i) a gene (cls) encoding a cardiolipin synthase enzyme in both VREfs and VREfm, involved in cell membrane homeostasis and ii) a VREfs homolog of the liaF gene, which is part of a three-component gene system involved in the bacterial cell envelope response to antimicrobials and antimicrobial peptides. Thus, we aim to a) investigate the contribution of mutations in the above genes (cls in both VREfs and VREfm and liaF in VREfs) to DAP-resistance, and b) evaluate strategies to optimize the use of DAP for VRE by testing the effect of escalating doses of DAP and combination therapies of DAP with i) ampicillin (for VREfs), and ii) with tigecycline or rifampin (for VREfm), in preventing emergence of DAP-R using a murine model of infective endocarditis. We anticipate that these studies will contribute to a deeper understanding of the role of CM phospholipid homeostasis and cell envelope regulation in the development of antibiotic resistance and antimicrobial peptide action and will certainly facilitate the preservation of DAP as a useful antibiotic to treat VRE infections in the future.