Molecular Mechanisms Of Daptomycin Resistance In Enterococci

描述(申请人提供)：抗生素耐药细菌的出现是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，万古霉素耐药肠球菌(VRE)是美国各地医院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VRE感染的治疗；即利奈唑胺和奎努普汀-达福普汀(Q/D)。然而，这两种药物在严重感染中的治疗效果不佳，副作用频繁发生，以及对利奈唑胺和Q/D耐药的VRE分离株的出现和广泛传播，阻碍了这两种药物的使用。达托霉素(DAP)是一种脂肽类抗生素，其杀伤机制涉及与细菌细胞膜的相互作用，这种作用依赖于钙离子。DAP是目前唯一具有抗VRE活性的杀菌抗生素。虽然DAP没有FDA批准的治疗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，这些患者通常病情严重，免疫系统有重要损害，因此临床医生经常被迫使用DAP。DAP在VRE治疗中的非标记性使用在几个实例中导致了DAP耐药(DAP-R)的发展，从而使临床情况进一步恶化。我们此次拨款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潜在靶点，目的是保护DAP对VRE的疗效。基于对DAP敏感和耐DAP粪肠球菌(VREf)和粪肠球菌(VREfm)VRE临床分离株的比较全基因组、CM和细胞膜超微结构分析所收集的信息，我们发现了两个极有可能参与DAP-R-R发展的基因：i)VREf和VREfm中编码心磷脂合成酶的基因(Cls)，参与细胞膜的动态平衡；ii)LIAF基因的VREfs同源基因，它是参与细菌包膜对抗菌素和抗菌肽反应的三组分基因系统的一部分。因此，我们的目标是：a)研究上述基因突变(VREf和VREfm中的CLS和VREf中的LIAF)对DAP耐药的贡献，以及b)通过测试递增剂量的DAP和DAP与I)氨苄西林(VREf)和ii)替格环素或利福平(VREfm)在预防DAP-R出现方面的效果来评估优化DAP治疗VRE的策略。我们预计，这些研究将有助于更深入地了解CM磷脂稳态和细胞膜调节在抗生素耐药性和抗菌肽作用中的作用，并将有助于保存DAP作为未来治疗VRE感染的有用抗生素。

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.