Carbonic anhydrase inhibition as a target for antibiotic synergy in enterococci

总结 粪肠球菌是一种医院相关的机会致病菌， 发病率和死亡率。多药耐药肠球菌的发生率不断增加， 需要替代治疗策略来对抗这些病原体。在本提案中，我们描述了 一株罕见庆大霉素高敏感性大肠杆菌的分离和鉴定粪菌菌株， 从感染性心内膜炎病人身上培养出来的我们进行了体外抗性选择， 这个E。faecalis菌株以产生显示野生型的一步（即单突变）突变体 庆大霉素敏感性水平。一步突变体的全基因组测序表明， 亲本菌株中庆大霉素敏感性增高是由于突变破坏了E. 粪α-碳酸酐酶。另外，我们观察到碳酸酐酶抑制剂 乙酰唑胺和庆大霉素一起显示出协同活性，抑制野生 E型。粪菌菌株这一发现使我们假设碳酸酐酶的破坏 可使E.粪菌，以杀死与氨基糖苷类。为了确定这一现象的机械基础， 协同作用，我们将研究是否破坏E。粪便α-碳酸酐酶导致 通过质子动力依赖性转运增加庆大霉素摄取和/或增加膜 渗透性（目标1）。此外，我们还将研究氨基糖苷类和 化学多样的碳酸酐酶抑制剂对同基因E。粪菌菌株表达 不同碳酸酐酶基因型（Aim 2）。这是第一次， 碳酸酐酶破坏和细菌膜通透性将是 研究了本课题的成功完成将增加我们对E.粪便生物学， 同时也为开发有前途的新组合提供了重要的试验数据 治疗肠球菌感染的患者。

SUMMARY Enterococcus faecalis is a hospital-associated opportunistic pathogen that causes infections with high morbidity and mortality. An increasing occurrence of multidrug-resistant enterococci has driven the need for alternative treatment strategies to combat these pathogens. In this proposal, we describe the isolation and characterization of an unusual gentamicin hypersusceptible E. faecalis strain that was cultured from a patient with infective endocarditis. We performed an in vitro resistance selection with this E. faecalis strain to generate one-step (i.e. single mutation) mutants that displayed wild type gentamicin susceptibility levels. Whole-genome sequencing of the one-step mutants showed that gentamicin hypersusceptibility in the parent strain was caused by a mutation that disrupted the E. faecalis alpha-carbonic anhydrase. Separately, we observed that the carbonic anhydrase inhibitor acetazolamide and gentamicin together displayed synergistic activity at inhibiting the growth of wild type E. faecalis strains. This finding has led us to hypothesize that disruption of carbonic anhydrases can sensitize E. faecalis to killing with aminoglycosides. To determine the mechanistic basis of this synergy, we will examine whether disruption of the E. faecalis alpha-carbonic anhydrase causes increased gentamicin uptake via proton motive force-dependent transport and/or increased membrane permeability (Aim 1). In addition, we will investigate differential synergy between aminoglycosides and chemically diverse carbonic anhydrase inhibitors against isogenic E. faecalis strains expressing different carbonic anhydrase genotypes (Aim 2). This is the first time that a connection between carbonic anhydrase disruption and bacterial membrane energization or permeability will be investigated. Successful completion of this project will increase our understanding of E. faecalis biology, while also providing important pilot data toward the development of a promising new combination therapy for patients with enterococcal infections.