Carbonic anhydrase inhibition as a target for antibiotic synergy in enterococci

摘要 粪肠球菌是一种与医院相关的条件致病菌，可引起高致病性感染。 发病率和死亡率。多重耐药肠球菌的不断增加促使 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 一株罕见的庆大霉素过敏性粪肠球菌的分离和鉴定 从一位感染性心内膜炎患者身上培养出来。我们进行了体外抗药性筛选 这株粪肠球菌产生一步(即单一突变)突变体，显示野生型 庆大霉素敏感性水平。一步突变体的全基因组测序显示 庆大霉素在亲本菌株中的过敏性是由破坏E. 粪便中的α-碳酸氢酶。另外，我们观察到碳酸酐酶抑制剂 乙酰唑胺和庆大霉素联用抑制野生型沙门氏菌的生长 粪肠球菌类型。这一发现使我们假设碳酸酐酶的破坏 能使粪肠球菌对氨基糖苷类致死敏感。要确定这一点的机制基础 协同效应，我们将研究粪肠球菌α-碳酸氢酶的破坏是否会导致 通过质子动力依赖的转运和/或增加膜增加庆大霉素的摄取 渗透率(目标1)。此外，我们还将研究氨基糖苷类药物与其他药物的差异协同作用。 不同化学成分的碳酸酐酶抑制剂对表达同源粪肠球菌的抑制作用 不同碳酸酐酶基因型别(目标2)。这是第一次将 碳酸氢酶的破坏和细菌膜的通电或通透性 调查过了。该项目的成功完成将增加我们对粪肠球菌生物学的了解， 同时也为开发有前景的新组合提供了重要的试点数据 肠球菌感染患者的治疗。

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.