The LiaFSR system and antimicrobial peptide resistance in enterococci

摘要 抗药性细菌的出现是21世纪人类健康面临的最大威胁之一 世纪。特别是，万古霉素耐药肠球菌（VRE）是最具挑战性的微生物之一， 临床环境。事实上，耐万古霉素的屎肠球菌已被传染病委员会指定为耐万古霉素的屎肠球菌。 美国疾病协会认为，“超级细菌”之一，迫切需要新的治疗方法。 达托霉素（DAP），一种具有强效体外杀菌活性的细胞膜靶向脂肽抗生素 抗VRE的抗生素已经成为对抗这些微生物的关键“一线”抗微生物剂。然而，在这方面， 在治疗过程中产生耐药性是一个令人畏惧的挑战。DAP抗性的主要介导因子是 肠球菌是一组基因（命名为liaFSR），编码一个三组分调控系统 参与协调细胞被膜对抗生素和抗微生物肽的适应性反应。 LiaR是系统的反应调节剂（LuxR型），其活性似乎由LiaF调节， LiaS（组氨酸激酶）。然而，LiaF在肠球菌中的具体调节作用尚不清楚。 此外，我们已经鉴定了一组三个基因，它们是LiaR反应的介导物（命名为 liaXYZ）。LiaX是一种表面暴露和分泌的蛋白质，似乎是LiaR的主要协调者。 通过负调节LiaFSR系统介导细胞膜反应，控制细胞膜 磷脂重塑（与DAP抗性相关的表型）。此外，LiaX的N-末端 与青霉素结合蛋白5（肠球菌中细胞壁合成所需的关键酶）相互作用， β-内酰胺类药物的存在），并可能介导“跷跷板”效应（对β-内酰胺类药物过敏， DAP抗性的形成）。LiaY和LiaZ是由LiaX调控的两种跨膜蛋白。 我们的数据表明，LiaYZ是DAP抗性所必需的，LiaY可能是导致变化的原因。 在细胞膜磷脂结构中。因此，我们建议的首要假设是， 了解LiaFSR和LiaXYZ协调细胞膜反应的机制， 抗生素将为抗菌肽耐药性的分子机制提供新的见解， 细菌的适应性，可以利用新的治疗干预措施。我们计划发展我们的 实验方法在三个主要的具体目标。在sp.目的I中，我们将研究LiaF的作用， 一种跨膜蛋白，似乎在反应调节因子的激活中起主要和独特的作用 肠球菌中的LiaR。在具体目标II中，我们将重点关注LiaX作为主效应器的特性。 细胞包络自适应反应。具体目标III将评估LiaYZ作为DAP耐药介质的作用 和细胞膜重塑，假设这种作用是通过与 心磷脂合酶，一种主要的磷脂酶。我们希望能为一种新的生化物质提供证据 这是细胞对抗生素反应的范例，也可能是药物开发的新靶点。

ABSTRACT The emergence of antibiotic-resistant bacteria is one of the greatest threats to human health in the 21st century. In particular, vancomycin-resistant enterococci (VRE) are one of the most challenging organisms in clinical settings. Indeed, vancomycin-resistant Enterococcus faecium have been designated by the Infectious Disease Society of America as one of the “superbugs” against which new therapies are urgently needed. Daptomycin (DAP), a cell membrane-targeting lipopeptide antibiotic with potent in vitro bactericidal activity against VRE, has become a key “front-line” antimicrobial agent against these organisms. However, development of resistance during therapy is a daunting challenge. The major mediator of DAP resistance in enterococci is a cluster of genes (designated liaFSR) that encode a three-component regulatory system involved in orchestrating the cell envelope adaptive response to antibiotics and antimicrobial peptides. LiaR is the response regulator (LuxR-type) of the system whose activity seems to be regulated by LiaF and LiaS (histidine kinase). However, the specific regulatory role of LiaF in enterococci is unknown. Additionally, we have identified a cluster of three genes that are mediators of the LiaR response (designated liaXYZ). LiaX is a surface exposed and secreted protein that appears to be the main orchestrator of the LiaR- mediated cell membrane response by negatively regulating the LiaFSR system, controlling cell membrane phospholipid remodeling (a phenotype associated with DAP resistance). Additionally, the N-terminus of LiaX interacts with penicillin-binding protein 5 (a key enterococcal enzyme required for cell-wall synthesis in the presence of β-lactams) and is likely to mediate the “see-saw” effect (hypersusceptibility to β-lactams upon developing of DAP resistance). LiaY and LiaZ are two transmembrane proteins that are regulated by LiaX. Our data indicate that LiaYZ are required for DAP resistance and that LiaY is likely responsible for changes in cell membrane phospholipid architecture. Thus, the overarching hypothesis of our proposal is that understanding the mechanisms by which LiaFSR and LiaXYZ orchestrate the cell membrane response against antibiotics would provide novel insights into the molecular mechanisms of antimicrobial peptide resistance and bacterial adaptation that could be exploited with novel therapeutic interventions. We plan to develop our experimental approach in three major specific aims. In Sp. Aim I, we will investigate of the role of LiaF, a transmembrane protein that seems to play a major and distinct role in the activation of the response regulator LiaR in enterococci. In Specific Aim II, we will focus on the characterization of LiaX as the master effector of the cell envelope adaptive response. Specific Aim III will assess the role LiaYZ as mediators of DAP resistance and cell membrane remodeling under the assumption that such effect is mediated through interactions with cardiolipin synthase, a major phospholipid enzyme. We expect to provide evidence for a novel biochemical paradigm to the cell envelope response to antibiotics and, potentially, new targets for drug development.