The LiaFSR system and antimicrobial peptide resistance in enterococci

摘要 抗药性细菌的出现是21世纪对人类健康的最大威胁之一 世纪。尤其是耐万古霉素的肠球菌(VRE)是最具挑战性的细菌之一 临床环境。事实上，对万古霉素耐药的粪肠球菌已经被感染性疾病 美国疾病学会被认为是迫切需要新疗法来对抗的“超级细菌”之一。 达托霉素(DAP)--一种具有较强体外杀菌活性的细胞膜靶向脂肽抗生素 抗VRE，已成为对抗这些微生物的关键的一线抗菌剂。然而， 在治疗过程中产生耐药性是一项艰巨的挑战。DAP抗性的主要介体 肠球菌是编码三组分调控系统的一组基因(指定为ilifsr)。 参与协调细胞被膜对抗生素和抗菌肽的适应性反应。 说谎者是系统的反应调节器(LuxR类型)，其活动似乎受LIAF和 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，我们已经确定了一个由三个基因组成的簇，它们是说谎者反应的中介(指定为 利亚XYZ)。LianX是一种表面暴露和分泌的蛋白质，似乎是说谎者的主要指挥- 负性调节LianFSR系统、调控细胞膜介导细胞膜应答 磷脂重塑(与DAP抵抗相关的表型)。此外，LianX的N-末端 与青霉素结合蛋白5(一种关键的肠球菌酶)相互作用 β-内酰胺类药物的存在)，并可能介导拉锯效应(对β-内酰胺类药物的过敏反应 DAP抗性的发展)。Liay和LianZ是受LianX调控的两种跨膜蛋白。 我们的数据表明，LianYZ是DAP抗性所必需的，Liay可能是导致变化的原因 在细胞膜上有磷脂结构。因此，我们提案的首要假设是 了解LianFSR和LianXYZ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和 细菌适应，可通过新的治疗干预措施加以利用。我们计划发展我们的 实验方法主要有三个具体目标。在Sp.目的I，我们将调查LIAF的作用，a 跨膜蛋白似乎在反应调节因子的激活中起着主要和独特的作用 肠球菌病的骗子。在《特定目标2》中，我们将重点研究辽西作为主要效应者的特征。 细胞包络的适应性反应。SPARTIAM III将评估连云港作为DAP抵抗的中介体的作用 和细胞膜重塑的假设下，这种影响是通过相互作用 心磷脂合成酶，一种主要的磷脂酶。我们希望为一种新的生物化学提供证据 从细胞包膜到对抗生素的反应，以及潜在的药物开发新靶点。

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.