How do superbugs hide from the host? Understanding the mechanisms of innate immune evasion in the opportunistic pathogen Enterococcus faecalis

所有肠球菌都产生一种表面多糖，称为肠球菌多糖抗原（EPA），在发病过程中起关键作用（Mistou et al., 2016）。EPA是由两个基因簇编码的：(i) 18个非常保守的基因，编码核心合成机制；（ii） 10-20个基因在不同菌株之间变化，负责多糖主干的修饰。我们的研究结果表明，装饰基因的突变体表现出形态缺陷，对β -内酰胺的易感性改变，并且在斑马鱼感染模型中不再具有毒性（Prasjnar等人，2013；Smith等人，2018）。我们假设EPA在细胞壁合成中参与蛋白质的细胞表面展示中起主要作用，并且这种聚合物的修饰在发病过程中介导了先天免疫逃避。本项目旨在研究EPA对粪肠杆菌耐药性和毒力的影响。将设定三个目标。探索EPA在细胞生长和抗微生物药物耐药性中的作用。我们将使用现有的不再或有条件地产生EPA的粪肠杆菌重组菌株来研究这种聚合物对细胞壁结构和动力学的贡献。我们还将探讨EPA如何调节靶向细胞包膜的抗菌剂的能力(例如。（β -内酰胺、阳离子肽、胆盐）结合并穿透细胞壁。探讨EPA在发病过程中的作用。我们将研究(i) EPA及其装饰如何在发病过程中调节巨噬细胞和中性粒细胞的识别，使用体外吞噬实验和斑马鱼感染模型；（ii）研究EPA对宿主细菌种群动态的作用。我们将测试可溶性EPA是否可以作为诱饵分子来逃避吞噬作用。探索肠球菌在感染过程中触发的细胞信号通路。我们将在原代人和小鼠巨噬细胞中使用体外吞噬实验来识别形成宿主对肠球菌感染反应的信号转导级联。

All enterococci produce a surface polysaccharide called the enterococcal polysaccharide antigen (EPA) that plays a key role during pathogenesis (Mistou et al., 2016). EPA is is encoded by two gene clusters: (i) 18 genes extremely conserved, encoding a core synthetic machinery and (ii) 10-20 genes variable from one strain to another, responsible for the decoration of the polysaccharide backbone. Our results indicate that mutants in the decoration genes display morphological defects, an altered susceptibility to beta-lactams and are no longer virulent in a zebrafish model of infection (Prasjnar et al., 2013; Smith et al., 2018). We hypothesize that EPA plays a major role for the cell surface display of proteins involved in cell wall synthesis and that the decoration of this polymer mediates innate immune evasion during pathogenesis.The aim of this project is to investigate how EPA contributes to E. faecalis antimicrobial resistance and virulence. Three objectives will be set.1. Explore the role of EPA in cell growth and resistance to antimicrobials. We will use available E. faecalis recombinant strains which no longer or conditionally produce EPA to investigate the contribution of this polymer to cell wall structure and dynamics. We will also explore how EPA modulates the capacity of antimicrobials targeting the cell envelope (eg., beta-lactams, cationic peptides, bile salts) to bind and penetrate the cell walls.2. Investigate the role of EPA during pathogenesis. We will study (i) how EPA and its decoration modulate recognition by macrophages and neutrophils during pathogenesis, using both in vitro phagocytosis assays and the zebrafish model of infection and (ii) investigate the role of EPA on bacterial population dynamics in the host. We will test if soluble EPA can be used as a decoy molecule to evade phagocytosis.3. Explore the cell signalling pathway triggered by enterococci during infection. We will use in vitro phagocytosis assay in primary human and murine macrophages to identify signal transduction cascades that shape the host response to enterococcal infections.