Context-specific modulation of Clostridioides difficile virulence by Vancomycin-resistant Enterococcus faecium

艰难梭菌的毒素表达和孢子形成是特定环境的现象， 来自周围环境的信号，包括肠的局部营养环境。长期 本实验室的目标是确定细菌-细菌和宿主相互作用的机制基础， 调节C.很难本建议的总体目标是：㈠确定机制 的扩散抑制剂C.耐万古霉素屎肠球菌产生的艰难生长 (VRE)和（ii）确定C.艰难孢子形成。 中心假设是VRE调节C。多种环境依赖性的艰难毒力 这些机制取决于可用的碳源。该项目的基本原理是， 确定这两个物种之间的相互作用机制将允许开发抗- VRE治疗限制了CDI的可能性。为了实现这两个具体目标， 目的：1）明确碳源依赖抑制C.艰难的VRE。 基于初步数据，工作假设是：在存在碳源的情况下， VRE、C.低于pH阈值和碳的操纵会抑制艰难生长 来源在体外和VRE定殖动物中将调节C.艰难的毒力在这些实验中， 确定培养基将用于筛选一组碳源、肠球菌、乳酸菌和 艰难梭菌菌株，以确定条件调节生长，这些数据将用于开发 优化的VRE-C小鼠模型。艰难感染目标2）定义与接触相关的交互 VRE抑制C.很难根据初步数据，工作假设是： 在缺乏碳源的情况下，VRE抑制C.通过不可扩散的调制器难以区分 并且在这种状态下抑制孢子形成将增加由C.很难在这些合作中， 将使用培养测定和荧光显微镜来确定是否抑制孢子形成。 介导的接触和在什么发育阶段孢子形成被抑制。C. 将使用艰难梭菌来确定孢子形成遗传程序的哪个组分受VRE影响。 申请人认为，拟议的研究具有创新性，因为它特别侧重于 两种胃肠道病原体之间的相互作用以及肠道条件如何影响平衡 毒力性状之间的关系拟议的研究是重要的，因为它将提供一个背景， 了解C.在VRE定殖动物中的艰难毒力，这反映了常见的 这两种细菌在人类患者中的共定植。

Toxin expression and sporulation by Clostridioides difficile are context-specific phenomena that integrate signals from the surrounding environment including the local nutrient milieu of the intestine. The long-term goal of this laboratory is to define the mechanistic basis of bacteria-bacteria and host interactions that modulate virulence of C. difficile. The overall objectives in this proposal are to (i) determine the mechanism of a diffusible inhibitor of C. difficile growth produced by Vancomycin-resistant Enterococcus faecium (VRE) and (ii) define the mechanism of a contact-dependent inhibition of C. difficile sporulation by VRE. The central hypothesis is that VRE modulates C. difficile virulence by multiple context-dependent mechanisms that vary depending on the available carbon source. The rationale for this project is that determination of mechanisms of interaction between the two species will allow the development of anti- VRE treatments that limit the potential for CDI. To attain the overall objectives these two specific aims will be pursued: Aim 1) Identify the mechanism of carbon source-dependent suppression of C. difficile by VRE. Based on preliminary data, the working hypothesis is: in the presence of carbon sources that are converted to organic acids by VRE, C. difficile growth is suppressed below a pH threshold and manipulation of carbon sources in vitro and in VRE colonized animals will modulate C. difficile virulence. In these experiments a defined medium will be used to screen a panel of carbon sources, enterococci, lactic acid bacteria and C.difficile strains to determine conditions modulate growth and these data will be used to develop an optimized mouse model of VRE-C. difficile infection. Aim 2) Define the contact-dependent interaction with VRE that inhibits sporulation by C. difficile. Based on preliminary data, the working hypothesis is: in the absence of a carbon source, VRE inhibits entry into sporulation by C. difficile by a non-diffusible modulator and inhibition of sporulation in this state will increase the level of toxin produced by C. difficile. In these co- culture assays and fluorescence microscopy will be used to determine if the suppression of sporulation is mediated by contact and at what developmental stage sporulation is suppressed. Genetic selection in C. difficile will be used to determine which component of the sporulation genetic program is affected by VRE. The research proposed is innovative in the applicant’s opinion because it focuses specifically on interactions between two gastrointestinal pathogens and how conditions in the gut influence the balance between virulence traits. The proposed research is significant because it will provide a context for understanding the potential for C. difficile virulence in VRE-colonized animals, which mirrors the frequent co-colonization by these two bacteria in human patients.