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

艰难梭状芽胞杆菌的毒素表达和产孢量是整合了 来自周围环境的信号，包括肠道的局部营养环境。长期的 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 调节艰难梭菌的毒力。这项建议的总体目标是：(一)确定机制 耐万古霉素屎肠球菌产艰难梭菌扩散性抑制物的研究 (VRE)和(Ii)定义了VRE对艰难梭菌产孢量的依赖接触抑制的机制。 核心假设是VRE通过多个上下文依赖的方式调节艰难梭菌的毒力 根据可用碳源的不同而变化的机制。这个项目的基本原理是 确定两个物种之间的相互作用机制将允许发展抗 VRE治疗限制了CDI的可能性。为了实现总目标，这两个具体目标将 目的：1)确定VRE对艰难梭菌的碳源依赖抑制机制。 根据初步数据，工作假设是：在存在转化为碳源的情况下 VRE，艰难梭菌对有机酸的生长被抑制在pH阈值以下和碳的操纵 在体外和在VRE定植动物中的来源将调节艰难梭菌的毒力。在这些实验中， 将使用定义的培养基来筛选一组碳源、肠球菌、乳酸菌和 艰难梭菌用于确定调节生长的条件，这些数据将用于开发一种 优化的VRE-C小鼠模型。艰难梭菌感染。目标2)定义依赖于接触的交互 抑制艰难梭菌产孢量的VRE。根据初步数据，工作假设是：在 在没有碳源的情况下，VRE通过非扩散调节器抑制艰难梭菌进入产孢期 在这种状态下抑制产孢会增加艰难梭菌产生的毒素水平。在这些合作中- 培养试验和荧光显微镜将被用来确定是否抑制产孢量 通过接触和在什么发育阶段抑制产孢子来调节。甘蓝属植物的遗传选择 艰难梭菌将被用来确定VRE影响产孢子遗传程序的哪一组分。 申请人认为，拟议的研究是创新的，因为它专门关注 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 在毒力特征之间。这项拟议的研究具有重要意义，因为它将为 了解艰难梭菌在VRE定植动物中的潜在毒力，这反映了频繁的 在人类患者体内由这两种细菌共同定植。

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.