The impact of dietary metals on the gut microbiome and Clostridium difficile infection

 描述(申请人提供)：艰难梭菌是一种革兰氏阳性芽胞形成细菌，可引起多种胃肠道疾病。在过去的十年中，与艰难梭菌感染(CDI)相关的发病率、严重性和成本显著增加。用常规抗生素治疗感染的困难，复发感染率的增加，以及超强毒力菌株的出现，突显了研究新的治疗策略的必要性。肠道的破坏促进了CDI的启动 微生物组，最常见的是通过广谱抗菌治疗，使艰难梭菌能够定植和生长。然而，非抗生素相关的CDI病例的比率有很好的记录，并在稳步上升。这表明，许多未知因素可能会影响CDI的易感性。在这项建议中，我们将探讨营养金属锌(锌)对CDI的影响。在感染期间，获得营养金属对细菌复制和毒力因子的产生产生了深远的影响。为了利用这一点，宿主 在一种被称为营养免疫的过程中，会产生限制金属供应的因素。钙保护素(CP)是一种这样的蛋白质因子，它通过结合锌和锰(Mn)的能力而具有强大的抗菌性能。令人惊讶的是，很少有人研究CP介导的锌饥饿在CDI中的作用。在感染期间影响锌利用率的另一个因素是饮食。饮食中锌水平的改变与免疫系统功能下降和对各种感染的易感性增加有关；然而，关于 饮食中金属水平的变化如何影响肠道微生物群。此外，膳食锌对CDI易感性的影响还有待确定。我们假设：(I)膳食金属水平的变化深刻影响肠道微生物群的组成，这种重塑影响艰难梭菌的易感性；(Ii)钙保护素(CP)介导的金属封存对于限制艰难梭菌的生长、致病和持久性是必不可少的；(Iii)Diicil通过参与金属摄取、代谢和解毒的专用基因系统的表达来适应营养金属水平的变化。我们计划通过一系列综合的具体目标来检验这三个假说。首先，我们将定义饮食中营养金属水平的变化如何影响小鼠肠道微生物群和敏感性CDI(目标1)。接下来，我们将确定CP介导的金属饥饿在CDI中的作用(目标2)。最后，我们将确定艰难梭菌在高或低n条件下生长和毒力所需的基因产物(目标3)。这些实验将阐明改变膳食锌水平对肠道微生物群和CDI的影响。此外，艰难梭菌参与营养金属动态平衡的基因产物的特征将为未来专注于了解艰难梭菌如何在宿主内获得营养的研究奠定基础。总之，这项建议的结果将为开发治疗CDI的新疗法策略奠定基础。

 DESCRIPTION (provided by applicant): Clostridium difficile is a gram-positive, spore-forming bacterium that causes a wide range of gastrointestinal disorders. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased significantly. Difficulties in treating infections with conventional antibiotics, increasing rates f recurrent infection, and the emergence of hyper-virulent strains underscore the need for investigating new therapeutic strategies. Initiation of CDI is facilitated by disruption of the gut microbiome, most commonly mediated by broad-spectrum antimicrobial treatment, which enables C. difficile colonization and outgrowth. However, the rate of non-antibiotic associated CDI cases are well documented and steadily on the rise. This suggests that a wide range of unexplored factors likely influence susceptibility to CDI. In this proposal, we will explore the impact of the nutrient metal, Zinc (Zn), on CDI. During infection, access to nutrient metals profoundly impacts bacterial replication and virulence factor production. To exploit this, the host produces factors that limit metal availability in a process termed nutritional immunity. One such protein factor, calprotectin (CP), has strong antimicrobial properties mediated by its ability to bind Zn and manganese (Mn). Surprisingly, little work has been done to characterize the contribution of CP-mediated Zn starvation in CDI. Another factor likely impacting Zn availability during infection is diet. Altered dietary Zn levels are associated with decrease immune system function and increased susceptibility to various infections; however, there is a paucity of data on how altered dietary metal levels affect the gut microbiome. Furthermore, the impact of dietary Zn on susceptibility to CDI has yet to be defined. We hypothesize that (i) alterations in dietary metal levels profoundly impact the composition of the gut microbiome and this remodeling affects the susceptibility to C. difficile, (ii) calprotectin (CP) mediated metal sequestration is essential for limiting growth, pathogenesis, and persistence of C. difficile, and (iii) C. difficil adapts to variations in nutrient metal levels through the expression of dedicated gene systems involved in metal uptake, metabolism, and detoxification. We plan to test these three hypotheses through a series of integrated Specific Aims. First, we will define how dietary alterations in nutrient metal levels affect the murine gut microbiome and susceptibility CDI (Aim 1). We will next determine the role for CP-mediated metal starvation in CDI (Aim 2). Finally, we will identify C. difficile gene products that are required for growth and virulence in high or low n conditions (Aim 3). These experiments will elucidate the impact of altered dietary Zn levels on the gut microbiome and CDI. Furthermore, characterization of C. difficile gene products involved in nutrient metal homeostasis will lay the groundwork for future studies focused on understanding how C. difficile acquires nutrients within the host. Together, results from this proposal will lay the groundwork for the development of novel therapeutics strategies for treatment of CDI.