Plasmid encoded biofilm formation by the intestinal Bacteroidales and its importance in community ecology and resilience

项目摘要/摘要 肠道微生物区系在人类健康中发挥着重要作用，因此成为人们极感兴趣的焦点 治疗干预的目标。然而，我们在机制层面上对生态环境的理解 而塑造这个多样化和密集殖民生态系统的进化力量仍然脆弱。值得注意的是， 尽管我们知道水平基因转移在肠道中普遍存在，但我们只知道 从表面上看，这些交换的基因中的大多数的不同角色以及这个保存库如何影响 社区动态。同样，人们对社区复原力背后的机制知之甚少。这 对于最丰富的革兰氏阴性肠道微生物群类杆菌来说，这个问题尤其耐人寻味 成员，可以稳定地殖民几十年。这项提案将调查生物膜的重要性。 由拟杆菌科形成的群落生态和韧性，侧重于共生体 巨质粒pMMCAT，使获得它的菌株能够形成生物膜。这一质粒是 例外是因为人内高频率地转移到多个类杆菌属物种和其 无处不在，建筑保守，在全球人类群体中。一些研究表明，粘膜 健康人体中的生物膜很少见，但关于其他位置或独立生物膜的信息很少。我 假设在一个群落中的许多物种之间共享这个巨型质粒，能够形成 多物种生物膜，并在社区合作中发挥作用，特别是通过增加社区 恢复力。为了验证这一假设，或者理解pMMCAT的替代角色，我将系统地 在单个品系(AIM)的培养和诺生菌小鼠中鉴定该质粒所赋予的表型 1)或在一些菌株携带它的不同类杆菌群中(目标2)。了解生态环境的作用 对于pMMCAT，我将评估其所赋予的表型是否被共存的菌株协同或抑制 以及是否所有、只有一些其他菌株受益，或者没有其他菌株受益。对结肠中的生物膜进行可视化处理 根据压力脉冲，我将使用两种不同的方法来保存肠道群落的空间结构 并使用生物膜基质特异性染色。我随后将研究pMMCAT的进化动力学 在这些财团中转移(目标3)。为此，我将直接量化和可视化培养中的质粒转移 在灵知生菌的小鼠身上。我还将量化携带pMMCAT和表达生物膜形成的成本 基因。我将评估引入作弊品系的影响，这种品系不会支付健康成本 生产生物膜基质公益产品。最后，我将跟踪pMMCAT在十二年中的演变 在之前被发现携带pMMCAT菌株的四名人类志愿者中有四年。这是对 肠道中形成质粒编码生物膜的动力学和机制将改善我们的电流 了解肠道生态及其在人类种群中的最新变化，并将提供更多 为健康利益而进行有针对性的微生物群干预的垫脚石。

Project Summary/Abstract The intestinal microbiota plays a major role in human health and is therefore the focus of significant interest as a target for therapeutic interventions. However, our understanding at the mechanistic level of the ecological and evolutionary forces shaping this diverse and densely colonized ecosystem is still tenuous. Notably, although we know that horizontal gene transfer is pervasive in the gut community, we understand only superficially the different roles of the majority of these exchanged genes and how this repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying community resiliency. This question is particularly intriguing for the Bacteroidales, the most abundant Gram-negative gut microbiome members, which can stably colonize for decades. This proposal will investigate the importance of biofilm formation by the Bacteroidales for community ecology and resilience, focusing on the conjugative megaplasmid pMMCAT which enables biofilm formation in the strains that acquire it. This plasmid is exceptional because of the high frequency of intrapersonal transfer to multiple Bacteroidales species and its ubiquity, with conserved architecture, across global human populations. Some studies suggest that mucosal biofilms in healthy humans are rare, but there is little information about other locations or unattached biofilms. I hypothesize that this megaplasmid, shared among many species in a community, enables the formation of multi-species biofilms and plays a role in community cooperation, notably through increasing community resilience. To test this hypothesis, or otherwise understand alternative roles of pMMCAT, I will systematically characterize the phenotypes conferred by this plasmid in culture and in gnotobiotic mice in a single strain (aim 1) or in different Bacteroidales consortia where some strains carry it (aim 2). To understand the ecological role of pMMCAT, I will evaluate if its conferred phenotypes are synergized or inhibited by the co-resident strains and whether all, only some, or none of the other strains benefit. To visualize biofilms in the colon prior to and following a stress pulse, I will use two different methods to preserve the spatial structure of the gut community and use a biofilm matrix-specific stain. I will subsequently examine the evolutionary dynamics of pMMCAT transfer in these consortia (aim 3). To this end, I will directly quantify and visualize plasmid transfer in culture and in a gnotobiotic mouse. I will also quantify the cost of carrying pMMCAT and expressing biofilm formation genes. I will evaluate the impact of the introduction of a cheater strain that doesn’t pay this fitness cost of producing the biofilm matrix public good. Finally, I will track the evolution of pMMCAT over the course of twelve years in four human volunteers previously found to have pMMCAT-harboring strains. This dissection of the dynamics and mechanisms underlying plasmid-encoded biofilm formation in the gut will improve our current understanding of intestinal ecology and its recent changes in human populations, and will provide one more stepping stone towards targeted microbiome interventions for health benefits.