Defining the Intracellular Growth Niche of Foodborne Listeria monocytogenes

摄入受单核细胞增生李斯特菌（Lm）污染的食物会导致人类疾病， 从轻微的自限性胃肠炎到危及生命的败血症和脑膜脑炎。具体 影响疾病严重程度的因素还不清楚，我们对肠道阶段的了解， 特别是，黑热病是非常有限的。我们最近开发了一种食源性感染的小鼠模型， 研究Lm与肠道天然免疫细胞的相互作用。在初步研究中，我们发现， 肠道中的大部分Lm是细胞外的。这是一个出乎意料的结果，因为细胞内生长 并在细胞间传播而不遇到细胞外环境通常被认为是 这些兼性胞内细菌的主要毒力策略。LplA 1缺陷型Lm， 在细胞内复制可以很容易地侵入肠道粘膜，并在底层建立感染。 固有层，但没有持续以及野生型Lm，并在感染后三天有严重的 肠系膜淋巴结（MLN）扩散缺陷。这表明细胞内生长不是 肠道感染的初始阶段所需的，但复制在一些尚未确定的细胞类型中， 随着感染的进行肠道变得越来越重要。多色流式细胞术可以 区分八种不同的骨髓来源的吞噬细胞的子集是独特的肠道。初步 这里提供的数据证实，肠组织含有至少一种细胞类型， Lm的细胞内生长，并排除了Ly 6Chi单核细胞和所有三个常规树突状细胞亚群。 细胞作为细胞内生态位。该提案的主要目标是确定肠道中的细胞类型， 支持Lm的细胞内生长，并确定肠细胞类型的先天免疫应答， 主要与细胞内或细胞外Lm相互作用。我们假设Lm最初 主要是它们不能有效入侵或生存的细胞类型， 感染后，细菌转变为一种细胞类型，作为一个受保护的细胞内生长的小生境。在目标1中， 候选的肠髓样细胞类型将被分选纯化，直接离体感染， 细胞内定位和复制。在目标2中，流式细胞术将用于鉴定Lm相关细胞 在回肠和结肠的固有层和粘膜下层以及排出这些中的每一个的MLN中， 组织（SI-MLN和LI-MLN）中的细胞，以追踪侵入小鼠肠粘膜的Lm的命运。在目标3中，我们 通过测量以下细胞的产生来确定所有八个肠髓样细胞亚群的初始反应： 已知由宿主细胞表面结合或胞质受体触发的细胞因子。这些探索性 研究将通过定义肠道中发生的早期事件来填补该领域的关键知识空白， Lm的食物传播。!

Ingestion of Listeria monocytogenes (Lm)-contaminated food results in human disease ranging in severity from mild, self-limiting gastroenteritis to life-threatening septicemia and meningoencephalitis. The specific factors that influence disease severity are not well understood, and our knowledge of the intestinal phase of listeriosis, in particular, is severely limited. We recently developed a mouse model of foodborne infection to study the interaction of Lm with intestinal innate immune cells. In preliminary studies, we found that the majority of Lm in the gut were extracellular. This was an unexpected result, because intracellular growth and spread from cell-to-cell without encountering the extracellular milieu are generally regarded as the primary virulence strategies for these facultative intracellular bacteria. LplA1-deficient Lm that were unable to replicate intracellularly could readily invade the gut mucosa and establish infection in the underlying lamina propria, but did not persist as well as wildtype Lm, and by three days post-infection had a severe defect in spreading to the mesenteric lymph nodes (MLN). This suggests that intracellular growth is not required for the initial stages of intestinal infection, but replication in some as-yet-unidentified cell type in the gut becomes increasingly more important as the infection proceeds. Multicolor flow cytometry can discriminate eight different subsets of myeloid-derived phagocytes that are unique to the gut. Preliminary data provided here verified that intestinal tissue contained at least one cell type that could support intracellular growth of Lm, and ruled out Ly6Chi monocytes and all three subsets of conventional dendritic cells as the intracellular niche. The primary goal of this proposal is to identify the cell type(s) in the gut that support intracellular growth of Lm and to define the innate immune response of intestinal cell types that that interact primarily with either intracellular or extracellular Lm. We hypothesize that Lm initially interact primarily with cell types that that they cannot efficiently invade or survive in, and that later in the course of infection, the bacteria shift to a cell type that serves as a protected intracellular growth niche.!In Aim 1, four candidate intestinal myeloid cell types will be sort purified, infected directly ex vivo and assayed for both intracellular localization and replication. In Aim 2, flow cytometry will be used to identify Lm-associated cells in the lamina propria and submucosa of the ileum and colon as well as the MLN that drain each of these tissues (SI-MLN and LI-MLN) to track the fate of Lm that invade the gut mucosa in mice. In Aim 3, we will define the initial response of all eight subsets of intestinal myeloid cells by measuring the production of cytokines known to be triggered by either host cell surface bound or cytosolic receptors. These exploratory studies will fill a key knowledge gap in the field by defining the early events that occur in the gut during foodborne transmission of Lm. !