Defining the Intracellular Growth Niche of Foodborne Listeria monocytogenes

定义食源性单核细胞增生李斯特菌的细胞内生长生态位

基本信息

批准号：
10113535
负责人：
SARAH E. F. D'ORAZIO
金额：
$ 22.95万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10113535
关键词：
Anatomy Bacteremia Bacteria Biological Assay Blood Bypass C57BL/6 Mouse Cell surface Cells Colon Cytosol Data Defect Dendritic Cells Dependence Disease Outbreaks E-Cadherin Eating Elderly Environment Epithelial Event Exposure to Feces Flow Cytometry Food Food Contamination Food Supply Gastroenteritis Gastrointestinal tract structure Gentamicins Goals Growth Gut Mucosa Hospitalization Immune Immune system Immunoassay In Vitro Incidence Individual Infection Ingestion Innate Immune Response Interleukin-12 Interleukin-18 Interleukin-6 Intestinal Diseases Intestines Intravenous Invaded Knowledge Lamina Propria Large Intestine Lead Life Ligation Listeria monocytogenes Listeriosis M cell Measures Mediating Meningoencephalitis Minor Modeling Mus Myelogenous Myeloid Cells Neuraxis Phagocytes Phase Population Process Production Resistance Route Sepsis Septicemia Severities Severity of illness Small Intestines Stains Submucosa Surface Symptoms Systemic infection Therapeutic Intervention Time Tissues Virulence Work cell type cytokine cytosolic receptor design draining lymph node enteric infection experience experimental study extracellular feeding foodborne foodborne infection foodborne pathogen human disease ileum in vivo macrophage mesenteric lymph node monocyte mortality mouse model neonate pathogen prevent response transmission process

项目摘要

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. !

摄入李斯特菌单核细胞增生（LM）污染的食物导致人类疾病的严重程度范围从轻度，自限制的胃肠炎到威胁生命的败血病和脑膜脑炎。具体影响疾病严重程度的因素尚不清楚，我们对特别是李斯特氏病受到严重限制。我们最近开发了一种食源性感染的小鼠模型研究LM与肠道免疫细胞的相互作用。在初步研究中，我们发现肠道中的大多数LM都是细胞外的。这是一个意外的结果，因为细胞内生长并从细胞到细胞传播而不遇到细胞外环境通常被视为这些细胞内细菌的主要毒力策略。 LPLA1缺陷LM无法在细胞内复制可以轻易入侵肠粘膜并在下面的感染中建立感染 lamina propria，但不像LM那样持久，感染后三天有严重扩散到肠系膜淋巴结（MLN）的缺陷。这表明细胞内生长不是肠道感染的初始阶段需要，但在某些尚未确定的细胞类型中复制随着感染的进行，肠道变得越来越重要。多色流式细胞仪可以区分肠道所特有的八个不同的髓样衍生的吞噬细胞。初步的此处提供的数据证实，肠组织至少包含一种可以支持的细胞类型 LM的细胞内生长，排除Ly6chi单核细胞和所有三个子集的常规树突状子集细胞为细胞内小众。该提案的主要目的是确定肠道中的细胞类型支持LM的细胞内生长并定义肠细胞类型的先天免疫反应主要与细胞内或细胞外LM相互作用。我们假设LM最初相互作用主要是用它们无法有效入侵或生存的细胞类型，而在此期间之后感染，细菌转移到一个细胞类型，作为受保护的细胞内生长生长位。！在AIM 1中，四个候选肠髓样细胞类型将被排序纯化，直接感染了离体并分析了两者细胞内定位和复制。在AIM 2中，流式细胞仪将用于鉴定与LM相关的细胞在叶片和结肠的lamina lamina propria和suber子中，以及沥干这些的MLN 组织（Si-MLN和Li-MLN）跟踪侵入小鼠肠粘膜的LM的命运。在AIM 3中，我们将通过测量的产生已知的细胞因子是由宿主细胞表面结合或胞质受体触发的。这些探索性研究将通过定义肠道中发生的早期事件在现场的关键知识差距 LM的食源性传播。呢