Francisella tularensis Interactions with Airway Epithelial Cells

土拉热弗朗西丝氏菌是一种人畜共患细菌病原体，在数百个国家引起致命感染。 不同的动物物种。人类感染是由昆虫媒介传播、直接物理传播、 接触受感染的动物，摄入受污染的食物或水，以及吸入雾化的 有机体所有接触途径都可导致传播性感染，但发病率最高， 死亡率与吸入性感染有关。我们的目标是了解宿主 病原体相互作用定义肺土拉菌病。为此，我们在体内开发了 和体外模型研究F.土拉热菌与不同肺细胞类型的相互作用。与这些模型 我们发现F.土拉菌在组织和肺泡巨噬细胞内侵入和复制， 树突细胞、嗜中性粒细胞和肺泡II型（AT-II）上皮细胞。入侵和 AT-II细胞内的复制代表了细菌病原体的新环境小生境。目标1 我们将描述F。土拉菌与AT-Ⅱ细胞的相互作用，重点是细菌的运输及其 对AT-II细胞生理学的影响。筛选转座子插入突变体库，鉴定菌株 在AT-II细胞内复制缺陷的细胞。一个这样的突变体有一个转座子插入在一个 我们称之为regA基因。regA基因序列是弗朗西斯属物种所特有的。Rega 在进入AT-II细胞时诱导表达180倍，在进入J774细胞时诱导表达6倍。 转座子突变体侵入但未能在AT-II细胞中复制，但它保留了在AT-II细胞中复制的能力。 在巨噬细胞内复制，而regA缺失突变体在AT-II细胞或 巨噬细胞在缺失和插入regA中，许多基因的表达都发生了改变 通过微阵列和2D-凝胶分析确定的突变菌株。.受影响的五种基因 RegA在TC-1细胞中生长的野生型细菌中被诱导，并且在其他遗传学中也被鉴定。 筛选与毒力相关的基因。基于这些和其他数据，我们提出RegA是 参与对细胞内环境的感知和适应反应。在目标2中， 检查RegA的属性和功能，并确定其表达的机制 是受管制的。在目的3中，5个基因座编码具有未知功能的蛋白质，其表达为 受RegA影响的患者将接受遗传和生化分析，以确定其在以下方面的作用： 弗朗西斯菌胞内生长。

Francisella tularensis is a zoonotic bacterial pathogen that causes fatal infections in hundreds of different animal species. Human infections result from transmission by insect vectors, direct physical contact with infected animals, ingestion of contaminated food or water, and inhalation of aerosolized organisms. All contact routes can result in disseminated infections, but the highest morbidity and mortality is associated with inhalation ¿ initiated infection. Our goal is to understand the host pathogen interactions that define pulmonary tularemia. Towards that end we have developed in vivo and in vitro models to study F. tularensis interactions with different lung cell types. With these models we have found that F. tularensis invades and replicates within tissue and alveolar macrophages, dendritic cells, neutrophils and alveolar type II (AT-II) epithelial cells following inhalation. Invasion and replication within AT-II cells represents a novel environmental niche for a bacterial pathogen. In Aim 1 we will characterize F. tularensis interactions with AT-II cells, focusing on bacterial trafficking and its impact on AT-II cell physiology. A transposon insertion mutant library was screened to identify strains that were deficient for replication within AT-II cells. One such mutant had a transposon insertion in a gene we termed regA. The regA gene sequence was unique to Francisella species. RegA expression was induced 180-fold upon entry into AT-II cells, and 6-fold upon entry into J774 cells. The transposon mutant invaded but failed to replicate in AT-II cells, however it retained the ability to replicate within macrophages, whereas regA deletion mutants did not replicate in either AT-II cells or macrophages. The expression of a number of genes was altered in both deletion and insertion regA mutant strains as determined by microarry and 2D-gel analysis. . Five of the genes affected by RegA were induced in wild type bacteria grown in TC-1 cells, and were also identified in other genetic screens for virulence-associated genes. Based on these and other data we propose that RegA is involved in the sensing and adaptation response to the intracellular environment. In Aim 2 we will examine the properties and function of, RegA, and determine the mechanism by which its expression is regulated. In Aim 3, 5 loci encoding proteins with unknown function and whose expression is affected by RegA will be subjected to genetic and biochemical analysis to determine their roles in Francisella intracellular growth.