Francisella tularensis Interactions with Airway Epithelial Cells

项目负责人/主要研究者（最后一名、第一名、中间名）： Kawula，托马斯H. 3 R56 AI069339-01A2 摘要土拉热弗朗西丝菌是一种人畜共患细菌性病原体，可引起数百种不同动物的致命性感染。人类感染的原因是通过昆虫媒介传播、与受感染动物直接身体接触、摄入受污染的食物或水以及吸入雾化的生物体。所有的接触途径都可能导致播散性感染，但最高的发病率和死亡率与吸入性感染有关。我们的目标是了解宿主病原体的相互作用，确定肺兔热病。为此，我们开发了体内和体外模型来研究F。土拉热菌与不同肺细胞类型的相互作用。利用这些模型，我们发现F.土拉菌在吸入后侵入组织和肺泡巨噬细胞、树突细胞、嗜中性粒细胞和肺泡II型（AT-II）上皮细胞内并在其中复制。AT-II细胞内的入侵和复制代表了细菌病原体的新环境生态位。在目标1中，我们将描述F。土拉菌与AT-II细胞的相互作用，重点是细菌运输及其对AT-II细胞生理学的影响。筛选转座子插入突变体文库以鉴定AT-II细胞内复制缺陷的菌株。其中一个突变体在我们称为regA的基因中插入了转座子。regA基因序列是弗朗西斯属物种所特有的。RegA表达在进入AT-II细胞后诱导180倍，在进入J774细胞后诱导6倍。转座子突变体入侵，但未能在AT-II细胞中复制，但它保留了在巨噬细胞内复制的能力，而regA缺失突变体在AT-II细胞或巨噬细胞中均不复制。通过微阵列和2D-凝胶分析确定，在缺失和插入regA突变株中，许多基因的表达发生了改变。.受RegA影响的五个基因在TC-1细胞中生长的野生型细菌中被诱导，并且在其他遗传筛选中也被鉴定为毒力相关基因。基于这些和其他数据，我们提出，RegA是参与传感和适应细胞内环境的反应。在目标2中，我们将研究RegA的性质和功能，并确定其表达调控的机制。在目标3中，将对编码具有未知功能的蛋白质并且其表达受RegA影响的5个位点进行遗传和生化分析，以确定它们在弗朗西斯菌胞内生长中的作用。

Program Director/Principal Investigator (Last, First, Middle): Kawula, Thomas H. 3 R56 AI069339-01A2 ABSTRACT 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.