Molecular mechanisms of Francisella tularensis pathogenesis & immunity

土拉热弗朗西丝氏菌是一种高度传染性的革兰氏阴性球杆菌， 人畜共患病兔热病，是A类病原体。了解F. 土拉热病以对抗可能的威胁是显而易见的。土拉菌病的一个标志是 在保护性细胞介导的免疫反应开始之前，细菌在哺乳动物宿主中生长。 哺乳动物宿主被赋予许多抗微生物效应子功能。因此，F. tularensis 已经进化出了破坏宿主防御的机制令人惊讶的是，这种小细菌可以感染它的 通过各种不同的感染途径感染宿主，每种感染途径涉及不同的宿主组织部位， 不同的微环境。考虑到F.土拉热病毒通过多种组织感染宿主 我们的假设是，除了一般生存和生长所需的一组核心基因外， 在体内，F.土拉热菌具有特定组织或微生境所需的额外基因。所以我们 总体目标是确定新的核心和组织特异性毒力因子在F。土拉热。上 目的，我们将确定组织特异性（例如肺，脾和皮肤特异性）F。土拉菌毒力因子 我们建立的基于微阵列的阴性选择方法， 和皮内接种途径。在第二个和第三个目标中，我们将验证 新的毒力因子，并在我们的小鼠感染模型中表征分子机制， 体外组织培养试验。 该项目与该计划中的其他Francisella项目具有协同作用，因为它将使我们能够 直接比较马库斯·霍维茨博士实验室获得的遗传和蛋白质组学分析结果， 利用F.土拉热亚种tularensis、LVS和F. novicida与我们的体内阴性选择结果。 由于我们将使用相同的转座子突变体文库用于我们的体内和体外测定，因此我们将使用相同的转座子突变体文库进行体内和体外测定。 非常快速的基于微阵列的检测方法，我们将确定新的弗朗西斯因子， 以极其高效的方式宿主蛋白质。

Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia and is a Category A agent. The need for understanding the molecular basis for F. tularensis disease in order to combat possible threats is evident. A hallmark of tularemia is the ability of the bacterium to grow in mammalian hosts before the onset of a protective cell-mediated immune response. Mammalian hosts are endowed with numerous antimicrobial effector functions. Accordingly, F. tularensis has evolved mechanisms to subvert host defenses. It is very striking that this small bacterium can infect its host via a variety of different infection routes, each of which involves a different host tissue site with a vastly different microenvironment. Given that F. tularensis is so successful at infecting its host via multiple tissue sites, our hypothesis is that in addition to a core set of genes that are needed for general survival and growth in vivo, F. tularensis possess additional genes that are required in specific tissues or microniches. Thus, our overarching goal is to identify novel core and tissue-specific virulence factors in F. tularensis. In the first aim, we will identify tissue-specific (e.g. lung-, spleen, and skin-specific) F. tularensis virulence factors using our well-established microarray-based negative selection methodology following intranasal, intraperitoneal and intradermal routes of inoculation. In the second and third aims, we will validate the tissue-specificity of novel virulence factors and characterize the molecular mechanisms in our mouse models of infection and in vitro in tissue culture assays. This project is synergistic with the other Francisella project in the Program in that it will allow us to directly compare the results of genetic and proteomic analyses obtained by Dr. Marcus Horwitz's laboratory utilizing F. tularensis subsp. tularensis, the LVS and F. novicida with our in vivo negative selection results. Since we will be using the same transposon mutant library for our in vivo and in vitro assays, followed by our very rapid microarray-based detection method, we will identify novel Francisella factors that interact with host proteins in an extremely efficient manner.