Mechansisms of innate immune evasion by Francisella tularensis

图拉氏方济氏菌是一种兼性的巨噬细胞内病原体，也是引起 图拉热症。尽管很明显，吸入只有十个图拉氏亚种。图拉尔人可能是致命的，而且 巨噬细胞是体内这种生物的主要储存库，Ft如何逃脱消除只是 开始被定义了。在摄取的几个小时内，Ft就会冲破吞噬体膜， 在细胞质中复制。我们最近的数据首次表明，全毒力Ft株SchuS4 在比先前认识到的更早的途径阶段干扰吞噬小体成熟 从一个新的隔间中逃脱，该隔间排除了晚期内体标记Rab7，甘露糖-6-磷酸 受体、溶二磷脂酸和组织蛋白酶S，尽管局部积累LAMP-1。这一切的产生 隔室需要活的、MglA阳性的Ft，是通过这种病原体抑制的能力实现的 蛋白激酶C-a(PKCA)在感染细胞中的活性。因此，我们的数据确定了PKCA 作为已知的第一个被Ft靶向以逃避细胞内杀伤的宿主因子。以下项目也需要PKCA 先天防御的其他方面，以及我们的初步数据支持MHC II类抗原的假设 呈现和氧化防御机制也可能受损。同时，Ft如何被攻破 吞噬体膜尚不清楚，我们提出的证据支持一种模型，在该模型中， 接头和Rab家族GTP酶在吞噬小体溶解和细菌逃逸到 胞浆。从细菌的角度来看，几乎没有鉴定出弗朗西斯氏菌的毒力因子，它们的 行动机制仍不明朗。我们最近开发了一种Tn5转座子突变系统 Francisella，我们已经用来鉴定FTL0347和FTL1542的突变体，它们编码一个假设的 膜透性酶和酰辅酶A合成酶。已知的毒力因子的表达是 在这些突变体中都显著减少，初步特征表明这些基因是 分别为吞噬小体逃逸和抑制吞噬细胞呼吸爆发所需。另外， 我们构建了我们的转座子的垃圾(转座子位置)(杂交)衍生物，它将允许我们 识别人类巨噬细胞进入Ft、生长和存活所需的新基因，因此，我们的 具体目的是：1)阐明Ft介导的抑制作用的机制和功能后果 巨噬细胞中的PKCA信号转导；以及2)鉴定和鉴定新的图拉氏狸殖吸虫基因。 利用转座子诱变逃避巨噬细胞的天然防御，包括垃圾。

Francisella tularensis is a facultative intracellular pathogen of macrophages and the causative agent of tularemia. Although it is clear that inhalation of as few as ten F. tularensis subsp. tularensis can be fatal, and that macrophages are the major reservoir of this organism in vivo, how Ft evades elimination is only beginning to be defined. Within a few hours of uptake, Ft breaches the phagosome membrane and replicates in the cytosol. Our recent data demonstrate for the first time that fully virulent Ft strain SchuS4 disrupts phagosome maturation at an earlier stage of the pathway than has previously been appreciated and escapes from a novel compartment that excludes the late endoosome markers Rab7, mannose-6-phosphate receptor, lysobisphosphatidic acid and cathepsin S, despite local accumulation of lamp-1. Generation of this compartment requires live, MglA-positive Ft and is achieved via the ability of this pathogen to inhibit profoundly the activity of protein kinase C-a (PKCa) throughout infected cells. Thus, our data identify PKCa as the first host factor known to be targeted by Ft to evade intracellular killing. PKCa is also required for other aspects of innate defense, and our preliminary data support the hypothesis that MHC class II antigen presentation and oxidative defense mechanisms may also be impaired. At the same time, how Ft breaches the phagosome membrane is unclear, and we present evidence to support a model in which clathrin adapters and Rab family GTPases play important roles in phagosome dissolution and bacterial escape to the cytosol. From the bacterial perspective, few Francisella virulence factors have been identified and their mechanisms of action remain obscure. We recently developed a Tn5 transposon mutagenesis system for Francisella that we have used to identify mutants in FTL0347 and FTL1542 which encode a hypothetical membrane permease and acyl-CoA synthetase, respectively. Expression of known virulence factors is markedly reduced in both these mutants and preliminary characterization suggests that these genes are required for phagosome escape and inhibition of the phagocyte respiratory burst, respectively. Additionally, we constructed a TraSH (Transposon Site (Hybridization) derivative of our transposon that will allow us to identify novel genes required for Ft entry, growth and survival in human macrophages, Accordingly, our Specific Aims are: 1) to elucidate the mechanisms and functional consequences of Ft-mediated inhibition of PKCa signaling in macrophages; and 2) to identify and characterize novel F. tularensis genes required for evasion of innate macrophage defenses using transposon mutagenesis, including TRASH.