Mechansisms of innate immune evasion by Francisella tularensis

土拉热弗朗西丝氏菌是巨噬细胞的兼性细胞内病原体，是巨噬细胞感染的病原体。 兔热病尽管很明显吸入10华氏度。土拉热亚种土拉热可能是致命的， 巨噬细胞是这种生物体在体内的主要储存库，Ft如何逃避消除只是 开始被定义。在摄取的几个小时内，Ft突破吞噬体膜， 在细胞质中复制。我们最近的数据首次表明，完全毒力Ft菌株SchuS 4 在途径的较早阶段破坏吞噬体成熟， 从排除晚期内体标记物Rab 7（甘露糖-6-磷酸）的新隔室中逃逸 受体，溶血双磷脂酸和组织蛋白酶S，尽管局部积累的灯-1。产生该 隔室需要活的MglA阳性Ft，并且通过这种病原体抑制 蛋白激酶C-a（PKC α）的活性在整个感染细胞中的变化。因此，我们的数据确定PKCa 作为已知被Ft靶向以逃避细胞内杀伤的第一宿主因子。PKCa也需要 其他方面的先天防御，我们的初步数据支持的假设，MHC II类抗原 呈递和氧化防御机制也可能受损。与此同时，Ft如何突破 吞噬体膜尚不清楚，我们提出的证据支持一个模型，其中网格蛋白 衔接子和Rab家族GTPases在吞噬体溶解和细菌逃逸到噬菌体中起重要作用。 胞质液从细菌的角度来看，很少有弗朗西斯氏菌属的毒力因子被鉴定出来，并且它们的 作用机制仍然不清楚。我们最近开发了一个Tn 5转座子诱变系统， 我们已经用它来鉴定FTL 0347和FTL 1542中的突变体，这些突变体编码一个假设的 膜透性酶和酰基辅酶A合成酶。已知毒力因子的表达是 在这两种突变体中显著降低，初步表征表明这些基因是 分别是吞噬体逃逸和抑制吞噬细胞呼吸爆发所需的。此外，本发明还 我们构建了转座子的TraSH（转座子位点（杂交）衍生物， 鉴定人巨噬细胞中Ft进入、生长和存活所需的新基因。 具体的目的是：1）阐明Ft介导的抑制的机制和功能后果， 巨噬细胞中的PKCa信号传导;和2）鉴定和表征新的F.土拉菌所需的基因 使用转座子诱变逃避先天巨噬细胞防御，包括TRASH。

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.