Mechanisms of Innate Immune Evasion by Mycobacterium Tuberculosis

项目总结 结核分枝杆菌(Mtb)是结核病(TB)的病原体和主要致病因子。 全球范围内死于细菌感染的原因。Mtb的成功源于它的规避能力 巨噬细胞的降解。最近的研究表明，巨噬细胞通过 两条不同的溶酶体转运途径，涉及标记了Lc3的细胞器(2，3)。外食是一个过程 通过它，标记了LC3的双膜细胞器捕获和降解入侵的微生物。LC3-关联 吞噬作用(LAP)类似于异体吞噬，但不涉及双膜，需要NADPH 氧化物酶和活性氧(ROS)，这不是吞噬所必需的。这些溶酶体 降解途径由刺激病原体识别受体(PRRs)的微生物配体激活。 为什么激活了许多PRR的MTB未能引发与LC3相关的实质性事件 吞噬溶酶体贩运不被理解。我们广泛的初步数据有力地表明，CPSA和 结核分枝杆菌分泌的未知蛋白，特别是阻止LAP。我们假设CPSA干扰了 激活NADPH氧化酶，从而阻止ROS的产生和LAP介导的Mtb向 溶酶体。与我们的假设一致，我们发现缺乏cpsa的结核分枝杆菌菌株表现出戏剧性。 与LAP的LC3标记物增强共定位，并且它们在巨噬细胞和 老鼠。此外，NADPH氧化酶和LAP特殊需要的蛋白质是 巨噬细胞杀死cpsa突变体。CPSA包含LytR-CPSA-PSR(LCP)结构域，通常 在革兰氏阳性生物体中发现。在肺炎链球菌和枯草芽孢杆菌中，LCP结构域结合 磷酸化的多异戊二烯类脂。我们用晶体结构和S。 肺炎杆菌LCP蛋白，发现Mtb CPSA中所有的磷脂结合残基都是保守的。 此外，我们还发现CPSA可以与人T细胞白血病病毒I型结合蛋白1(TAX1BP1)结合， 和核点蛋白52 kDa(NDP52)。TAX1BP1和NDP52是参与链接的Paralog 将细菌运往自噬机器。因此，我们假设CPSA的抑制能力 NADPH氧化酶和LAP依赖于结合的脂磷酸盐和宿主蛋白TAX1BP1和NDP52。至 检验我们的假设，我们将(1)研究巨噬细胞杀死cPSA突变体的途径，(2) 描述CPSA蛋白的作用机制，以及(3)评估其先天的重要性 体内的免疫逃逸机制。结合起来，我们的研究将阐明一种新的免疫机制 最可怕的病原体之一的逃避。通过研究结核分枝杆菌利用的分子机制 破坏宿主细胞的功能，我们将进行基本的观察，这将有助于 改善结核分枝杆菌的治疗方法和疫苗。

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis (TB) and the leading cause of death worldwide from a bacterial infection. The success of Mtb stems from its ability to evade degradation by macrophages. Recent studies have revealed that macrophages clear microorganisms through two distinct lysosomal trafficking pathways that involve LC3-marked organelles (2, 3). Xenophagy is a process by which LC3-marked, double-membrane organelles capture and degrade invading microbes. LC3-associated phagocytosis (LAP) is similar to xenophagy, but does not involve a double membrane and requires NADPH oxidase and reactive oxygen species (ROS), which are not necessary for xenophagy. These lysosomal degradative pathways are activated by microbial ligands that stimulate pathogen recognition receptors (PRRs). The reason why Mtb, which activates numerous PRRs, fails to provoke substantive LC3-associated phagolysosomal trafficking is not understood. Our extensive preliminary data strongly suggest that CpsA, an uncharacterized protein secreted by Mtb, specifically blocks LAP. We hypothesize that CpsA interferes with the activation of NADPH oxidase, thereby blocking the generation of ROS and the LAP-mediated delivery of Mtb to the lysosome. Consistent with our hypothesis, we found that Mtb strains lacking cpsA exhibit dramatically enhanced colocalization with the LC3 marker of LAP and that they are highly attenuated in macrophages and mice. Moreover, NADPH oxidase and the proteins specifically required for LAP are necessary for macrophages to kill the cpsA mutant. CpsA contains a LytR-CpsA-Psr (LCP) domain, which is commonly found in Gram-positive organisms. In Streptococcus pneumoniae and Bacillus subtilis, the LCP domain binds phosphorylated polyisoprenoid lipids. We modeled the structure of Mtb CpsA using the crystal structures an S. pneumoniae LCP protein and found that all of the lipid phosphate-binding residues are conserved in Mtb CpsA. In addition, we found that CpsA can bind the human T-cell leukemia virus type I binding protein 1 (TAX1BP1), and nuclear dot protein 52 kDa (NDP52). TAX1BP1 and NDP52 are paralogs that are involved in linking bacterial cargo to the autophagy machinery. Thus, we hypothesize that the ability of CpsA to inhibit the NADPH oxidase and LAP depends upon binding lipid phosphate and host proteins TAX1BP1 and NDP52. To test our hypotheses, we will (1) study the pathway by which macrophages kill the cpsA mutant, (2) characterize the mechanism of action of the CpsA protein, and (3) evaluate the importance of this innate immune evasion mechanism in vivo. Combined, our studies will elucidate a novel mechanism of immune evasion by one of the most formidable pathogens. By studying the molecular mechanisms Mtb utilizes to sabotage host cellular functions, we will make fundamental observations that will aid in the development of better therapeutics and vaccines for Mtb.