Molecular and Cellular Pathogenesis of Legionella

 描述(申请人提供)：在水环境中，嗜肺军团菌(LP)在阿米巴内繁殖，但在传播给人类后会引起肺炎，发病率和死亡率都很高。在阿米巴和人巨噬细胞中，LP在含有军团菌的空泡(LCV)内增殖，该空泡避免了溶酶体的融合，并被内质网(ER)重塑。LCV的生物发生由Dot/ICM IV型分泌系统控制，该系统向宿主细胞注入~300蛋白效应器，调节无数细胞过程，使LP能够在细胞内增殖。这种大电池的LP效应器，如AnkB和AnkH，很少是细胞内感染所需的。为了在宿主细胞中实现生物学功能，许多注射的细菌效应器需要翻译后 各种主机的改装。天冬酰胺(ASN)羟化修饰真核蛋白质的翻译后修饰会影响其蛋白质-蛋白质相互作用，在炎症性病灶的低氧条件下已被广泛研究。目前尚不清楚是否有任何注射的细菌效应物被宿主天冬酰胺羟基酶FIH翻译后修饰。在许多已被FIH修饰的人Ankyrin重复序列蛋白(Ank)中，已发现ASN羟化基序。我们的生物信息学初步数据已经在LP的AnkB和AnkH效应器的ankyrin重复序列中确定了ASN羟化基序，并且我们表明这两个效应器都被宿主FIH天冬酰胺羟基酶修饰。我们发现宿主FIH被迅速招募到LCV膜上，其中AnkH和AnkB是唯一定位的。我们的电子基因组分析已经在LP的其他八个易位效应器中确定了ASN羟化基序。重要的是，RNAi介导的敲除或化学抑制FIH可消除LP的细胞内增殖，并促进LCV与溶酶体的融合。因此，我们的假设是：宿主介导的ASN羟化是FIH介导的LP效应物在LCV的生物发生和LP的细胞内增殖中所必需的。为了验证我们的假设，我们的具体目的是研究以下几个方面：i)AnkH的ASN羟化及其在细胞内感染中的作用；ii)AnkB的ASN羟化及其在细胞内感染中的作用；以及iii)Oter LP效应分子的ASN羟化及其在LCV的生物发生中的作用。当我们的研究完成后，我们将知道AnkH的生物学功能和宿主靶标，并将确定AnkB的泛素化底物。宿主FIH介导的ASN羟化在AnkH和AnkB生物学功能中的作用将被揭示。这一点意义重大，因为这两个效应器都是液泡内增殖所需的极少数300个LP效应器中的两个。我们将报告我们的新发现，即通过含有病原体的液泡获得FIH复合体，以及它在修饰几种LP效应器中的作用。我们将揭示FIH介导的几个LP效应器的ASN羟化在LCV逃避溶酶体中的作用。我们对LP与初级hMDM的相互作用以及FIH对宿主翻译后修饰的特异性操纵的研究具有创新性，不仅对LP，而且对其他病原体都具有广泛的影响和意义。这一点从我们发现的耶尔森氏菌YopM效应子的ASN羟化，以及志贺氏菌的IpaH4.5效应子和分枝杆菌的EsxF效应子的潜在ASN羟基化中可见一斑。

 DESCRIPTION (provided by applicant): In the aquatic environment, Legionella pneumophila (Lp) proliferates within amoeba but upon transmission to humans it causes pneumonia with high morbidity and mortality. Within amoeba and human macrophages, Lp proliferates within the Legionella-containing vacuole (LCV), which evades lysosomal fusion and is remodeled by the endoplasmic reticulum (ER). Biogenesis of the LCV is controlled by the Dot/Icm type IV secretion system, which injects into the host cell ~300 protein effectors that modulate a myriad of cellular processes to enable intracellular proliferation of Lp. Very few of this large battery o Lp effectors, such as AnkB and AnkH, are required for the intracellular infection. To attain biological function in the host cell, many injected bacterial effectors require post- translational modification by various host machineries. Post-translational modification of eukaryotic proteins by Asparagine (Asn) hydroxylation impacts their protein-protein interaction, and has been widely studied during hypoxic conditions in inflammatory foci. It is not known whether any injected bacterial effector is post-translationally modified by the host asparagine hydroxylase, FIH. The Asn hydroxylation motif has been identified within many human Ankyrin repeats-containing proteins (Ank) that have been shown to be modified by FIH. Our bioinformatics preliminary data have identified an Asn hydroxylation motif within the ankyrin repeats of the AnkB and AnkH effectors of Lp, and we show that both effectors are modified by the host FIH Asparagine hydroxylase. We show that the host FIH is rapidly recruited to the LCV membrane, where AnkH and AnkB are exclusively localized. Our in silico genomic analyses have identified the Asn hydroxylation motif in eight other translocated effectors of Lp. Importantly, RNAi-mediated knockdown or chemical inhibition of FIH abolishes intracellular proliferation of Lp and promotes fusion of the LCV to the lysosomes. Therefore, our hypothesis is: host-mediated Asn hydroxylation of Lp effectors by FIH is required for their functions in biogenesis of the LCV and intracellular proliferation of Lp. To test our hypothesis, our specific aims are to characterize th following: I) Asn hydroxylation of AnkH and its role in the intracellular infection; II) Asn hydroxylation of AnkB and its role in the intracellular infection; and III) Asn hydroxylation of oter Lp effectors and its role in biogenesis of the LCV. Upon completion of our proposed studies, the biological function and host targets of AnkH will be known, and the ubiquitination substrates of AnkB will be identified. The role of host FIH-mediated Asn hydroxylation in the biological function of AnkH and AnkB will be uncovered. This is significant, since both effectors are two of very few of the battery of ~300 Lp effectors to be required for intra-vacuolar proliferation. We wil report on our novel discovery of the acquisition of the FIH complex by a pathogen-containing vacuole and its role in modification of several Lp effectors. We will uncover the role of FIH-mediated Asn hydroxylation of several Lp effectors in lysosomal evasion by the LCV. Our proposed studies on Lp-primary hMDMs interaction and specific manipulation of the host post- translational modification by FIH are innovative with broad high impact and significance not only for Lp but for other pathogens. This is evident from our discovery of Asn hydroxylation of the YopM effector of Yersinia, and potential Asn hydroxylation of the IpaH4.5 effector of Shigella, and the EsxF effector of Mycobacterium.