Farnesylated effectors of Legionella

军团菌法尼基化效应子

基本信息

批准号：
8665875
负责人：
Yousef A Abu Kwaik
金额：
$ 18.75万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-24 至 2015-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant) Bacterial pathogens utilize type III-VII translocation systems to inject a large cadre of protein effectors into the host cell leading to modulation of cellular processes and manifestation of disease. Although many injected bacterial effectors are targeted into host cellular membranes to attain biological function, the mechanisms of membrane targeting of injected bacterial effectors are not well understood. We have recently described a novel mechanism for anchoring an injected bacterial effector into host membranes through host-mediated farnesylation, which covalently links a protein to a 15-carbon "farnesyl" lipid moiety. Farnesylation of proteins is a conserved eukaryotic post-translational lipidation of conserved cysteine residue within the C-terminal teterapeptide "CaaX" motif of a protein. Farnesylation of hydrophilic proteins, such as Ras, enable the lipidated protein to be anchored into the lipid bi-layer of membranes to exhibit biological function. Within amoeba and human cells, the Legionnaires' disease bacterium, Legionella pneumophila (Lp), resides and proliferate within an ER-remodeled Legionella-containing vacuole (LCV), which evades lysosomal fusion. Remodeling the LCV within amoeba and human cells is dependent on the Dot/Icm type IV secretion system, which injects ~300 effectors into the host cell. We have shown that the Ankyrin B (AnkB) Dot/Icm-injected effector of Lp is anchored to the LCV membrane through host-mediated farnesylation of its "CaaX" motif, which is indispensable for Lp intracellular proliferation. Our preliminary data show that the host farnesylation enzymes are recruited to the cytosolic side of the LCV membrane, which is novel among intra-vacuolar pathogens. The cytosolic side of the LCV membrane is highly enriched with other farnesylated proteins, in addition to AnkB. In silico genomic analyses of Lp genomes have identified 6 Lp genes encoding novel proteins that harbor a C-terminal CaaX motif. The 6 encoded novel proteins are translocated into the host cell by the Dot/Icm system, and have been designated as prenylated effectors of Legionella (Pel). When the Pels are ectopically expressed in human cells, evidence suggests that the effectors associate with cellular membranes, but this membrane localization is perturbed upon: 1) inhibition of host farnesylation; or 2) substitution of the conserved cysteine residue (pel C-A) within the CaaX motif of the Pels. Importantly, a pelB null mutant and a pelB 502C-A substitution mutant exhibit similar severe defects in intracellular proliferation in macrophages. Therefore, our hypothesis is: The injected novel Pel effectors are farnesylated by the host cell farnesylation machinery that targets them to specific cellular membranes to exhibit their functions required for intracellular proliferation of Lp. To test the hypothesis, our specific aims are to determine: I) Host mediated farnesylation and membrane-anchoring of the Pel effectors; and 2) Role of the pel effectors and their farnesylation in the intracellular infection by Lp. The Significance of our studies: 1) Lp is a major cause of pneumonia; 2) The proposed studies will enhance our knowledge of Lp-host interaction through deciphering the role of novel translocated effectors in the intracellular infection; 3) Since our in silico genomic analyses show prevalence of the C-terminal CaaX motif in effectors of other pathogens with type III-VII translocated systems, our studies will stimulate investigations into a new paradigm in other pathogens; and 4) Our findings will contribute to our understanding the role of farnesylation in various human diseases, such as Ras-mediated malignancies. Innovation of the proposed studies come from: 1) the hypothesis; 2) hijacking the host farnesylation machinery by the pathogen-containing vacuole, which is highly enriched with farnesylated proteins; 3) Novelty of the 6 Pels Dot/Icm-translocated effectors, indicating novel functions in cellular modulations; 4) Our multidisciplinary approach of deciphering role of host-mediated lipidation of the Pel effectors combined with their biological functions in the intracellular infection by Lp; and 5) The major role for PelB and its C-terminal CaaX motif in the intracellular infection of human macrophages.

描述（由申请人提供）细菌病原体利用 III-VII 型易位系统将大量蛋白质效应子注入宿主细胞，从而导致细胞过程的调节和疾病的表现。尽管许多注射细菌效应子被靶向宿主细胞膜以获得生物学功能，但注射细菌效应子的膜靶向机制尚不清楚。我们最近描述了一种通过宿主介导的法尼基化将注射的细菌效应物锚定到宿主膜上的新机制，该法尼基化将蛋白质与 15 个碳的“法尼基”脂质部分共价连接。蛋白质的法尼基化是蛋白质 C 端四肽“CaaX”基序内保守半胱氨酸残基的保守真核翻译后脂化。亲水蛋白（例如 Ras）的法呢基化使脂化蛋白能够锚定到膜的脂质双层中，从而表现出生物学功能。在阿米巴原虫和人类细胞中，军团病细菌嗜肺军团菌 (Lp) 驻留在内质网改造的含有军团菌的液泡 (LCV) 中并增殖，该液泡可逃避溶酶体融合。变形虫和人类细胞内 LCV 的重塑依赖于 Dot/Icm IV 型分泌系统，该系统将约 300 个效应子注入宿主细胞。我们已经证明，注射 Lp 的锚蛋白 B (AnkB) Dot/Icm 效应子通过宿主介导的“CaaX”基序法尼基化锚定在 LCV 膜上，这对于 Lp 细胞内增殖是必不可少的。我们的初步数据表明，宿主法尼基化酶被招募到 LCV 膜的胞质侧，这在液泡内病原体中是新颖的。除了 AnkB 之外，LCV 膜的胞质侧还富含其他法尼基化蛋白。 Lp 基因组的计算机基因组分析已鉴定出 6 个 Lp 基因，它们编码带有 C 末端 CaaX 基序的新型蛋白质。这 6 个编码的新蛋白通过 Dot/Icm 系统易位到宿主细胞中，并被指定为军团菌 (Pel) 的异戊二烯化效应子。当 Pels 在人类细胞中异位表达时，有证据表明效应子与细胞膜相关，但这种膜定位受到以下因素的干扰：1) 抑制宿主法尼基化；或 2) Pels CaaX 基序内保守半胱氨酸残基 (pel C-A) 的取代。重要的是，pelB 无效突变体和 pelB 502C-A 替代突变体在巨噬细胞的细胞内增殖中表现出类似的严重缺陷。因此，我们的假设是：注射的新型 Pel 效应子由宿主细胞法尼基化机制进行法尼基化，该机制将它们靶向特定的细胞膜，以展示 Lp 细胞内增殖所需的功能。为了检验这个假设，我们的具体目标是确定： I) 宿主介导的法尼基化和 Pel 效应器的膜锚定； 2) pel 效应子及其法尼基化在 Lp 细胞内感染中的作用。我们研究的意义： 1）Lp是肺炎的一个主要原因； 2）拟议的研究将通过破译新型易位效应器在细胞内感染中的作用来增强我们对LP-宿主相互作用的认识； 3) 因为我们的计算机基因组分析显示由于 C 端 CaaX 基序在具有 III-VII 型易位系统的其他病原体的效应器中普遍存在，我们的研究将激发对其他病原体新范式的研究； 4) 我们的研究结果将有助于我们了解法尼基化在各种人类疾病中的作用，例如 Ras 介导的恶性肿瘤。本研究的创新点来自于：1）假设； 2）通过含有病原体的液泡劫持宿主法尼基化机制，该液泡富含法尼基化蛋白质； 3) 6 Pels Dot/Icm 易位效应器的新颖性，表明细胞调制中的新功能； 4) 我们采用多学科方法破译宿主介导的 Pel 效应子脂化作用及其在 Lp 细胞内感染中的生物学功能； 5) PelB 及其 C 端 CaaX 基序在人巨噬细胞的细胞内感染中的主要作用。