Farnesylated effectors of Legionella

军团菌法尼基化效应子

• 批准号: 8589705
• 负责人: Yousef A Abu Kwaik
• 金额: $ 21.15万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/8589705
• 关键词: Abbreviations; Amoeba genus; Ankyrins; Bacteria; Biological Process; C-terminal; Carbon; Cell physiology; Cells; Cellular Membrane; Co-Immunoprecipitations; Computer Simulation; Cysteine; Data; Defect; Disease; Enzymes; Exhibits; Genes; Genome; Genomics; Human; Infection; Investigation; Knowledge; Legionella; Legionella pneumophila; Legionnaires' Disease; Link; Lipids; Malignant Neoplasms; Mediating; Membrane; Pneumonia; Prevalence; Proliferating; Protein Farnesylation; Proteins; Recruitment Activity; Role; Side; System; Testing; Transferase; Type IV Secretion System Pathway; Vacuole; farnesylation; human disease; innovation; interdisciplinary approach; macrophage; mutant; novel; pathogen; protein farnesyltransferase; public health relevance

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物注入宿主细胞，导致细胞过程的调节和疾病的表现。尽管许多注射的细菌效应子靶向进入宿主细胞膜以获得生物学功能，但是注射的细菌效应子的膜靶向机制还没有很好地理解。我们最近描述了一种新的机制，通过宿主介导的法尼基化，共价连接一个蛋白质的15-碳“法尼基”脂质部分锚定到宿主细胞膜的注射细菌效应。蛋白质的法尼基化是蛋白质C-末端四肽“CaaX”基序内保守的半胱氨酸残基的保守的真核翻译后脂化。亲水性蛋白质如Ras的法尼基化使得脂化蛋白质能够锚定到膜的脂质双层中以表现出生物学功能。 在变形虫和人类细胞内，军团病细菌，嗜肺军团菌（Lp），驻留在ER-重塑的含军团菌的空泡（LCV）内并在其中增殖，其逃避溶酶体融合。阿米巴和人类细胞内LCV的重塑依赖于Dot/Icm IV型分泌系统，该系统将约300个效应物注入宿主细胞。我们已经证明，锚蛋白B（Ank B）Dot/Icm注射的Lp效应器通过宿主介导的CaaX基序法尼基化锚定在LCV膜上，这是Lp细胞内增殖所必需的。 我们的初步数据显示，宿主法尼基化酶被募集到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膜的胞质侧高度富集有其他法尼基化蛋白。Lp基因组的计算机基因组分析已经鉴定了6个编码具有C-末端CaaX基序的新型蛋白质的Lp基因。编码的6个新蛋白通过Dot/Icm系统被转运到宿主细胞中，并已被指定为军团菌（Pel）的异戊烯化效应子。当Pel在人细胞中异位表达时，证据表明效应子与细胞膜缔合，但这种膜定位在以下情况下受到干扰：1）抑制宿主法尼基化;或2）取代Pel的CaaX基序内的保守半胱氨酸残基（pel C-A）。重要的是，pelB无效突变体和pelB 502 C-A置换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效应物被宿主细胞法尼基化机制法尼基化，所述宿主细胞法尼基化机制将它们靶向特定的细胞膜以表现出Lp的细胞内增殖所需的功能。为了验证这一假设，我们 具体的目的是确定：I）宿主介导的法尼基化和Pel效应物的膜锚定;和2）Pel效应物及其法尼基化在Lp的细胞内感染中的作用。 本研究的意义：1）Lp是肺炎的主要原因; 2）拟议的研究将通过破译新的易位效应子在细胞内感染中的作用，增强我们对Lp-宿主相互作用的认识; 3）由于我们的计算机基因组分析显示， C-末端CaaX基序在具有III-VII型易位系统的其他病原体的效应子中的流行，我们的研究将刺激对其他病原体的新范例的调查;和4）我们的发现将有助于我们理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。 本研究的创新点在于：1）提出了一个新的假说; 2）通过富含法尼基化蛋白的含病原体的液泡劫持宿主的法尼基化机制; 3）发现了6个Pels Dot/Icm易位效应子的新奇性，表明它们在细胞调节中具有新的功能; 4）我们的多学科方法解释宿主介导的Pel效应物的脂化作用及其在Lp细胞内感染中的生物学功能; PelB及其C端CaaX基序在人巨噬细胞胞内感染中的主要作用。