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，从而调节细胞过程和疾病的表现。尽管许多注射的细菌效应子被靶向宿主细胞膜以获得生物学功能，但尚不清楚注射细菌效应子的膜靶向的机理。我们最近描述了一种新型机制，该机制通过宿主介导的Farnesylation将注入的细菌效应子锚定在宿主膜中，该机制将蛋白质连接到15-碳“ Farnesyl”脂质部分。蛋白质的Farnesylation是一种保守的真核后脂化脂质，对蛋白质的C末端双肽“ CAAX”基序中保守的半胱氨酸残基的翻译后脂质化。诸如RAS之类的亲水性蛋白质的法尼化使脂质的蛋白质能够固定在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中，军团疾病细菌，肺炎军团菌（LP）居住在含ER的含军团菌的液泡（LCV）中，逃避了溶酶体融合。在变形虫中重塑LCV和人类细胞中的LCV取决于DOT/ICM IV型分泌系统，该系统将约300个效应子注入宿主细胞中。我们已经表明，LP的Ankyrin B（ANKB）点/ICM注射效应子通过其“ CAAX”基序的宿主介导的Farnesylation固定在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，将宿主的Farnesylation酶募集到LCV膜的胞质侧，该膜是空中性病原体中新颖的。除了ANKB外，LCV膜的胞质侧具有高度富含其他法尼蛋白的蛋白质。在对LP基因组的硅基因组分析中，已经鉴定出具有含有C末端CAAX基序的新型蛋白质的6个LP基因。通过点/ICM系统将6种编码的新型蛋白转移到宿主细胞中，并已被指定为Legionella（PEL）的先前效应子。当PEL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，但是该膜定位在以下方面受到干扰：1）抑制宿主Farnesylation；或2）在PEL的CAAX基序内取代保守的半胱氨酸残基（PEL C-A）。重要的是，pelb无效突变体和Pelb 502C-A取代突变体在巨噬细胞的细胞内增殖中表现出相似的严重缺陷。因此，我们的假设是：注入的新型PEL效应子是由宿主细胞Farnesylation机械靶向的，它将其靶向特定的细胞膜，以表现出其LP细胞内增殖所需的功能。为了检验假设，我们具体目的是确定：i）宿主介导的pEL效应子的法尼化和膜锚定； 2）PEL效应子及其在LP细胞内感染中的作用。我们研究的意义：1）LP是肺炎的主要原因； 2）拟议的研究将通过破译新型易位效应子在细胞内感染中的作用来增强我们对LP-host相互作用的了解； 3）自从我们的硅基因组分析显示以来我们的研究将刺激对其他病原体中新的范式的研究，在其他病原体的效应子中，C末端CAAX基序的患病率是III-VII型易位系统的效应子。 4）我们的发现将有助于我们理解Farnesyration在各种人类疾病中的作用，例如RAS介导的恶性肿瘤。拟议研究的创新来自：1）假设； 2）通过含病原体的液泡劫持宿主的法尼化机制，该液泡高度富含法尼蛋白； 3）6骨点/ICM转移效应子的新颖性，表明细胞调节中的新功能； 4）我们的多学科方法是宿主介导的PEL效应子的脂肪的解密作用，并结合了LP的细胞内感染中其生物学功能； 5）PELB及其C末端CAAX基序的主要作用在人类巨噬细胞的细胞内感染中。