Farnesylated effectors of Legionella

描述(申请人提供)细菌病原体利用III-VI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。虽然许多注射的细菌效应器被靶向宿主细胞膜以获得生物学功能，但注射的细菌效应器的膜靶向机制还不是很清楚。我们最近描述了一种新的机制，通过宿主介导的法尼化将注射的细菌效应器锚定到宿主膜上，法尼化将蛋白质共价连接到15个碳的“法尼基”脂质部分。蛋白质法尼化是蛋白质C端五肽CAAX基序中保守的半胱氨酸残基的翻译后保守脂肪化。亲水性蛋白质的法尼斯化，如RAS，使脂化的蛋白质能够锚定在膜的脂质双层中，从而发挥生物学功能。 在阿米巴和人类细胞内，军团病细菌嗜肺军团菌(Lp)驻留并在内质网改造的含军团菌空泡(LCV)内增殖，该空泡避免溶酶体融合。阿米巴和人类细胞内LCV的重塑依赖于Dot/ICM IV型分泌系统，该系统向宿主细胞注入约300个效应器。我们已经证明，Ankyrin B(Ankyrin B，Ankyrin B)Dot/ICM注射的LP效应分子通过宿主介导的CAAX基序的法尼化作用被锚定在LCV膜上，这是LP细胞内增殖所必需的。 我们的初步数据表明，宿主法尼化酶被招募到LCV膜的胞液侧，这在空泡内病原体中是新的。除AnkB外，LCV膜的胞液侧高度富含其他法尼化蛋白。在Lp基因组的电子基因组分析中，已经鉴定出6个Lp基因编码含有C端CAAX基序的新蛋白。这6个编码的新蛋白通过Dot/ICM系统转运到宿主细胞中，被指定为军团菌的前烯基化效应因子(Pel)。当PEL在人细胞中异位表达时，证据表明效应器与细胞膜有关，但这种膜定位受到以下因素的干扰：1)抑制宿主的法尼化；或2)PELS的CAAX基序中保守的半胱氨酸残基(PEL C-A)的替代。重要的是，在巨噬细胞的细胞内增殖中，一个pelB零突变体和一个pelB 502C-A替代突变体表现出相似的严重缺陷。因此，我们的假设是：注射的新型PEL效应器被宿主细胞的法尼化机制法尼化，该机制将它们靶向特定的细胞膜，以展示它们在LP细胞内增殖所需的功能。为了检验这一假设，我们的 具体目的是确定：1)宿主介导的PEL效应器的法尼化和膜锚定；2)PEL效应器及其法尼化在LP细胞内感染中的作用。 我们研究的意义：1)LP是肺炎的主要原因；2)拟议的研究将通过破译新的易位效应因子在细胞内感染中的作用来增强我们对LP-宿主相互作用的了解；3)由于我们的电子基因组分析显示 随着C端CAAX基序在其他具有III-VI型易位系统的病原体的效应器中的流行，我们的研究将促进对其他病原体的新范式的研究；以及4)我们的发现将有助于我们理解法尼化在各种人类疾病中的作用，如RAS介导的恶性肿瘤。 建议研究的创新来自：1)假设；2)通过含有病原体的液泡劫持宿主的法尼化机制，高度富含法尼化的蛋白质；3)6个Pels Dot/ICM易位的效应器的新颖，表明在细胞调节中的新功能；4)我们的多学科方法破译宿主介导的PEL效应器的脂化作用及其在LP细胞内感染中的生物学功能；以及5)PelB及其C端CAAX基序在人巨噬细胞内感染中的主要作用。

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.