Cellular Biology Of Host/parasite Interactions

宿主/寄生虫相互作用的细胞生物学

• 批准号: 9354716
• 负责人: David (Ted) Hackstadt
• 金额: $ 115.43万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354716
• 关键词: Agriculture; Amino Acids; Area; Bacteria; Bacterial Proteins; Basic Science; Binding; Biochemical; Biological Assay; Blindness; Cell membrane; Cells; Cellular biology; Characteristics; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chlamydophila pneumoniae; Chlamydophila psittaci; Cholesterol; Chronic; Chronic Disease; Coiled-Coil Domain; Communities; Cytoplasm; Cytosol; Development; Disease; Domestic Fowls; Economics; Endocytosis; Endosomes; Epidemiology; Epithelium; Eukaryotic Cell; Event; Face; Genetic; Genetic Transcription; Golgi Apparatus; Growth; Human; Immunologics; In Vitro; Industry; Infection; Inflammation; Inguinal lymph node group; Intervention; Length; Link; Livestock; Lymphogranuloma Venereum; Malignant neoplasm of cervix uteri; Mediating; Membrane; Membrane Fusion; Membrane Microdomains; Membrane Proteins; Methods; Microinjections; Microtubule-Organizing Center; Microtubules; Modification; Molecular and Cellular Biology; Mutation; N-ethylmaleimide-sensitive protein; Parasites; Pasteurella pseudotuberculosis; Pathway interactions; Pelvic Inflammatory Disease; Phenotype; Plasmids; Pneumonia; Process; Protein Biosynthesis; Proteins; Proteomics; Recombinant Proteins; Recycling; Regulation; SNAP receptor; Sexually Transmitted Diseases; Shuttle Vectors; Signal Transduction; Site; Specificity; Sphingomyelins; Staging; Study Section; System; Systemic infection; Trachoma; Transferrin; Translations; Type III Secretion System Pathway; United States; Vacuole; Vesicle; Weight; Yersinia; genetic manipulation; human disease; improved; interest; member; mutant; novel; obligate intracellular parasite; pathogen; pathogenic bacteria; promoter; protein protein interaction; receptor; response; tool; trafficking

Members of the genus Chlamydia are bacterial obligate intracellular parasites of eukaryotic cells. They constitute an important group of pathogenic bacteria that are responsible for multiple medically significant conditions. The species Chlamydia trachomatis is comprised of at least fifteen serologically defined groups or "serovars" that are associated with human diseases. Trachoma, the world's leading cause of infectious blindness, is caused by serovars A, B, Ba, and C. Chlamydial sexually transmitted disease (STD) is the most common reportable disease in the United States. Serovars D though K are most commonly associated with STDs. The more serious sequelae of these diseases, blindness from trachoma and pelvic inflammatory disease from chlamydial STD, are immunopathological responses to chronic or repeated infections. While trachoma and sexually transmitted infections are primarily localized to the mucosal epithelium, a more systemic infection, lymphogranuloma venereum (LGV), caused by C. trachomatis serovars L1, L2, and L3, is also a sexually transmitted infection that causes inflammation of the inguinal lymph nodes. C. pneumoniae, is a common cause of community acquired pneumonia and is currently of interest due to possible associations with a variety of chronic diseases. C. psittaci is a zoonotic disease that infects many different types of poultry and livestock thus is of economic importance to agricultural industries and is occasionally transmitted to humans. Chlamydiae undergo their entire intracellular developmental cycle within a parasitophorous vacuole, termed an inclusion, that is unique among intracellular parasites. Chlamydiae are endocytosed into a tightly membrane-bound vesicle which grows throughout the developmental cycle to accommodate an increasing number of intracellular bacteria. The chlamydial inclusion, unlike vacuoles containing other intracellular pathogens, is not interactive with endocytic vesicular trafficking pathways but is instead fusogenic with an incompletely understood exocytic pathway which delivers sphingomyelin and cholesterol from the Golgi apparatus to the plasma membrane. Although all species of Chlamydia intersect this pathway, no other intracellular parasites have yet been found to similarly interact with this host vesicular trafficking pathway. Sequestration of chlamydiae within a vesicle that intersects an exocytic pathway is hypothesized to provide a unique, protected intracellular niche in which the chlamydiae replicate. Entry into this pathway is an active process on the part of the chlamydiae as both de novo transcription and translation are required. Virtually all of these interactions are specific and localized to the inclusion. This specificity strongly suggests modification of the exposed inclusion membrane. Examples of cis-acting modifications to the nascent inclusion membrane include: evasion of lysosomal fusion, interactions with microtubules to deliver the nascent inclusion to the peri-Golgi region and microtubule organizing center, initiation of fusion with exocytic vesicular traffic from the Golgi apparatus, and recruitment of, but not fusion with, recycling endosomes containing transferrin and its receptor. Many of these interactions are temporally associated with the exposure of inclusion membrane proteins to the host cell cytoplasm by a chlamydial type III secretion system. C. trachomatis expresses up to fifty predicted inclusion membrane proteins characterized by a long, bilobed hydrophobic domain of approximately 40 amino acids in length. Incs are exposed on the cytosolic face of the inclusion membrane and thus are likely candidates for factors controlling interactions with the host cell. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. Here we describe a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III dependent manner from Y. pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first thirty amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. We employed recently developed genetic tools to verify localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of 50 Incs identified 10 which were previously unverified Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by co-localization with p-Src. Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they are not Incs. Collectively our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will further our understanding of how this pathogen maintains its unique intracellular niche and mediates interactions with the host. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of non-fusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for chlamydia with a method for complementation of site-specific mutants.

衣原体属的成员是真核细胞的细菌专性细胞内寄生虫。 它们构成了一组重要的病原菌，可导致多种具有医学意义的病症。沙眼衣原体物种由至少十五个与人类疾病相关的血清学定义的群体或“血清型”组成。 沙眼是世界上导致传染性失明的主要原因，由 A、B、Ba 和 C 血清型引起。衣原体性传播疾病 (STD) 是美国最常见的报告疾病。 血清型 D 至 K 最常与 STD 相关。 这些疾病的更严重的后遗症，沙眼引起的失明和衣原体性传播疾病引起的盆腔炎，是对慢性或反复感染的免疫病理反应。 虽然沙眼和性传播感染主要局限于粘膜上皮，但由沙眼衣原体 L1、L2 和 L3 血清型引起的性病淋巴肉芽肿 (LGV) 是一种更全身性的感染，也是一种性传播感染，会导致腹股沟炎症淋巴结。 肺炎衣原体是社区获得性肺炎的常见病因，由于可能与多种慢性疾病有关，目前引起人们的关注。 鹦鹉热衣原体是一种人畜共患疾病，可感染许多不同类型的家禽和牲畜，因此对农业具有重要的经济意义，并且偶尔会传播给人类。 衣原体在寄生液泡（称为包涵体）内经历整个细胞内发育周期，这在细胞内寄生虫中是独一无二的。 衣原体被内吞到紧密膜结合的囊泡中，该囊泡在整个发育周期中生长以容纳越来越多的细胞内细菌。 与含有其他细胞内病原体的液泡不同，衣原体包涵体不与内吞性囊泡运输途径相互作用，而是与不完全了解的胞吐途径融合，该途径将鞘磷脂和胆固醇从高尔基体传递到质膜。 尽管所有衣原体物种都与该途径相交，但尚未发现其他细胞内寄生虫与该宿主囊泡运输途径有类似的相互作用。 假设将衣原体隔离在与胞吐途径相交的囊泡内，以提供衣原体复制的独特的、受保护的细胞内生态位。 对于衣原体而言，进入该途径是一个活跃的过程，因为需要从头转录和翻译。 事实上，所有这些相互作用都是特定的并且局限于包容性。 这种特异性强烈表明对暴露的包涵体膜进行了修饰。 对新生包涵体膜进行顺式作用修饰的例子包括：逃避溶酶体融合、与微管相互作用以将新生包涵体递送至高尔基体周围区域和微管组织中心、与来自高尔基体的胞吐囊泡运输启动融合，以及募集含有转铁蛋白及其受体的再循环内体，但不与之融合。 许多这些相互作用在时间上与衣原体 III 型分泌系统将包涵膜蛋白暴露于宿主细胞细胞质相关。 沙眼衣原体表达多达 50 种预测的包涵膜蛋白，其特征是长度约为 40 个氨基酸的长双叶疏水结构域。 Incs暴露在包涵膜的胞质面上，因此可能是控制与宿主细胞相互作用的因子的候选者。 衣原体与宿主细胞的许多相互作用取决于细菌蛋白质的合成以及这些蛋白质可能暴露于细胞质。 由于缺乏衣原体遗传工具，以前检查分泌蛋白的研究需要使用异源细菌系统。 衣原体基因操作的最新进展现在允许用质粒转化细菌。 在这里，我们描述了一种穿梭载体系统 pBOMB4，它允许在组成型或条件启动子控制下表达重组蛋白。 我们发现，包涵膜蛋白 IncD 以 III 型依赖性方式从假结核杆菌中分泌，并且也在感染细胞中从沙眼衣原体中分泌，在感染细胞中它适当地定位于包涵膜。 含有分泌信号的前三十个氨基酸被截短的 IncD 不再分泌并被细菌保留。 分泌蛋白的胞浆暴露可以通过使用 CyaA、GSK 或显微注射测定来确认。 NrdB 是一种预计保留在细菌内的蛋白质，它确实定位于衣原体。 此外，我们还发现，衣原体效应蛋白 CPAF 通过 Sec 依赖性途径分泌到宿主细胞胞浆中，当从该系统表达时，也会进入胞浆。 我们采用最近开发的遗传工具来验证先前未定位到包涵膜的预测 Incs 的定位。 50 个 Incs 的表达确定了 10 个之前未经验证的 Incs。一个新的 Inc 和 3 个先前描述的 Inc 定位于包涵膜微域，与 p-Src 共定位证明了这一点。 一些预测的 Incs 并未定位于包涵体膜，而是仍然与细菌相关。使用耶尔森氏菌作为替代宿主，我们证明其中许多不是通过 III 型分泌来分泌的，进一步表明它们不是 Incs。总的来说，我们的结果强调了遗传工具在证明衣原体分泌方面的实用性。旨在阐明效应子功能的进一步机制研究将进一步了解这种病原体如何维持其独特的细胞内生态位并介导与宿主的相互作用。 IncA 是一种包涵膜蛋白，编码两个与真核 SNARE（可溶性 N-乙基马来酰亚胺敏感因子附着受体）基序同源的卷曲螺旋结构域。最近的生化证据表明，由 SNARE 样结构域 1 (SLD-1) 和部分 SNARE 样结构域 2 (SLD-2) 组成的功能核心是多重感染中沙眼衣原体包涵体特征性同型融合所必需的。细胞。 为了验证 IncA 在衣原体同型融合中的重要性，我们生成了 incA::bla 突变体。 incA 的插入失活导致非融合内含物的形成，这种表型通过与全长 IncA 的互补而被完全挽救。 同型包涵融合的拯救依赖于由SLD-1和部分SLD-2组成的功能核心的存在。总的来说，这些结果证实了鉴定 IncA 功能域的体外膜融合测定，并通过位点特异性突变体的互补方法扩展了可用于衣原体的遗传工具。