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义务细胞内寄生虫。 它们构成了一组重要的致病细菌，这些细菌负责多种医学上的重要疾病。该物种沙眼衣原体由至少十五种与人类疾病相关的血清学定义组或“血清”组成。 三方瘤是传染失明的主要原因，是由血清A，B，Ba和C. g属性传播疾病（STD）引起的，是美国最常见的报告疾病。 血清虽然K虽然k最常见于性病。 这些疾病的更严重的后遗症，沙眼瘤的失明和衣原体性病的骨盆炎性疾病，是对慢性或重复感染的免疫病理反应。 虽然沙洲瘤和性传播感染主要位于粘膜上皮，但由C. c. c. trachomatis serovars L1，L1，L2和L3引起的更全身性感染，淋巴神经瘤（LGV）也是一种性传播感染，也是一种性传播感染，这是引起性传播的感染。 肺炎梭菌是社区获得性肺炎的常见原因，目前由于可能与多种慢性疾病有关。 C. psittaci是一种人畜共患病，它感染了许多不同类型的家禽和牲畜，因此对农业产业至关重要，偶尔会传播给人类。 衣原体在寄生虫液泡中经历了整个细胞内发育周期，称为包容性，在细胞内寄生虫中是独一无二的。 衣原体被内吞成紧密的膜结合囊泡，该囊泡在整个发育周期中生长，以适应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，衣原体的包含与内吞囊泡运输途径没有相互作用，而是fusogenic却具有未完全理解的外co型途径，从而传达了鞘磷脂和胆固醇从高脂蛋白剂到果仁糖从plasmammambrane中。 尽管所有种类的衣原体都与这一途径相交，但尚未发现其他细胞内寄生虫类似地与该宿主的水泡贩运途径相似。 假设在囊泡中的衣原体隔离囊泡，以提供独特的，受保护的细胞内生态位，其中衣原体复制。 进入该途径是衣原体的一个积极过程，因为需要从头转录和翻译。 几乎所有这些相互作用都是特定的，并且本地化为包容性。 该特异性强烈表明对暴露的纳入膜的修饰。 对新生纳入膜进行顺式作用修饰的实例包括：逃避溶酶体融合，与微管的相互作用，以向高尔基细胞区域和微管组织中心提供新生的包容性，与外粒囊泡融合的融合启动，但与Golgi的融合，但不是与Golgi的转移和招募，但不是，但不是Recrition flosing flosing flosing flosing fromperife flosing of froming soff from frop，and Recrition of Recrition of from from，它的受体。 这些相互作用中的许多相互作用在时间上与通过衣原体III型分泌系统暴露于纳入膜蛋白暴露于宿主细胞细胞质。 沙眼梭状芽孢杆菌最多表达五十个预测的夹杂膜蛋白，其特征在于长度约40个氨基酸的长，双弯曲的疏水结构域。 INC暴露在包含膜的胞质面上，因此可能是控制与宿主细胞相互作用的因素的候选者。 衣原体与宿主细胞的许多相互作用取决于细菌蛋白的合成以及这些蛋白质可能暴露于细胞质中。 由于缺乏衣原体的遗传工具，先前检查分泌蛋白质的研究需要使用异源细菌系统。 现在，衣原体的遗传操纵的最新进展可以使细菌用质粒转化。 在这里，我们描述了一个穿梭矢量系统PBOMB4，该系统允许在本构或条件启动子控制下表达重组蛋白。 我们表明，包含膜蛋白INCD以Y. pseudotuberculsosis的III型分泌，并在感染细胞中从沙眼梭状芽胞盘中分泌，在该细胞中它适当地定位于包含膜。 含有分泌信号的前三十个氨基酸被截断的INCD不再分泌，并被细菌保留。 可以使用CYAA，GSK或显微注射测定法确认分泌蛋白的胞质暴露。 NRDB确实定位于衣原体，预计将保留在细菌中。 此外，我们已经表明，通过SEC依赖性途径将衣原体效应蛋白CPAF分泌到宿主细胞细胞质中，也从该系统表达时也可以访问胞质醇。 我们采用了最近开发的遗传工具来验证以前尚未定位在包含膜的预测INC的定位。 50 Incs的表达确定了10，这是先前未经验证的INC的表达。一个新颖的INC和3先前描述的INC被定位为包含膜微区域，这是通过与P-SRC共定位的。 几个预测的INCS没有定位于包含膜，而是与细菌有关。我们以Yersinia为代理宿主，证明其中许多不是通过III型分泌分泌的，进一步表明它们不是Incs。我们的结果共同凸显了遗传工具的实用性，以证明衣原体的分泌。旨在阐明效应子功能的进一步的机械研究将进一步了解这种病原体如何保持其独特的细胞内生态位并介导与宿主的相互作用。 INCA是一种包含膜蛋白，它编码与真核网络同源（可溶性N-乙基米拉米酰胺敏感因子附着受体）基序同源的两个盘绕螺旋结构域。最近的生化证据表明，由圈状结构域1（SLD-1）组成的功能核心和SNARE样结构域2（SLD-2）的一部分是多种感染细胞中沙眼包含的特征性同型融合所必需的。 为了验证印加在衣原体中同种型融合中的重要性，我们产生了一个inca :: bla突变体。 INCA的插入灭活导致形成非舒张包含物，这种表型通过与全长INCA的补充完全挽救了这种表型。 同型包容融合的拯救取决于由SLD-1和SLD-2的一部分组成的功能核的存在。这些结果共同证实了在体外膜融合测定中鉴定INCA功能结构域并扩展可用于衣原体的遗传工具，并采用一种补充位点特异性突变体的方法。