Examination of eukaryotic SNARE syntaxin 6 localization to the chlamydial inclusi

检查真核 SNARE 语法蛋白 6 对衣原体包涵体的定位

• 批准号: 8105775
• 负责人: Elizabeth Ann Rucks
• 金额: $ 15.77万
• 依托单位: UNIVERSITY OF SOUTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8105775
• 关键词: Africa; Antibiotic Resistance; Antibiotics; Asia; Bacteria; Bacterial Sexually Transmitted Diseases; Binding; Biology; Blindness; Cell Wall; Cell membrane; Cell model; Cells; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Cholesterol; Clinical; Data; Development; Epithelial Cells; Eukaryotic Cell; Goals; Golgi Apparatus; Growth; Human; Immune response; Infection; Intercept; Lipids; Mediating; Membrane; Microbiology; Modeling; Nutrient; Organism; Pathway interactions; Peptide Signal Sequences; Process; Protein Binding; Protein Biosynthesis; Proteins; Protocols documentation; Research; Retrieval; Role; SNAP receptor; Signal Transduction; Small Interfering RNA; Sphingolipids; Sphingomyelins; TGN38 protein; Technology; Tyrosine; United States; Vacuole; Vesicle; base; design; knock-down; lipid transport; microbial; pathogen; prevent; protein protein interaction; public health relevance; receptor; syntaxin 4; syntaxin 6; trafficking

DESCRIPTION (provided by applicant): Microbial manipulation of host SNARE machinery is an emerging field in cellular microbiology. My research focuses on the mechanisms that the obligate intracellular bacterium Chlamydia trachomatis employs to hijack sphingolipid metabolites from the host cell. C. trachomatis is an obligate intracellular pathogen that replicates within a parasitophorous vacuole termed an inclusion. Sphingolipids are required for the integrity of the inclusion membrane (IM) and are also incorporated into the chlamydial cell wall, indicating their importance in both chlamydial development and viability. The mechanisms by which the IM and, thus, chlamydiae obtain eukaryotic lipids are poorly understood. Previous studies have shown that the inclusion intercepts Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To examine Golgi-derived vectoral trafficking to the chlamydial inclusion, a polarized epithelial cell model was developed. Consistent with previous studies, SM was retained within chlamydial-infected cells and associated with purified C. trachomatis elementary bodies. The retention of SM by chlamydiae correlates with a disruption of basolateral SM trafficking, suggesting that chlamydiae preferentially intercept basolaterally-directed, SM-containing exocytic vesicles. This proposal is designed to elucidate mechanisms of chlamydial lipid acquisition by specifically investigating the role of a trans-Golgi associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein syntaxin 6 in this process. Preliminary data demonstrate that syntaxin 6 localizes to the chlamydial inclusion. This process requires chlamydial protein synthesis and is conserved across species, similar to chlamydial recruitment and retention of SM. Our central hypothesis is that syntaxin 6 mediates chlamydial lipid acquisition. In Specific Aim 1, we will determine the role of syntaxin 6 in chlamydial SM acquisition. To this end, we will examine SM trafficking to the inclusion with established intracellular lipid trafficking protocols in syntaxin 6 knock down cells, identify additional lipids that syntaxin 6 may be trafficking to the inclusion, and identify chlamydial binding partners for syntaxin 6. The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma membrane retrieval signal (YGRL). In Specific Aim 2, we will characterize the role of the syntaxin 6 YGRL motif in the targeting of proteins to the chlamydial inclusion. To this end, we will examine if other eukaryotic proteins containing the YGRL motif localize to the chlamydial inclusion and if addition of the YGRL causes retargeting of other eukaryotic proteins to the inclusion. Importantly, these studies will define a mechanism for the elusive, but critical process of chlamydial lipid acquisition. Additionally, these studies may identify a eukaryotic signal sequence that targets eukaryotic proteins to an intracellular bacterial parasitophorous vacuole. Public Health Relevance: The obligate intracellular pathogen Chlamydia trachomatis, a serious human pathogen, requires host-derived lipids, such as sphingomyelin, for survival. These studies will examine the role of syntaxin 6 in the poorly understood process of chlamydial lipid acquisition and define a mechanism for lipid acquisition.

描述(申请人提供)：微生物操纵宿主诱捕机械是细胞微生物学中的一个新兴领域。我的研究重点是专性胞内细菌沙眼衣原体从宿主细胞劫持鞘脂代谢物的机制。沙眼衣原体是一种专有的细胞内病原体，在被称为包涵体的寄生空泡中复制。鞘磷脂是包涵体膜(IM)的完整性所必需的，也被结合到衣原体细胞壁中，表明它们在衣原体发育和生存中的重要性。IM和衣原体获得真核脂的机制还知之甚少。以前的研究表明，这种包合物可以截留高尔基体衍生的含有鞘磷脂(SM)和胆固醇的胞外囊泡。为了研究高尔基体向衣原体包涵体的矢量转运，建立了极化上皮细胞模型。与以前的研究一致，SM被保留在衣原体感染的细胞中，并与纯化的沙眼衣原体基本体有关。衣原体对SM的滞留与基侧SM运输的中断有关，这表明衣原体优先截取基侧定向的、含SM的胞外小泡。这项建议旨在通过专门研究反式高尔基体相关的N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)蛋白Synaxin 6在这一过程中的作用来阐明衣原体脂质获得的机制。初步数据表明，Synaxin 6定位于衣原体包涵体。这一过程需要衣原体蛋白质的合成，并且在物种之间是保守的，类似于衣原体对SM的招募和保留。我们的中心假设是，Synaxin 6介导了衣原体脂质的获得。在特定的目标1中，我们将确定Synaxin 6在衣原体SM获得中的作用。为此，我们将在Synaxin 6击倒细胞中利用已建立的细胞内脂质运输协议来检查SM向包涵体的转运，识别Synaxin 6可能向包涵体转运的额外脂类，并确定Synaxin 6的衣原体结合伙伴。Synaxin 6到衣原体包涵体的定位需要酪氨酸基序或质膜检索信号(YGRL)。在特定的目标2中，我们将表征Synaxin 6 YGRL基序在将蛋白质靶向衣原体包涵体中的作用。为此，我们将检查其他包含YGRL基序的真核蛋白是否定位于衣原体包涵体，以及添加YGRL是否会导致其他真核蛋白重定向到包涵体。重要的是，这些研究将确定衣原体脂质获取这一难以捉摸但关键的过程的机制。此外，这些研究可能确定真核信号序列，将真核蛋白靶向细胞内细菌寄生虫性液泡。 公共卫生相关性：专性细胞内病原体沙眼衣原体是一种严重的人类病原体，需要宿主衍生的脂类，如鞘磷脂，才能生存。这些研究将检验Synaxin 6在鲜为人知的衣原体脂质获得过程中的作用，并确定脂质获得的机制。