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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

基本信息

批准号：
8249802
负责人：
Elizabeth Ann Rucks
金额：
$ 10.64万
依托单位：
UNIVERSITY OF SOUTH DAKOTA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8249802
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

项目摘要

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（由申请人提供）：宿主陷阱机制的微生物操纵是细胞微生物学中的一个新兴领域。我的研究重点是专性细胞内细菌沙眼衣原体从宿主细胞中劫持鞘脂代谢产物的机制。C.沙眼衣原体是一种专性细胞内病原体，其在称为内含物的寄生虫空泡内复制。鞘脂是完整的包涵体膜（IM）所必需的，也被纳入衣原体细胞壁，表明它们在衣原体发育和生存能力的重要性。IM和衣原体获得真核脂质的机制知之甚少。以前的研究表明，包含拦截高尔基体衍生的胞吐囊泡含有鞘磷脂（SM）和胆固醇。为了研究高尔基体衍生的载体贩运衣原体包涵体，极化上皮细胞模型的开发。与以前的研究一致，SM保留在衣原体感染的细胞内，并与纯化的C。沙眼原体保留SM的衣原体与基底外侧SM贩运的中断，这表明衣原体优先拦截基底外侧定向的，含SM的胞吐囊泡。这个建议的目的是阐明衣原体脂质收购的机制，具体调查的作用，一个trans-Golgi相关的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白syntaxin 6在这个过程中。初步数据表明，syntaxin 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，并在不同物种之间保守，类似于衣原体的招募和SM的保留。我们的中心假设是，syntaxin 6介导衣原体脂质收购。在具体目标1中，我们将确定syntaxin 6在衣原体SM收购中的作用。为此，我们将研究SM贩运纳入与已建立的细胞内脂质贩运协议在syntaxin 6敲低细胞，确定额外的脂质，syntaxin 6可能被贩运的包容性，并确定衣原体结合的合作伙伴syntaxin 6。syntaxin 6定位于衣原体包涵体需要酪氨酸基序或质膜修复信号（YGRL）。在具体目标2中，我们将描述突触融合蛋白6 YGRL基序在将蛋白质靶向衣原体包涵体中的作用。为此，我们将检查其他含有YGRL基序的真核蛋白是否定位于衣原体内含物，以及YGRL的添加是否会导致其他真核蛋白重新靶向该内含物。重要的是，这些研究将为衣原体脂质获得的难以捉摸但关键的过程确定一种机制。此外，这些研究可能会确定一个真核生物的信号序列，靶向真核生物蛋白质的细胞内细菌寄生虫空泡。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，需要宿主来源的脂质（如鞘磷脂）才能生存。这些研究将探讨syntaxin 6在衣原体脂质获得过程中的作用，并确定脂质获得的机制。