Examination of eukaryotic SNARE syntaxin 6 localization to the chlamydial inclusi

描述（由申请人提供）：微生物操纵宿主SNARE机械是细胞微生物学的新兴领域。我的研究重点是专性细胞内细菌沙眼衣原体劫持宿主细胞鞘脂代谢物的机制。沙眼衣原体是一种专性细胞内病原体，在被称为包涵体的寄生液泡内复制。鞘脂是包涵膜（IM）完整性所必需的，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中的重要性。IM和衣原体获得真核脂质的机制尚不清楚。先前的研究表明，包涵体拦截含有鞘磷脂（SM）和胆固醇的高尔基衍生的胞外泡。为了检验高尔基衍生的载体运输到衣原体包涵体，开发了极化上皮细胞模型。与先前的研究一致，SM保留在衣原体感染的细胞内，并与纯化的沙眼衣原体有关。衣原体对SM的保留与基底外侧SM运输的中断有关，这表明衣原体优先拦截基底外侧定向的、含有SM的胞外囊泡。本研究旨在通过研究反式高尔基相关可溶性n -乙基丙烯酰亚胺敏感因子附着蛋白受体（SNARE）蛋白合蛋白6在衣原体脂质获取过程中的作用来阐明衣原体脂质获取的机制。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合成，并且在物种间是保守的，类似于衣原体的招募和SM的保留。我们的中心假设是syntaxin 6介导衣原体脂质获取。在Specific Aim 1中，我们将确定syntaxin 6在衣原体SM获得中的作用。为此，我们将在syntaxin 6敲除细胞中使用已建立的细胞内脂质运输协议来检查SM运输到包涵体，确定syntaxin 6可能运输到包涵体的其他脂质，并确定syntaxin 6的衣原体结合伙伴。syntaxin 6定位到衣原体包涵体需要酪氨酸基序或质膜检索信号（YGRL）。在Specific Aim 2中，我们将描述syntaxin 6 YGRL基序在衣原体包涵体蛋白靶向中的作用。为此，我们将研究含有YGRL基序的其他真核蛋白是否定位在衣原体包涵体上，以及添加YGRL是否会导致其他真核蛋白重靶向到包涵体上。重要的是，这些研究将定义一个难以捉摸的机制，但衣原体脂质获取的关键过程。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液泡。

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.