Chlamydial lipid acquisition and host response

描述(申请人提供)：虽然衣原体感染可以用抗生素治疗，但目前的预防策略未能减少新的感染和随后的后遗症。因此，有必要进一步了解衣原体在宿主体内的生存机制，以确定有效的靶点来预防感染，增加检测的简便性或干扰/控制衣原体的生长。衣原体在宿主体内的生存最重要的是有机体获得和利用宿主细胞衍生的脂类的能力。在宿主细胞内，EBS在一个被称为衣原体包涵体的膜结合的空泡中分化为RBS。这种包合物截取了高尔基体衍生的胞外囊泡的一个子集，其中含有鞘磷脂和胆固醇。利用一个极化的上皮细胞模型来研究脂类和蛋白质向包涵体的定向运输，我们证明了衣原体包涵体优先拦截基底部靶向的胞外囊泡，这表明沙眼衣原体与 基地侧贩运机制的一个子集。随后的研究表明，反式高尔基体陷阱蛋白Synaxin 6以一种在衣原体物种中保守的方式与衣原体包涵体共定位，需要衣原体蛋白质的合成，并利用真核信号序列YGRL。YGRL信号序列将Synaxin 6从质膜返回到反式高尔基体；该信号序列对Synaxin 6定位到包涵体的要求表明Synaxin 6在包涵体膜上和包涵体外循环。最近，我们证明了第二种反式高尔基体陷阱蛋白-。根据Synaxin 6和Synaxin 10的定位，我们认为这些蛋白可能与不同的蛋白质池相互作用，从而影响它们在衣原体包涵体上的功能。这一提议的驱动假设是，衣原体招募Synaxin 6和10来运输脂质往返于包涵体和生物体本身；因为衣原体生物体中宿主脂类的准确组成与衣原体在感染期间限制宿主防御的能力有关。在目标1中，我们将询问蛋白质-蛋白质相互作用和合成素6和10在包涵体上的运输动力学。回收活动将首次在衣原体包涵体膜上进行检查。在目标2中，我们将把特定的合成素蛋白的定位与衣原体脂质组成的明显变化联系起来，然后通过检测脂类改变的衣原体生物对特定细胞信号通路的激活的影响，将衣原体脂组成的变化与宿主的反应联系起来。这项建议中的研究将使用新的成像技术来检查衣原体获取的离散机制。值得注意的是，我们将询问并入衣原体细胞壁的真核脂如何影响宿主对感染的反应。

DESCRIPTION (provided by applicant): Although chlamydial infections are treatable with antibiotics, current prevention strategies fail to reduce new infections and subsequent sequelae. Hence, there is a great need to further understand chlamydial survival mechanisms within the host to identify efficacious targets to prevent infection, increase the ease of detection or interrupt/control chlamydial growth. Paramount to chlamydial survival within the host is the organism's ability to obtain and utilize host cell-derived lipids. Within the host cell, EBs differentiate into RBs in a membrane-bound vacuole termed the chlamydial inclusion. The inclusion intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin and cholesterol. Utilizing a polarized epithelial cell model to study directional trafficking of lipidsand proteins to the inclusion, we demonstrated that the chlamydial inclusion preferentially intercepts basolaterally targeted exocytic vesicles, suggesting that C. trachomatis specifically interact with a subset of basolateral trafficking machinery. Subsequent studies demonstrated that trans-Golgi SNARE protein syntaxin 6 colocalizes with the chlamydial inclusion in a manner that is conserved across chlamydial species, requires chlamydial protein synthesis and utilizes a eukaryotic signal sequence, YGRL. The YGRL signal sequence returns syntaxin 6 to the trans-Golgi from the plasma membrane; the requirement of this signal sequence for syntaxin 6 localization to the inclusion suggests that syntaxin 6 cycles on and off the inclusion membrane. Recently, we demonstrated that a second trans-Golgi SNARE protein, syntaxin 10 colocalizes to the chlamydial inclusion in a manner distinct from the localization of syntaxin 6. Syntaxin 10 colocalizes to the inclusion in association with Golgi structural proteins, indicating that insteadof docking vesicles to the inclusion, it may dock Golgi-structures. Based on the localization of both syntaxin 6 and syntaxin 10, we propose that these proteins may be interacting with distinct pools of proteins thereby affecting their function at the chlamydial inclusion. The driving hypothesis of this proposal is that Chlamydia recruit syntaxin 6 and 10 to traffic lipids to or fro the inclusion and the organisms themselves; as the precise composition of host lipids in chlamydial organisms is linked to the ability of Chlamydia to limit host defense during an infection. In Aim 1, we will interrogate protein-protein interactions and the trafficking dynamics f syntaxins 6 and 10 at the inclusion. For the first time, recycling events will be examined at the chlamydial inclusion membrane. In Aim 2, we will correlate the localization of specific syntaxin proteins with distinct changes in chlamydial lipid composition, and then, link the changes in chlamydial lipid composition to host response by examining the effect of lipid-altered chlamydial organisms on the activation of specific cell signaling pathways. Studies in this proposal will examine discrete mechanisms of chlamydial acquisition, using novel imaging techniques. Significantly, we will interrogate how eukaryotic lipids that are incorporated into the chlamydial cell wall impact host response to infection.