Chaperone Mining of the Chlamydia Type Three Secretion System

描述（申请人提供）：沙眼衣原体是性传播疾病的主要原因。了解这种病原体如何在宿主细胞中侵入和复制是我们设计有效治疗方法的基础，可以帮助预防衣原体感染。最近有研究表明衣原体具有3型分泌（TTS）系统的基因。在其他细菌中，TTS系统是病原体共有的一个强大的毒力成分，使它们能够破坏和入侵宿主。TTS系统由几种蛋白组成，这些蛋白以高度调控的方式相互作用。衣原体没有一个经典的遗传系统来研究这个复杂的系统，因此这是一项具有挑战性的任务。然而，所有TTS系统的一个共同元素是TTS伴侣蛋白，它是与其他TTS关键元素（即结构蛋白、效应蛋白和调节蛋白）相互作用的关键。我们计划利用TTS伴侣蛋白来鉴定和表征其他关键的衣原体TTS蛋白。一旦确定了这些蛋白的作用，即效应子、转座子或转录调节剂，将被确定。特别是在Aim 1中，我们将使用TTS伴侣作为下拉试验和细菌双杂交系统中的诱饵，使用靶向和全基因组克隆。为了进一步表征所鉴定的蛋白质，在Aim 2中，我们将建立蛋白质与衣原体发育周期的时间关系。将利用RT-PCR、光镜和免疫电镜对蛋白质在整个发育周期中的出现时间和位置进行表征。我们还将通过确定它们是否可以由异源TTS系统分泌来确定所鉴定的蛋白质是否具有TTS效应分子的特征。在其他病原体中，与TTS伴侣相互作用的蛋白质已被证明在TTS的转录调控中发挥作用。然而，对衣原体TTS的调控尚不清楚，因此我们必须首先建立转录组织，以确定所鉴定的蛋白质是否在衣原体TTS的转录调控中起作用。这将在Aim 3中完成：定位启动子并确定负责转录的RNA聚合酶的形式；通过结合预测的调控元件识别转录调控因子；最后，测试Aim 1中鉴定的蛋白质作为转录调节因子的潜力。完成这些目标将有助于找到并将TTS拼图的各个部分组合在一起，以确定该系统如何在衣原体中发挥作用。预防和控制衣原体感染是一项重大的公共卫生问题。了解衣原体TTS系统的关键要素应有助于制定破坏TTS系统的策略，最终目标是减轻或预防该病原体的感染。

DESCRIPTION (provided by applicant): Chlamydia trachomatis is a leading cause of sexually transmitted diseases. Understanding how this pathogen invades and replicates in host cells is fundamental to our efforts to design effective therapies that can aid in the prevention of Chlamydia infections. It has recently been shown that Chlamydia possesses genes for a type three secretion (TTS) system. In other bacteria TTS systems are a powerful virulence component shared by pathogens that allow them to subvert and invade the host. TTS systems are composed of several proteins that interact in a highly regulated manner. Chlamydia does not have a classic genetic system with which to study this complex system, thereby making this a challenging task. However, a common element to all TTS systems are the TTS chaperones which serve as a lynchpin interacting with other TTS key elements, namely, structural, effectors and regulatory proteins. We plan to exploit TTS chaperones in order to identify and characterize other key chlamydial TTS proteins. Once identified the role these proteins play, i.e. effectors, translocon or transcriptional regulator, will be established. Specifically in Aim 1 we will use TTS chaperones as bait in pull down assays and in a bacterial two-hybrid system, using targeted and genomic-wide cloning. To further characterize the proteins identified, in Aim 2 we will establish the temporal relationship of the proteins to the Chlamydia developmental cycle. Proteins will be characterized as to time of appearance and location throughout the developmental cycle, using RT-PCR, light and immuno-electron microscopy. We will also establish if proteins identified have a characteristic property of a TTS effector molecule by determining if they can be secreted by a heterologous TTS system. In other pathogens, proteins interacting with TTS chaperones have been shown to play a role in transcriptional regulation of TTS. However, nothing is known of the regulation of the Chlamydia TTS so we will first have to establish the transcriptional organization in order to determine whether the proteins identified have a role in the transcriptional regulation of the Chlamydia TTS. This will be accomplished in Aim 3 by: mapping promoters and determining the form of RNA polymerase responsible for transcription; identifying transcriptional regulators by binding to predicted regulatory elements; finally, testing the proteins identified in Aim 1 for their potential to function as a transcriptional regulator. Accomplishing these goals will aid in finding and putting the pieces of the TTS puzzle together to determine how this system functions in Chlamydia. Preventing and controlling Chlamydia infections is of great public health concern. Having an understanding of the critical elements of the Chlamydia TTS system should lead to the development of strategies to subvert the TTS system with the ultimate goal of attenuating or preventing infections with this pathogen.