Rab protein functions that impact Coxiella intracellular replication

 描述(申请人提供)：伯氏柯克斯体是一种革兰氏阴性细菌，是人类疾病Q热的病原体。这种细胞内的病原体已经进化出复杂的机制来逃避宿主细胞的防御。伯氏梭菌独特的一个重要特征是它能在宿主溶酶体的酸性和水解性环境中生存，并修改这个细胞器以产生一个支持复制的大液泡。为了实现这一点，C. 伯氏杆菌编码一个Dot/ICM IV型分泌系统，在感染过程中将100多个效应蛋白输送到宿主细胞的胞浆中。这些效应物调节宿主细胞的功能，包括操纵宿主囊泡运输途径，以促进含有伯氏梭菌的液泡的膜融合和扩张。含伯氏弧菌空泡(CCV)的生物发生和融合所需的宿主决定因素尚未明确定义；然而，由于Rab GTP酶协调宿主细胞内的囊泡运输，并且通常受细菌效应蛋白的调控，我们假设Rab蛋白是伯氏弧菌感染策略的中心。有数据表明，宿主细胞中特定Rab蛋白的沉默将干扰或增强伯氏梭菌的复制，这一假设得到了支持。在这里，我们建议进行研究，以确定Rab GTP酶在伯氏弧菌感染过程中的时空作用，并确定将溶酶体衍生的空泡转化为支持伯氏柯萨奇杆菌复制的独特细胞内隔室所需的Rab指导的过程。我们将干扰特定Rab蛋白的功能，并确定受Rab调控的CCV成熟阶段。我们还将确定在这些特定成熟阶段定位于CCV的RAB蛋白队列。总之，这将揭示伯氏梭菌运输到溶酶体衍生的液泡所需的Rab蛋白，液泡的酸化，Dot/ICM分泌系统的激活，以及细菌复制所需的宿主因子的输入。我们还将通过确定这些Rab蛋白如何影响CCV的生物发生和细胞内的生存，并测试细胞内感染所需的Burnetii效应蛋白是否具有影响这些Rab蛋白功能的活性，来关注对伯氏梭菌细胞内复制产生负面影响的Rab蛋白。这些研究将阐明用于创造支持伯氏梭菌复制的细胞内环境的宿主膜转运途径，并将识别在防御宿主细胞对抗细胞内病原体方面具有重要作用的Rab蛋白。

 DESCRIPTION (provided by applicant): Coxiella burnetii is a Gram-negative bacterium and causative agent of the human disease Q fever. This intracellular pathogen has evolved complex mechanisms for evading host cell defenses. One important feature that makes C. burnetii unique is that it survives within the acidic and hydrolytic environment of host lysosomes and modifies this organelle to create a large vacuole that supports replication. To accomplish this, C. burnetii encodes a Dot/Icm type IV secretion system that delivers over 100 effector proteins into the cytosol of the host cell during infection. These effectors modulate host cell functions, which include manipulating host vesicular transport pathways to promote membrane fusion and expansion of the vacuole containing C. burnetii. Host determinants required for biogenesis and fusion of the C. burnetii-containing vacuole (CCV) have not been clearly defined; however, because Rab GTPases coordinate vesicular transport within host cells, and are often modulated by bacterial effector proteins, we hypothesize that Rab proteins are central to the infection strategy used by C. burnetii. This hypothesis is supported by data showing that the silencing of specific Rab proteins in host cells will interfere or enhance C. burnetii replication. Here, we propose studies to determine the spatiotemporal roles for Rab GTPases during C. burnetii infection, and to determine the Rab-directed processes that are required to transform a lysosome-derived vacuole into a unique intracellular compartment that supports C. burnetii replication. We will disrupt the function of specific Rab proteins and determine the stage of CCV maturation that is regulated by the Rab. We will also define the cohort of Rab proteins that localize to the CCV during these specific maturation stages. Together, this will reveal the Rab proteins that are required for C. burnetii transport to a lysosome-derived vacuole, acidification o the vacuole, activation of the Dot/Icm secretion system, and the import of host factors required for bacterial replication. We will also focus on Rab proteins that negatively impact C. burnetii intracellular replication by determining how these Rab proteins affect CCV biogenesis and intracellular survival of C. burnetii and testing whether C. burnetii effector proteins required fo intracellular infection have activities that affect the function of these Rab proteins. These studis will elucidate host membrane transport pathways that are used to create the intracellular environment that supports C. burnetii replication and will identify Rab proteins that have important roles in defending host cells against intracellular pathogens.