Identification and Role of Type IV Effector Proteins in Coxiella burnetii

描述（由申请方提供）：贝氏柯克斯体主动表达效应物，这些效应物可能重塑宿主细胞以进行复制，并且对发病机制至关重要，可能是4型分泌系统依赖性（T4 SS），类似于嗜肺军团菌中的Dot/Icm分泌。鉴定了C.使用军团菌促进了贝氏体的发展，但研究表明， in C. Burnetii尚未完成。最近的进展，包括细胞外生长培养基，转座子诱变和稳定的穿梭载体提供了一个独特的机会，改变实验范式与此代理。目的是通过以下方式鉴定T4 SS依赖性效应子功能：1）鉴定新的T4 SS系统依赖性底物。工作假设是，全基因组筛选的ORF从C。Burnetii将鉴定大量的T4 SS底物（候选效应物）。2)建立所选T4 SS底物的潜在效应子功能。工作假设是，使用一系列体外和原位测定来鉴定定位、结合伴侣和细胞活性的假定T4 SS依赖性底物的检查将鉴定致病过程中的关键效应物。我们已经把一个主要的优先事项，在这个目标上的分子表征核本地化和Fic结构域含有效应。3)使用敲除诱变确定T4 SS底物在体外和体内毒力中的作用。工作假设是关键T4 SS底物的遗传失活将导致毒力降低。这些数据将用于扩大我们对Q热致病过程的理解，并确定抗C的靶点。贝氏疗法

DESCRIPTION (provided by applicant): Coxiella burnetii actively express effectors that likely remodel the host cell for replication and are essential for pathogenesis are likely type 4 secretion system-dependent (T4SS), similar to Dot/Icm secretion in Legionella pneumophila. Identification of T4SS substrates of C. burnetii has been facilitated using Legionella, but studies in C. burnetii have not been done. Recent advances including extracellular growth medium, transposon mutagenesis and a stable shuttle vector provide a unique opportunity to change the experimental paradigm with this agent. Objectives are to identify T4SS-dependent effector functions by: 1) Identification of novel T4SS system-dependent substrates. The working hypothesis is that genome-wide screens of ORFs from C. burnetii will identify a large collection of T4SS substrates (candidate effectors). 2) Establish a potential effector function for selected T4SS substrate. The working hypothesis is that examination of putative T4SS dependent substrates using a series of in vitro and in situ assays to identify localization, binding partner and cellular activity will identify critical effectors in the pathogenic process. We have placed a major priority in this aim on molecular characterization of nuclear localized and Fic-domain containing effectors. 3) Determine a role in virulence for T4SS substrates in vitro and in vivo using knockout mutagenesis. The working hypothesis is that genetic inactivation of key T4SS substrates will result in reduced virulence. These data will be used to expand our understanding of the pathogenic process in Q fever and identify targets for anti-C. burnetii therapy.