Identification and Role of Type IV Effector Proteins in Coxiella burnetii

描述(申请人提供)：伯氏柯克斯体积极表达效应物，可能重塑宿主细胞以进行复制，并对发病至关重要，可能是4型分泌系统依赖(T4SS)，类似于嗜肺军团菌的Dot/ICM分泌。利用军团菌鉴定伯氏梭菌T4SS底物是很方便的，但研究 在C.burnetii中还没有这样做。最近的进展，包括胞外生长介质、转座子诱变和稳定的穿梭载体，为改变这种药物的实验范式提供了独特的机会。目的是通过以下方式鉴定T4SS依赖的效应器功能：1)鉴定新的T4SS系统依赖的底物。工作假设是，从伯氏梭菌基因组范围内筛选ORF将识别大量T4SS底物(候选效应物)。2)为选定的T4SS底物建立潜在效应函数。工作假设是，通过一系列体外和原位检测T4SS依赖的底物来确定定位、结合伙伴和细胞活性，将确定致病过程中的关键效应因子。在这一目标中，我们已经把重点放在了核局部和含有Fic结构域的效应器的分子特征上。3)用基因敲除突变方法确定T4SS底物在体外和体内毒力中的作用。工作假设是，关键的T4SS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-C的靶点。Burnetii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.