Role of type IV secretion-mediated protein translocation in E. faecalis

描述（由申请方提供）：粪肠球菌是一种革兰氏阳性机会致病菌，可引起尿路和伤口感染、菌血症和感染性心内膜炎。像许多其他革兰氏阳性病原体一样，肠球菌在其基因组中携带移动的DNA元件，这些元件介导抗生素耐药性和其他毒力决定因素的传播，使医学界控制相关感染的努力复杂化。移动的DNA元件编码IV型分泌系统（T4 SS）;这些是负责将DNA底物递送至细菌靶细胞的多组分易位通道。在许多重要的革兰氏阴性病原体中，T4 SS不仅用于细菌间DNA转移，而且用于将效应蛋白注射到真核靶细胞中。在易位时，效应物破坏宿主细胞过程，从而允许入侵病原体的生长和增殖。最近的研究表明，革兰氏阴性菌和革兰氏阳性菌的T4 SS在其识别和易位同源底物穿过其细胞包膜的能力方面显示保守的作用机制。因此，令人惊讶的是，还没有研究检查革兰氏阳性病原体在感染期间使用T4 SS将效应蛋白递送至真核宿主细胞的潜力。在这里，我们建议测试一个假设，即革兰氏阳性细菌的T4 SS，像他们的革兰氏阴性对应物，有助于通过传递效应蛋白到真核靶细胞或环境的感染过程。 两个初步的发现有力地支持了我们的假设：i）我们已经获得了pCF 10编码的T4 SS从E.粪肠球菌供体细胞的大肠杆菌受体，和ii）由89-丝氨酸蛋白酶致病性岛（89 K派）编码的功能性T4 SS被证明是另一种革兰氏阳性病原体，猪链球菌的毒力所必需的。我们将联合收割机的克里斯蒂和丹尼小组的专业知识，以确定蛋白质底物，分泌信号，和宿主范围的蛋白质易位的两个E。faecalis T4 SS，一个由信息素应答质粒pCF 10编码，第二个由染色体153-kb致病岛（派）编码。我们将使用兔心内膜炎感染模型，将确定这些革兰氏阳性T4 SS的底物库的目标与感染测定相结合，以确定T4 SS介导的蛋白易位到E.粪便致病我们预计我们的调查将建立一个原则证明 T4 SS介导的蛋白质易位对革兰氏阳性病原体毒力的贡献。这样的结果将刺激新的研究计划，旨在破译机制和细胞间的蛋白质易位在感染E。粪菌和其他医学上重要的革兰氏阳性病原体。

DESCRIPTION (provided by applicant): Enterococcus faecalis is a Gram-positive opportunistic pathogen and causative agent of urinary tract and wound infections, bacteremia, and infectious endocarditis. Like many other Gram-positive pathogens, enterococci carry mobile DNA elements in their genomes that mediate the spread of antibiotic resistance and other virulence determinants, complicating efforts by the medical community to control associated infections. Mobile DNA elements code for type IV secretion systems (T4SS's); these are multicomponent translocation channels responsible for delivery of DNA substrates to bacterial target cells. In many important Gram-negative pathogens, T4SS's are employed not only for interbacterial DNA transfer but also for injection of effector proteins into eukaryotic target cell. Upon translocation, the effectors disrupt host cellular processes allowing for growth and proliferation of the invading pathogen. Recent studies suggest that T4SS's of Gram-negative and -positive bacteria display conserved mechanisms of action in their ability to recognize and translocate cognate substrates across their cell envelopes. Thus, it is surprising that no studies have yet examined the potential for Gram-positive pathogens to use T4SS's for delivery of effector proteins to eukaryotic host cells during infection. Here, we propose to test a hypothesis that T4SS's of Gram-positive bacteria, like their Gram-negative counterparts, contribute to the infection process through delivery of effector proteins into eukaryotic target cells or the milieu. Two initial findings strongly support our hypothesis: i) we have gained evidence for protein translocation by the pCF10-encoded T4SS from E. faecalis donor cells to Escherichia coli recipients, and ii) a functional T4SS encoded by an 89-kilobase pathogenicity island (89K PAI) was shown to be necessary for virulence of another Gram-positive pathogen, Streptococcus suis. We will combine the expertise of the Christie and Dunny groups to identify protein substrates, secretion signals, and host range for protein translocation by two E. faecalis T4SS's, one encoded by the pheromone-responsive plasmid pCF10 and the second by the chromosomal 153-kb pathogenicity island (PAI). We will integrate the goal of defining the substrate repertoire of these Gram-positive T4SS's with infection assays using a rabbit endocarditis infection model to define the contribution of T4SS-mediated protein translocation to E. faecalis pathogenesis. We anticipate that our investigations will establish a proof-of-principle for the contribution of T4SS-mediated protein translocation to virulence of a Gram-positive pathogen. Such an outcome will stimulate new research initiatives aimed at deciphering mechanistic and cellular consequences of intercellular protein translocation during infection by E. faecalis and other medically-important Gram-positive pathogens.