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

描述(申请人提供)：粪肠球菌是一种革兰氏阳性条件致病菌，可引起尿路和伤口感染、菌血症和感染性心内膜炎。与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可移动的DNA元素，这些DNA元素介导了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。可移动DNA元件编码IV型分泌系统(T4SS)；这些是负责将DNA底物输送到细菌靶细胞的多组分转位通道。在许多重要的革兰氏阴性病原菌中，T4SS不仅用于细菌间的DNA转移，还用于将效应蛋白注入真核靶细胞。在移位时，效应器扰乱宿主细胞的过程，允许入侵病原体的生长和增殖。最近的研究表明，革兰氏阴性和阳性细菌的T4SS在识别和跨细胞膜转运同源底物的能力方面显示出保守的作用机制。因此，令人惊讶的是，还没有研究检验革兰氏阳性病原体在感染期间使用T4SS将效应蛋白运送到真核宿主细胞的可能性。在这里，我们建议检验一个假设，即革兰氏阳性细菌的T4SS，像它们的革兰氏阴性细菌一样，通过将效应蛋白输送到真核靶细胞或环境中而促进感染过程。 两个初步发现有力地支持了我们的假设：i)我们已经获得证据表明编码pCF10的T4SS从粪肠球菌供体细胞到大肠杆菌受体细胞的蛋白质转位，以及ii)由千碱基致病岛(89K PAI)编码的功能性T4SS被证明是另一种革兰氏阳性病原体猪链球菌毒力所必需的。我们将结合Christie和Dunny团队的专业知识来确定两个粪肠球菌T4SS蛋白转位的蛋白质底物、分泌信号和宿主范围，一个由信息素反应质粒pCF10编码，另一个由染色体153-kb致病岛(PAI)编码。我们将把确定这些革兰氏阳性T4SS的底物谱系的目标与使用兔心内膜炎感染模型的感染检测结合起来，以确定T4SS介导的蛋白易位在粪肠球菌致病中的作用。我们预计，我们的调查将建立一个原则证明 T4SS介导的蛋白质易位对革兰氏阳性病原菌毒力的贡献。这一成果将刺激新的研究举措，旨在破译粪肠球菌和其他医学上重要的革兰氏阳性病原体感染期间细胞间蛋白转移的机制和细胞后果。

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.