Eukaryotic-type STK/STP-mediated modulation of enterococcal pathogenesis

描述(由申请人提供)：耐多药粪肠球菌医院感染的高流行率在医院环境中既是治疗又是感染控制挑战。显然需要使用新的方法来控制肠球菌的多药耐药问题。真核型丝氨酸/苏氨酸激酶(ESTK)和磷酸酶(ESTP)是新近发现的控制细菌生长、分裂、代谢和细菌表面蛋白、衣壳、毒素和毒力因子表达的原核生物调节蛋白。虽然最近有粪肠球菌对万古霉素耐药株的全基因组序列的信息，但还没有关于肠球菌ESTK(EfSTK)和ESTP(EfSTP)的性质和功能的信息。因此，这些蛋白在肠球菌毒力和新陈代谢中的重要性一般还没有被认识到。根据我们最近鉴定的A组链球菌(GAS)ESTK和ESTP的研究结果，我们推测肠球菌EFSTK和ESTP具有与GAS相似的功能，并在调节生长、代谢、细胞外基质结合表面蛋白和毒素的表达方面发挥重要作用，而毒素在肠球菌生物被膜的形成和心内膜炎的发病中起重要作用。为了验证这一假说，我们将克隆共转录的efstk和efstp基因，并制作相应的重组蛋白来研究它们的生化特性。为了了解这些假定的信号分子的生物学功能，将从三个具有不同致病/遗传特征的不同粪肠球菌菌株中创建EfSTK特异的敲除突变体。该基因的缺失对细菌的生长、形态、细胞分裂、粘着HepG2细胞的能力以及非生物表面生物膜的形成的影响将通过几种实验室分析来确定。利用qRT-PCR分析，将确定efSTK在调节选择性基因表达中的作用，这些基因负责细菌黏附、生物被膜的形成和毒力。最后，通过检测efSTK突变株对小鼠腹膜炎和大鼠心内膜炎动物感染模型的致病能力，阐明其对肠球菌毒力的影响。本研究的结果将使我们了解这些真核型信号蛋白的作用，不仅有助于更好地理解肠球菌的致病机制，而且有助于制定策略，开发针对粪肠球菌的新型化疗药物。 从(I)肠球菌ESTK(EfSTK)和ESTP(EfSTP)的生化特性、(Ii)等基因敲除肠球菌突变体的表型特征、(Iii)基于qRT-PCR的选定调控基因在这些突变体中的表达谱以及(Iv)这些突变体在动物感染模型中的毒力谱获得的知识将有助于更好地理解这些蛋白质在肠球菌致病机制、肠球菌多药耐药中的调控作用，并开发替代的化疗策略。

DESCRIPTION (provided by applicant): The high prevalence of nosocomial infections by multidrug-resistant Enterococcus faecalis presents both a treatment and an infection control challenge in the hospital setting. There is a clear demand to control enterococcal multi-drug resistance problems using newer approaches. Eukaryotic-type ser/thr-kinase (ESTK) and phosphatase (ESTP) are the recently recognized prokaryotic regulatory proteins that control the bacterial growth, division, metabolism, and expression of bacterial surface proteins, capsule, toxins, and virulence factors. Although information on the complete genome sequence of vancomycin-resistant strain of E. faecalis is recently available, there is no information on the nature and function of enterococcal ESTK (efSTK) and ESTP (efSTP). As a result, the importance of these proteins in enterococcal virulence and metabolism in general has not been appreciated. Based on the findings of group A Streptococcus (GAS) ESTK and ESTP that we recently characterized, we hypothesize that enterococcal efSTK and efSTP have functions akin to those described for GAS and play an important role in the regulation of growth, metabolism, expression of extracellular matrix-binding surface proteins, and toxins, which are important in the development of enterococcal biofilms and causation of endocarditis. To test this hypothesis, the contranscribing efstk and efstp genes will be cloned and corresponding recombinant proteins will be made to study their biochemical properties. To understand the biological functions of these putative signaling molecules, efSTK-specific knockout mutants will be created from three different E. faecalis strains with distinct pathogenic/genetic profiles. The impact of the deletion of this gene on bacterial growth, morphology, cell division, ability to adhere to HepG2 cells, and formation of biofilms on abiotic surfaces will then be determined using several laboratory assays. Using qRT-PCR assays, the role of efSTK in the regulation of the expression of selective genes that are responsible for the bacterial adherence, development of biofilms and virulence, will be determined. Finally, the impact on the enterococcal virulence will be elucidated by determining the ability of efSTK mutant to cause peritonitis in mouse and endocarditis in the rat animal infection models. The results obtained from this proposal will allow us to understand the role of these eukaryotic-type signaling proteins not only to better understand the mechanisms of enterococcal pathogenesis but also to help formulate strategies to develop novel chemotherapeutic agents against E. faecalis. Knowledge obtained from (i) the biochemical properties of enterococcal ESTK (efSTK) and ESTP (efSTP), (ii) phenotypic characteristics of isogenic knockout enterococcal mutants, (iii) qRT-PCR-based expression profiling of selected regulatory genes in these mutant and (iv) virulence profiling of these mutants in animal infection model will allow better understanding of the regulatory roles of these proteins in enterococcal pathogenesis, enterococcal multi-drug resistance, and to develop alternative chemotherapeutic strategies.