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

描述（由申请人提供）：多药耐药粪肠球菌引起的医院感染的高发，对医院环境中的治疗和感染控制都提出了挑战。有一个明确的需求，控制肠球菌多药耐药问题，采用新的方法。真核丝氨酸/苏氨酸激酶（ESTK）和磷酸酶（ESTP）是最近发现的原核调节蛋白，它们控制细菌的生长、分裂、代谢以及细菌表面蛋白、荚膜、毒素和毒力因子的表达。虽然最近有关于耐万古霉素的粪肠球菌菌株全基因组序列的信息，但没有关于肠球菌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代谢中的重要性尚未得到普遍认识。基于我们最近描述的A群链球菌（GAS） ESTK和ESTP的发现，我们假设肠球菌efSTK和efSTP具有类似于GAS的功能，并且在调节生长，代谢，细胞外基质结合表面蛋白和毒素的表达中发挥重要作用，这些在肠球菌生物膜的发育和心内膜炎的原因中起重要作用。为了验证这一假设，我们将克隆转录的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。为了了解这些可能的信号分子的生物学功能，将从三种具有不同致病/遗传谱的不同粪肠杆菌菌株中创建efstk特异性敲除突变体。该基因的缺失对细菌生长、形态、细胞分裂、粘附HepG2细胞的能力以及在非生物表面形成生物膜的影响将通过几个实验室分析来确定。利用qRT-PCR检测，efSTK在调控与细菌粘附、生物膜发育和毒力有关的选择性基因表达中的作用将被确定。最后，将通过测定efSTK突变体引起小鼠腹膜炎和大鼠动物感染模型心内膜炎的能力来阐明其对肠球菌毒力的影响。这项研究的结果将使我们了解这些真核型信号蛋白的作用，不仅可以更好地了解肠球菌的发病机制，而且有助于制定针对粪肠球菌的新型化疗药物的开发策略。

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.