Eukaryotic-type signaling mediates two-component regulation of GBS virulence

描述(由申请人提供)：无乳链球菌(B组链球菌，GBS)是一种p-溶血性革兰氏阳性球菌，可引起新生儿和成人的侵袭性感染。尽管在预防和治疗早发性新生儿疾病方面取得了重大进展，但与GBS相关的死产、早产、迟发性新生儿疾病和成人感染的发生率并未下降或预防。这些观察结果强调了预防GBS感染的替代策略的重要性。GBS在其发病周期中会遇到广泛的寄主环境。因此，病原体必须迅速适应不断变化的外部环境才能生存并成功感染。细菌双组分信号系统(TCS)是病原菌对外界/宿主环境进行适应性反应的关键信号系统。由传感器蛋白激酶CSRS和反应调节因子CsrR组成的TCS调节GBS细胞毒素P-H/C和cAMP因子的表达。我们的研究表明，GBS还编码两种真核类型的信号酶，包括调节毒力的传感器激酶Stk1和它的同源磷酸酶Stp1。这一建议旨在扩大我们对GBS中真核型信号的理解。我们的研究表明，真核型激酶Stk1以与TCS、CsrR/CSRS相反的方式调控GBS细胞毒素(P-H/C和cAMP因子)的表达。我们观察到Stk1需要CsrR来调节细胞毒素的表达，这表明在GBS中真核型信号和两组分信号之间存在联系。本研究的目的是阐明Stk1和CsrR在GBS适应性基因表达中的相互作用。在本提案中，我们还将定义分别调节Stk1和CsrR活性的同源信号组件Stp1和CSRs的作用。提出的三个具体目标将1)确定Stp1在Stk1调节CsrR活性中的作用；2)确定Stk1和CsrR之间的相互作用对CSRs激活CsrR能力的影响；3)确定Stk1调节CsrR活性的功能结果。结合遗传学、分子生物学、生物化学和蛋白质组学的方法来阐明GBS中真核型和两种组分信号机制之间的联系。这些研究将拓宽我们对GBS调节其适应性反应的信号机制的理解。这项工作将为未来研究GBS在不同宿主生态位中生存的适应性基因表达以及评估这些信号成分作为抗菌策略的靶标奠定基础。我们的发现也将对调节其他致病和非致病微生物适应性反应的新的信号机制产生广泛的影响。

DESCRIPTION (provided by applicant): Streptococcus agalactiae (Group B streptococci, GBS) are p-hemolytic, gram-positive cocci that cause invasive infections in human neonates and adults. Despite significant advances in prevention and treatment of early onset neonatal disease, rates of GBS-related still births, prematurity, late onset neonatal disease and adult infections are not decreased or prevented. These observations emphasize the importance of alternate strategies for prevention of GBS infections. GBS encounters a wide array of host environments during its disease cycle. Hence, it is essential for the pathogen to rapidly adapt to changing external environments to survive and establish successful infections. Bacterial two component signaling (TCS) systems are critical for pathogens to mediate their adaptive responses to the external/host environment. A TCS composed of a sensor kinase CsrS and a response regulator CsrR regulate expression of GBS cytotoxins i.e. p-hemolysin (P-H/C) and CAMP factor. Our studies have shown that GBS also encodes two eukaryotic-type signaling enzymes comprising a sensor kinase Stk1 and its cognate phosphatase Stp1 that regulate virulence. This proposal seeks to extend our understanding of eukaryotic-type signaling in GBS. Our studies show that the eukaryotic-type kinase Stk1 regulates the expression of GBS cytotoxins (P-H/C and CAMP factor) in a manner that is opposite to the TCS, CsrR/CsrS. Our observation that Stk1 requires CsrR for regulation of cytotoxin expression demonstrates a link between eukaryotic-type and two-component signaling in GBS. The objective of this proposal is to elucidate the interaction between Stk1 and CsrR for adaptive gene expression in GBS. In this proposal, we will also define the role of the cognate signaling components Stp1 and CsrS that regulate Stk1 and CsrR activity, respectively. The three specific aims proposed will 1) define the role of Stp1 on Stk1 regulation of CsrR activity 2) establish the effect of the interaction between Stk1 and CsrR on the ability of CsrS to activate CsrR and 3) determine the functional consequence of Stk1 regulation of CsrR activity. A combination of genetic, molecular, biochemical and proteomic approaches are proposed to elucidate the link between eukaryotic-type and two component signaling mechanisms in GBS. These studies will broaden our understanding on signaling mechanisms employed by GBS to mediate its adaptive responses. This work will provide the foundation for future studies on adaptive gene expression by GBS for survival in various host niches and for evaluation of these signaling components as targets in antimicrobial strategies. Our findings will also have widespread implications on novel mechanisms of signaling that regulate adaptive responses in other pathogenic and non-pathogenic microbes.