Two pathways for calcium signaling and virulence regulation in P. aeruginosa

摘要 铜绿假单胞菌是一种具有机会性的人类病原体，可引起严重的危及生命的感染。 囊性纤维化(CF)、心内膜炎、伤口、人工植入物和与医疗保健相关的患者 感染。铜绿假单胞菌致病力的多样性与一种突出的生理 生物体的适应性及其调节寄主反应的能力，部分是由于紧密协调的 基因表达的调控。因此，为了控制目前无法治愈的假单胞菌感染，它 对于产生协调病原菌毒力的调节电路的新知识至关重要 对寄主因素的反应。钙离子(Ca~(2+))是真核细胞内必不可少的信使， 调节重要的细胞过程。它聚集在CF患者的肺液和二尖瓣环内。 心内膜炎患者。宿主钙离子动态平衡的改变可能是毒力增强的触发因素 入侵的病原体。在支持方面，我们发现钙离子正向调节生物膜的形成、聚集和 铜绿假单胞菌几种毒力因子的产生。然而，这种现象的分子机制 监管是未知的。细胞内钙离子是否作为第二信使作用也是未知的。 原核生物就像在真核生物中一样。了解钙离子调节、信号转导和信号传导的机制 动态平衡将为控制铜绿假单胞菌的活性、毒力和与细菌的相互作用提供新的手段。 主持人。早些时候，我们鉴定了两个可能的钙结合蛋白EfhP和CARP，它们的突变导致 多种依赖钙离子的毒力和感染性缺陷。Efhp包含两个EF-Hand动机，已知 结合钙离子并通过构象变化传递钙信号。鲤鱼预计会形成一个贝塔螺旋桨 含有一个可能的植酸酶结构域。基于生物信息学和初步研究，我们假设 Efhp和鲤鱼提供了不同的钙信号转导途径，调节毒力和寄主- 病原菌相互作用对宿主内钙离子的响应。为了测试这一点，我们建议确定细胞 定位并确定这两个蛋白调控的结合伙伴和信号转导途径。我们 还将描述Efhp和Carp在铜绿假单胞菌与宿主相互作用中的作用，并定义其 参与急性和慢性感染的发展。通过利用三个OCRID核心的专业知识 ，我们将揭开钙离子信号的机制及其在调节铜绿假单胞菌能力中的作用。 在分子、细胞和组织层面上造成感染。这项研究具有很高的创新性 它将首次在实验上展示细菌中的钙信号，鉴定钙信号的成分 转导途径，并确定钙信号在铜绿假单胞菌体内致病中的作用。

Abstract Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, artificial implants, and in healthcare-associated infections. The versatility of P. aeruginosa pathogenicity is associated with an outstanding physiological adaptability of the organism and its ability to modulate host responses, due in part to a tightly coordinated regulation of gene expression. Therefore, to gain control over currently untreatable Pseudomonas infections, it is critically important to generate new knowledge of the regulatory circuits coordinating the pathogen virulence in response to host factors. Calcium ion (Ca2+) is an essential intracellular messenger in eukaryotic cells, regulating vital cellular processes. It accumulates in pulmonary fluids of CF patients and in mitral annulus of endocarditis patients. Alterations in the host Ca2+ homeostasis may serve as a trigger for enhanced virulence of invading pathogens. In support, we showed that Ca2+ positively regulates biofilm formation, swarming, and production of several virulence factors in P. aeruginosa. However, the molecular mechanisms of such regulation are not known. It is also not known whether intracellular Ca2+ plays role as a second messenger in prokaryotes as it does in eukaryotes. Understanding the mechanisms of Ca2+ regulation, signaling and homeostasis will provide novel means for controlling P. aeruginosa viability, virulence, and interactions with the host. Earlier, we identified two putative Ca2+-binding proteins EfhP and CarP, mutations in which cause multiple Ca2+-dependent defects in virulence and infectivity. EfhP contains two EF-hand motives, known to bind Ca2+ and relay Ca2+ signal through conformational changes. CarP is predicted to form a beta-propeller and has a putative phytase domain. Based on the bioinformatics and preliminary studies, we hypothesize that EfhP and CarP provide different routes of Ca2+ signal transduction regulating virulence and host- pathogen interactions in response to Ca2+ in a host. To test this, we propose to determine the cellular localization and identify binding partners and signal-transducing pathways regulated by the two proteins. We will also characterize the role of EfhP and CarP in P. aeruginosa interactions with a host, and define their involvement in the development of acute and chronic infections. By utilizing the expertise of three OCRID core facilities, we will unravel the mechanisms of Ca2+ signaling and its role in regulating the ability of P. aeruginosa to cause infections at the molecular, cellular, and organismal level. This research is highly innovative as for the first time it will experimentally demonstrate Ca2+ signaling in bacteria, identify the components of Ca2+ signal transduction pathways, and define the role of Ca2+ signaling in P. aeruginosa pathogenicity in vivo.