Ca2+-binding protein EfhP mediates Ca2+ regulation of Pseudomonas aeruginosa virulence and host-pathogen interactions.

钙离子（Ca 2+）是真核生物中最重要的细胞内和细胞间信使之一， 在那里它调节许多基本过程，包括先天免疫反应。因此，我们认为， 细胞内Ca2+稳态的异常与许多疾病有关，包括 肺部和心脏疾病通常与细菌感染有关。在 在细菌中，Ca2+已被证明影响各种生理过程，包括毒力。 然而，这种调节的分子机制仍然是难以捉摸的。识别和 对参与Ca2+调节和信号传导途径的蛋白质的表征不仅 为原核生物中Ca2+的信号传导作用提供了缺失的直接证据，但更多的是 重要的是，这将为Ca2+调节的宿主-病原体相互作用提供新的理解 引发传染病的发展。在本研究中，我们将研究 铜绿假单胞菌，一种兼性人类病原体，是导致严重 囊性纤维化（CF）、心内膜炎、伤口、烧伤和植入患者的慢性感染。早些时候 我们发现升高的Ca 2+可增强P. 铜绿。我们鉴定了一种推定的钙结合蛋白EfhP，它与 钙调素，并含有两个典型的EF-手图案。我们假设EfhP结合Ca2+， 并且在Ca2+信号传导和毒力调节中起关键作用。为了验证这些假设， 1)通过EfhP测量Ca2+结合并鉴定所需的氨基酸残基; 2）表征 EfhP Ca~（2+）结合在毒力因子调控中的作用; 3）表征EfhP的转录水平， 临床分离株和CF痰液中efhP对Ca2+、其他宿主相关因子的反应谱 4）鉴定由EfhP Ca2+结合触发的调节性磷酸化途径;以及 5)EfhP和Ca2+在铜绿假单胞菌-宿主相互作用中的作用。这项研究是 创新，因为它将是第一个研究钙结合蛋白，钙离子之间的关系， 信号传导和Ca2+调节的人类病原体的毒力非常重要。 了解Ca2+调节铜绿假单胞菌毒力的分子机制将有助于我们更好地了解铜绿假单胞菌的毒力。 促进目前对与Ca2+失衡相关的传染病的认识， 最终为开发新的抗微生物药物提供新的靶点。拟议研究 将由本科生和研究生组成的团队表演。它的目的是提供 为学生提供良好的培训环境，确保他们参与现代研究， 参加科学会议和专业网络， 同行评审期刊

Calcium (Ca2+) is one of the most important intra- and intercellular messengers in eukaryotes, where it regulates many essential processes, including innate immune responses. Therefore, abnormalities in cellular Ca2+ homeostasis have been implicated in many diseases, including pulmonary and heart diseases that are commonly associated with bacterial infections. In bacteria, Ca2+ has been shown to affect various physiological processes including virulence. However, the molecular mechanisms of this regulation are still elusive. Identification and characterization of proteins involved in Ca2+ regulatory and signalling pathways will not only provide a missing direct evidence of the signalling role of Ca2+ in prokaryotes, but more importantly, will provide a new understanding of Ca2+-regulated host-pathogen interactions triggering the development of infectious diseases. In the proposed research we will study Pseudomonas aeruginosa, a facultative human pathogen that is a leading cause of severe chronic infections in cystic fibrosis (CF), endocarditis, wound, burn, and implant patients. Earlier we showed that elevated Ca2+ enhances the production of virulence factors and infectivity in P. aeruginosa. We identified a putative Ca2+-binding protein, EfhP, which is homologous to calmodulin and contains two canonical EF-hand motifs. We hypothesize that EfhP binds Ca2+ and plays a key role in Ca2+ signaling and regulation of virulence. To test the hypotheses we will 1) measure Ca2+ binding by EfhP and identify the required amino acid residues; 2) characterize the role of EfhP Ca2+ binding in regulation of virulence factors; 3) characterize the transcriptional profile of efhP in response to Ca2+, other host-related factors, in clinical isolates and CF sputum samples; 4) identify the regulatory phosphorelay pathways triggered by EfhP Ca2+-binding; and 5) characterize the role of EfhP and Ca2+ in P. aeruginosa-host interactions. The research is innovative as it will be first to study the relationship between Ca2+-binding protein, Ca2+ signaling and Ca2+-regulated virulence in a human pathogen of extremely high importance. Understanding the molecular mechanisms by which Ca2+ regulates P. aeruginosa virulence will advance the current understanding of infectious diseases associated with Ca2+ imbalance and eventually provide novel targets for developing new antimicrobial drugs. The proposed research will be performed by teams of undergraduate and graduate students. It is designed to provide an excellent training environment for students by ensuring their involvement in modern research, their participation in scientific meetings and professional networking, and their publications in peer-reviewed journals.