Study of the PorX-PorY two-component system in Porphyromonas gingivalis

 描述(申请人提供)：牙龈卟啉单胞菌是一种革兰氏阴性厌氧细菌(类杆菌)，是牙周病的主要病原体。此外，越来越多的证据表明，感染牙龈假单胞菌可能会导致更严重的全身疾病，如心血管疾病和类风湿性关节炎。流行病学研究表明，这种口腔病原体可能会对人类健康产生广泛的影响。细菌病原体，包括牙龈假单胞菌，表达和分泌一系列毒力因子，使它们能够在人类宿主中引起疾病。尤其是，牙龈假单胞菌能分泌一种称为牙周疼痛的强蛋白水解酶，在牙周疾病的发病机制中被认为是致病因子，也就是对抗宿主的武器。一种新表征的IX型分泌系统PorSS，与其他革兰氏阴性细菌的特性不同，已被认为与这些牙龈痛的出口有关。二组分调控系统(TCS)提供了细菌中最普遍的信号转导系统。病原菌通过与感染小生境中存在的信号分子相互作用，并在感染过程中调节特定的基因集，包括毒力决定因素，从而利用不同的TCS来响应宿主环境，最终导致疾病。我们的初步结果表明，牙龈假单胞菌porX和porY基因座分别编码TCS组氨酸激酶(HK)和反应调节蛋白(RR)。这种不同寻常的TCS对细菌的毒力很重要，因为它激活了至少6个PorSS结构基因的转录。本项目旨在详细了解PorX-PorY系统在牙龈假单胞菌基因表达中的调控作用，包括其调控机制、对细菌毒力的贡献以及影响这一信号转导过程的新策略。由于这种TCS控制着这种临床上重要的口腔病原体的多种关键毒力因子的表达，我们将使用高通量分析来确定该调控系统控制的毒力决定因素，并揭示涉及的生理过程。我们将进行磷酸蛋白质组学分析，以研究PorX的磷酸酶活性如何主导磷酸传递机制来调节这种口腔细菌中的特定靶基因。此外，我们将讨论PorX中Chey样C末端结构域可能的双重功能，这可能是一个双组分系统的独特例子，它可以作为转录调节因子，同时作为膜上分泌机械的效应器。PorX调控的基因图谱与氯化血红素反应的HaeRS TCS的基因图谱部分重叠。因此，我们将确定PorX-PorY系统是否可以通过与调节器HAER串话来响应氯化血红素来建立一个集成的信号电路。这项研究将为理解牙龈假单胞菌孤儿蛋白PorX和PorY形成的一个不寻常的双组分系统的调控机制做出新的努力。

 DESCRIPTION (provided by applicant): Porphyromonas gingivalis is a Gram-negative anaerobic bacterium (bacteroides) and a major causative agent of periodontal diseases. In addition, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to rheumatoid arthritis. Epidemiological studies demonstrate that this oral pathogen could have broad range of human health impact. Bacterial pathogens, including P. gingivalis, express and secret an array of virulence factors, which enable them to cause disease in human host. Particularly, P. gingivalis secretes strong proteases called gingipains that are implicated as virulence factors, i.e., the arsenal to fight against the host, in periodontal pathogenesis. A newly characterized type IX secretion system, PorSS, distinct from those characterized in other Gram-negative bacteria, has been linked to export of these gingipains. Two-component regulatory systems (TCS) provide the most ubiquitous signal transduction systems in bacteria. Pathogenic bacteria employ varied TCSs in response to host environments by interacting with signal molecules present in infected niches and regulating specific sets of genes including virulence determinants during infection eventually leading to disease. Our preliminary results have shown that P. gingivalis porX and porY loci encode a TCS histidine kinase (HK) and response regulator (RR), respectively. This unusual TCS is important for bacterial virulence because it activates transcription of at least 6 PorSS structural genes. This project aims to provide a detailed understanding of the regulatory roles of the PorX-PorY system in P. gingivalis gene expression including its regulatory mechanisms of action, contribution to bacterial virulence and the novel strategies employed to influence this signal transduction process. Since this TCS controls the expression of multiple key virulence factors of this clinically important oral pathogen, we will use high- throughput assays to genome-wide characterize the gene profile to specify the virulence determinants controlled by this regulatory system and reveal the physiological processes involved. We will carry out phosphoproteomic analysis to study how the phosphatase activity of PorX would dominant a phosphorelay mechanism to regulate specific target genes in this oral bacterium. Furthermore, we will address possible dual function of the CheY-like C terminal domain in PorX, which may be a singular example of a two-component system that can function as a transcriptional regulator, meanwhile, as an effector on a secretion machinery on the membrane. The PorX-regulated gene profile partially overlaps with those by the hemin-responsive HaeRS TCS. Thus, we will determine whether the PorX-PorY system can build up an integrated signaling circuit by cross-talking with the regulator HaeR in response to hemin. This study will be a new effort for understanding regulatory mechanisms of an unusual two-component system formed by orphan proteins PorX and PorY from P. gingivalis.