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

 描述（由申请人提供）：牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌（拟杆菌），是牙周病的主要致病菌。此外，越来越多的证据表明，牙龈卟啉单胞菌感染可能导致更严重的全身性疾病，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，包括牙龈卟啉单胞菌，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。特别地，牙龈卟啉单胞菌分泌称为牙龈卟啉菌蛋白酶的强蛋白酶，其涉及毒力因子，即，在牙周病的发病机制中，对抗宿主的武库。一个新的特点IX型分泌系统，PorSS，不同于那些在其他革兰氏阴性菌的特点，已被链接到这些牙龈卟啉菌蛋白酶出口。双组分调节系统（TCS）是细菌中最普遍存在的信号转导系统。病原菌利用不同的TCS响应宿主环境，通过与感染的小生境中存在的信号分子相互作用，并在感染期间调节特定的基因组，包括毒力决定因子，最终导致疾病。我们的初步结果表明，牙龈卟啉单胞菌porX和porY基因座编码TCS组氨酸激酶（HK）和反应调节（RR），分别。这种不寻常的TCS对细菌毒力很重要，因为它激活至少6个PorSS结构基因的转录。本项目旨在详细了解PorX-PorY系统在牙龈卟啉单胞菌基因表达中的调控作用，包括其调控作用机制，对细菌毒力的贡献以及影响该信号转导过程的新策略。由于该TCS控制该临床上重要的口腔病原体的多个关键毒力因子的表达，我们将使用高通量测定来全基因组表征基因谱，以指定由该调节系统控制的毒力决定因子并揭示所涉及的生理过程。我们将进行磷酸化蛋白质组学分析，以研究PorX的磷酸酶活性如何主导磷酸化机制来调节这种口腔细菌中的特定靶基因。此外，我们将解决可能的双重功能的Chey-like C末端结构域的PorX，这可能是一个单一的例子，一个双组分系统，可以作为一个转录调节器，同时，作为一个效应器上的膜上的分泌机制。PorX调节的基因谱与氯化血红素反应性HaeRS TCS的基因谱部分重叠。因此，我们将确定PorX-PorY系统是否可以通过与调节器HaeR响应于氯化血红素的串扰来建立集成信号电路。本研究将为理解牙龈卟啉单胞菌孤儿蛋白PorX和PorY形成的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.