Studies on virulence regulation in Porphyromonas

牙龈卟啉单胞菌是一种黑色革兰氏阴性厌氧菌，被广泛认为是牙周病的重要病原。这种细菌表达几种可能导致其致病性的潜在毒力因子（例如，荚膜、LPS、菌毛、膜囊泡和水解酶）。另一个毒力因子，recA基因，赋予抵抗炎症性牙周袋的氧化应激环境。recA基因产物是DNA修复的关键蛋白，可保护牙龈假单胞菌免受由中性粒细胞和短暂空气暴露在牙周袋中产生的抗菌活性氧衍生物引起的DNA损伤。我们的实验室已经确定了两个基因，vimA和bcp，它们可能是recA转录单元的一部分，也可能在毒力中起作用。此外，牙龈卟啉单胞菌中vima介导的毒力调节可能代表了这种生物体中毒力因子的一种新的转录后调控。由于BCP同源物可能具有过氧化物酶功能，并且牙龈痛参与血红素积累，从而使H2O2失活，因此它可能被认为是生物体协调其氧化应激和蛋白水解活性的重要策略。recA基因座启动子在小鼠宿主感染期间是活跃的，这一观察结果进一步支持了这一重要性。此外，启动子活性受温度、铁和钙的影响，这些因素是已知的协调调节其他细菌毒力基因表达的因素。我们的观察结果，综合起来，可能表明复杂的recA位点在牙龈假单胞菌的生存和毒力中起重要作用。这是我们的假设，bcp-recA-vimA转录单位是重要的毒力和抗氧化应激保护。我们的总体目标是阐明vima介导的毒力调控的分子机制，并研究bcp-recA-vimA操纵子在牙龈假单胞菌抗氧化应激中的相对重要性。本研究的具体目的是：1)对牙龈卟卟菌W83中bcp-recA-vimA转录单元进行表征。这将包括：a)绘制转录起始位点；B)验证主起始位点上游的启动子序列；c)评价bcp基因对recA和vimA基因功能的影响；2)研究vimA基因突变对牙龈假单胞菌W83蛋白酶激活的功能意义；3)评价bcp-recA-vimA转录单元在氧化应激保护中的重要性。

Porphyromonas gingivalis, a black-pigmented, gram- negative anaerobe, is widely implicated as an important etiological agent of periodontal disease. This bacterium expresses several potential virulence factors (e.g., capsule, LPS, fimbriae, membrane vesicles, and hydrolytic enzymes) that may contribute to its pathogenicity. Another virulence factor, the recA gene, confers resistance to the oxidative stress environment of the inflammatory periodontal pocket. The recA gene product is a key protein in DNA repair that protects P. gingivalis from DNA damage induced by bactericidal reactive oxygen derivatives generated in the periodontal pocket by neutrophils and transient air exposure. Our laboratory has identified two genes, vimA and bcp, that may be part of the recA transcription unit and may also function in virulence. Further, the vimA-mediated virulence modulation in P. gingivalis, may represent a novel posttranscriptional regulation of virulence factors in this organism. Because the BCP homologue may have peroxidase function, and gingipains are involved in heme accumulation which can inactivate H2O2, it might be considered an important strategy for the organism to coordinate its oxidative stress and proteolytic activities. This importance is further supported by observation that the recA locus promoter is active during infection of the murine host. Moreover, the promoter activity is affected by temperature, iron and calcium which are factors known to coordinately regulate the expression of other bacterial virulence genes. Our observations, taken together, may suggest an important role for the complex recA locus in the survival and virulence of P. gingivalis. It is our hypothesis that the bcp-recA-vimA transcriptional unit is important for virulence and protection against oxidative stress. Our overall objective is to elucidate the molecular mechanism(s) for the vimA-mediated virulence regulation and examine the relative importance of the bcp-recA-vimA operon in oxidative stress resistance in P. gingivalis. Specific aims for the proposed research are: 1) To characterize the bcp-recA-vimA transcriptional unit in P. gingivalis W83. This will include: a) mapping the transcription initiation site; b) verifying the promoter sequence upstream of the primary start site; c) evaluating the effect of the bcp gene on the function on the recA and vimA genes; 2) To examine the functional significance of the vimA mutation on protease activation in P. gingivalis W83; and 3) To evaluate the importance of the bcp-recA-vimA transcriptional unit in oxidative stress protection.