Studies on virulence regulation in Porphyromonas

牙龈卟啉单胞菌是一种黑色素革兰氏阴性厌氧菌，被广泛认为是牙周病的重要病原菌。这种细菌表达几种潜在的毒力因子(例如，衣壳、内毒素、菌毛、膜小泡和水解酶)，这些因素可能与其致病性有关。另一种毒力因子，recA基因，对炎症牙周袋的氧化应激环境具有抵抗力。RecA基因产物是DNA修复中的关键蛋白质，可以保护牙龈假单胞菌免受中性粒细胞在牙周袋中产生的杀菌活性氧衍生物和短暂的空气暴露所引起的DNA损伤。我们的实验室已经确定了两个基因，viA和bcp，它们可能是recA转录单位的一部分，也可能在毒力中发挥作用。此外，VIMA介导的牙龈假单胞菌毒力调节，可能代表了该生物体中毒力因子的一种新的转录后调节。由于BCP同系物可能具有过氧化物酶功能，而牙龈痛参与了血红素的积累，从而可以灭活H_2O_2，因此它可能被认为是生物体协调其氧化应激和蛋白分解活性的重要策略。观察到recA基因启动子在小鼠宿主感染期间是活跃的，这进一步支持了这一重要性。此外，启动子的活性受到温度、铁和钙的影响，这些都是已知的协调调节其他细菌毒力基因表达的因素。综上所述，我们的观察结果可能表明，复杂的recA基因座在牙龈假单胞菌的生存和毒力中起着重要作用。我们的假设是，bcp-recA-VimA转录单位对毒力和对抗氧化应激具有重要作用。我们的总体目标是阐明VIMA介导的毒力调节的分子机制(S)，并研究BCP-recA-VIMA操纵子在牙龈假单胞菌氧化应激抵抗中的相对重要性。拟议研究的具体目的是：1)鉴定牙龈假单胞菌W83中的bcp-recA-VimA转录单位。这将包括：a)绘制转录起始点；b)验证初级起始点上游的启动子序列；c)评估bcp基因对recA和VimA基因功能的影响；2)检测VimA突变对牙龈假单胞菌W83中蛋白酶激活的功能意义；以及3)评估bcp-recA-VimA转录单位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.