Virulence factors of periodontopathogens

描述(申请人提供)：革兰氏阴性厌氧菌是牙周病的主要病原体。此外，它们还被证明与其他健康并发症有关，如心血管疾病、糖尿病和早产低出生体重婴儿。牙龈假单胞菌和前牙根霉菌。中间体缺乏合成氯化血红素的能力，必须从环境中获取营养。尽管在牙周病原体中获得氯化血红素很重要，但关于营养的摄取和调节机制方面的知识存在着重大差距。在牙龈假单胞菌W83的基因组上已经确定了几个编码氯化血红素运输蛋白的基因座，然而，这些基因座对该生物体中氯化血红素摄取的贡献尚不清楚。此外，由基因座编码的蛋白质的作用也没有很好的定义。我们已经确定了一个操纵子hmuYRSTUV，它是牙龈假单胞菌生长所必需的，它以血红蛋白-结合珠蛋白复合体为氯化血红素来源。操纵子的同源基因也存在于包括Prev在内的其他革兰氏阴性厌氧细菌的基因组中。我们的初步数据表明，HMU操纵子是铁调控的。尽管铁摄取调节剂Fur被证明在各种细菌中调节氯化血红素吸收位点的表达，但我们的初步研究表明，过氧化反应调节剂OxyR在HMU位点的表达调控中发挥作用。因此，我们将首先描述OxyR介导的调节机制。其次，我们将进一步确定HMU基因在牙龈假单胞菌吸收氯化血红素中的作用。我们将从比较HMU和另外两个存在于牙龈假单胞菌W83基因组上的氯化血红素吸收基因座IHT和TLR在该生物中氯化血红素吸收中的作用开始我们的表征。接下来，我们将检验这一假设，即HMU操纵子是牙周病原体获取氯化血红素的重要机制，并编码从宿主血球蛋白中提取氯化血红素分子并将其通过两层膜(进入细胞质)运输所需的蛋白质。因此，我们将确定HMU编码蛋白的细胞位置，检查这些蛋白相互作用以及与其他蛋白相互作用的能力，并确定该基因座编码的蛋白对牙龈假单胞菌W83中氯化血红素摄取的贡献。最后，为了扩大我们对氯化血红素在牙龈假单胞菌毒力中的作用的理解，我们将研究氯化血红素和血红素蛋白在这种细菌中对基因表达的作用。

DESCRIPTION (provided by applicant): Gram negative anaerobic bacteria are the major etiological agents of periodontal diseases. In addition, they have been shown to be associated with other health complications such as cardiovascular diseases, diabetes, and preterm low birth weight babies. Both P. gingivalis and Prev. intermedia lack the capacity to synthesize hemin and must acquire the nutrient from the environment. Despite the importance of hemin acquisition in the periodontopathogens, major gaps in knowledge exist regarding the mechanisms of uptake and regulation of the nutrient. Several loci encoding hemin transport proteins have been identified on the genome of P. gingivalis W83; however, the contribution of these loci to hemin uptake in this organism remains unknown. Also, the role of the proteins encoded by the loci is not well defined. We have identified an operon, hmuYRSTUV, required for growth of P. gingivalis with hemoglobin-haptoglobin complexes as a hemin source. A homolog of the operon is also present on the genome of other Gram-negative anaerobic bacteria including Prev. intermedia 17. Our preliminary data indicate that the hmu operon is iron regulated. Although the ferric uptake regulator, Fur, was demonstrated to regulate the expression of hemin uptake loci in variety of bacteria, our preliminary studies show the peroxide responsive regulator, OxyR, plays a role in regulation of expression of the hmu locus. Thus, first we will characterize the OxyR-mediated mechanism of regulation. Second, we will further define the role of the hmu locus in hemin uptake in P. gingivalis. We will start our characterization from comparison of the role of the hmu locus with the other two hemin uptake loci, iht and tlr, present on the genome of P. gingivalis W83 in hemin uptake in this organism. Next, we will test the hypothesis that the hmu operon is an important hemin acquisition mechanism in both periodontopathogens and encodes proteins necessary to extract the hemin molecule from host hemoproteins and transport the hemin across both membranes (into the cytoplasm). Thus we will determine the cellular location of the hmu - encoded proteins, examine the ability of the proteins to interact with each other and with other proteins, and define the contribution of proteins encoded by the locus to hemin uptake in P. gingivalis W83. Lastly, to broaden our understanding of the role of hemin in virulence of P. gingivalis, we will examine the role of hemin and hemoproteins on gene expression in this bacterium.