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。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。牙龈卟啉卟啉菌和Prev。中间体缺乏合成血红素的能力，必须从环境中获取营养。尽管血红蛋白获得在牙周病病原体中的重要性，但关于营养物质的摄取和调节机制的知识存在重大差距。在牙龈卟啉单胞菌W83基因组中发现了几个编码血红蛋白转运蛋白的位点；然而，这些基因座对血红蛋白摄取的贡献仍然未知。此外，由基因座编码的蛋白质的作用还没有很好地定义。我们已经确定了一个操作子hmuYRSTUV，它是牙龈卟啉卟啉菌生长所必需的，它的血红蛋白-触珠蛋白复合物是血红蛋白的来源。该操纵子的同源物也存在于其他革兰氏阴性厌氧菌的基因组中，包括Prev。媒介物17。我们的初步数据表明hmu操纵子是铁调控的。虽然铁摄取调节剂Fur被证明可以调节多种细菌血红蛋白摄取位点的表达，但我们的初步研究表明氧化应激反应调节剂OxyR在调节hmu位点的表达中起作用。因此，首先我们将描述氧合素介导的调控机制。其次，我们将进一步确定hmu位点在牙龈卟啉菌血红素摄取中的作用。我们将从比较hmu位点与其他两个血红素摄取位点（ht和tlr）在牙龈卟啉单胞菌W83基因组中血红素摄取中的作用开始我们的表征。接下来，我们将验证hmu操纵子是一个重要的血红蛋白获取机制的假设，并编码从宿主血红蛋白中提取血红蛋白分子并将血红蛋白跨膜运输（进入细胞质）所需的蛋白质。因此，我们将确定hmu编码蛋白的细胞位置，检查蛋白质相互作用和与其他蛋白质相互作用的能力，并确定由位点编码的蛋白质对牙龈卟啉卟啉菌W83血红蛋白摄取的贡献。最后，为了扩大我们对血红蛋白在牙龈卟啉单胞菌毒力中的作用的理解，我们将研究血红蛋白在牙龈卟啉单胞菌基因表达中的作用。我们预测，我们的研究将揭示血红蛋白获取的作用和机制，不仅在牙周病病原体，而且在其他厌氧菌。然后，这些知识可以用于设计干扰必需营养素获取的抗菌剂。

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 oxidative stress 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 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 on gene expression in this bacterium. We predict that our studies will shed light on the role and mechanisms of hemin acquisition not only in periodontopathogens but also in other anaerobic bacteria. Such knowledge may then be used to design antimicrobial agents that will interfere with the acquisition of the essential nutrient. PUBLIC HEALTH RELEVANCE: Iron and hemin have been shown to be indispensable nutrients as well as play a role in gene regulation in P. gingivalis. However, our understanding of the hemin-iron acquisition and regulation mechanisms of this organism is rudimentary. The cellular, molecular, and animal model approaches that we propose are expected to illuminate the mechanisms of hemin and iron acquisition. This information will serve as a platform for our future determination of the mechanisms of hemin/iron uptake. My work is guided by the conviction that a cellular and molecular understanding of iron/hemin metabolism in P. gingivalis will give way to novel translational approaches for both treating and preventing adult periodontal disease.