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.
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.
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