Biochemical characterization of Porphyromonas gingivalis HcpR

Virginia Commonwealth University, Richmond VA

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Abstract

? DESCRIPTION (provided by applicant): The obligate anaerobe Porphyromonas gingivalis is the etiological agent responsible for the progression of periodontal disease. The bacteria is localized to the oral cavity and periodontal pockets where it must withstand high levels of reactive nitrogen species generated by other oral flora and the host innate immune system. The mechanisms allowing for protection against such stress remain poorly understood. HcpR is a novel FNR-CRP family regulator that has been implicated in the regulation and coordination of the nitrosative stress response in P. gingivalis. Previously it has been shown that HcpR regulates the expression of Hcp, a putative hydroxylamine reductase. Growth of knockout strains of the hcpr gene were greatly reduced in the presence of reactive nitrogen species and did not survive with host cells. Biochemical analysis of HcpR reveals that it is a homo-dimer and binds the heme co-factor. Furthermore, it utilizes the heme co-factor to bind the diatomic gas molecule and reactive free radical nitric oxide (NO). To understand the mechanism HcpR utilizes to modulate gene expression we aim to characterize the DNA binding properties of HcpR and the role heme and NO play in DNA binding. Furthermore, we want to analyze the nitric oxide binding properties of HcpR in detail and determine which amino acid residues are directly involved in heme coordination and ligand binding using Raman spectroscopy. Finally, we aim to quantify the in vivo activation of HcpR in a heterologous E. coli system utilizing a fluorescent reporter gene. Beyond the research aspects of this training project, I aim to improve a number of career development skills. I seek to improve my grant writing skills, improve my presentation skills, and gain experience in the manuscript preparation process.