IADR Abstract Archives
HcpR, a Putative Regulator of the Porphyromonas gingivalis Nitrosative Stress Response
Objectives: Porphyromonas gingivalis, a keystone periodontal pathogen, must withstand high concentrations of nitrites to thrive in the oral cavity. The P. gingivalis mutant strain deficient in HcpR is highly susceptible to nitrosative stress and does not survive in the presence of host cells indicating that the HcpR regulator is important for pathogenicity and protection from nitrosative stress. Here we aim to characterize the regulon and biochemical mechanisms of HcpR.
Methods: To achieve this goal we have employed a combination of biochemical techniques such as UV-Vis spectroscopy, Resonance Raman spectroscopy, and SAXS to understand the biochemical attributes of HcpR and genetic techniques such as RNA-seq and qRT-PCR to characterize the regulon of HcpR.
Results: HcpR causes a drastic, dose dependent upregulation of PG0893, a putative hydroxylamine-reductase, when exposed to nitrite or nitric oxide. Whole transcriptome sequencing reveals that PG0893 is the only highly upregulated gene when exposed to nitrite. Binding of HcpR to DNA appears to be heme dependent. We constructed a homology model of HcpR based on an orthologue, Dnr, a heme binding sensor. The homology model is a homo-dimer composed of three domains – an N-terminal heme-binding domain, a dimerization helix, and a C-terminal DNA-binding domain. Analytical ultracentrifugation confirms the dimeric oligomerization of HcpR. We created an ab initio model using Small Angle X-ray Scattering (SAXS) and derived structural parameters to refine the homology model. Through various techniques, including resonance Raman spectroscopy, we show that HcpR binds heme in a specific manner. The presence of heme bound in the N-terminal domain allows HcpR to bind the diatomic gas molecule Nitric Oxide (NO) as shown by UV-Vis and Raman spectroscopy.
Conclusions: Up-regulation of PG0893 is HcpR dependent. HcpR is heme binding protein that utilizes its N-terminal domain to bind Nitric Oxide.
Methods: To achieve this goal we have employed a combination of biochemical techniques such as UV-Vis spectroscopy, Resonance Raman spectroscopy, and SAXS to understand the biochemical attributes of HcpR and genetic techniques such as RNA-seq and qRT-PCR to characterize the regulon of HcpR.
Results: HcpR causes a drastic, dose dependent upregulation of PG0893, a putative hydroxylamine-reductase, when exposed to nitrite or nitric oxide. Whole transcriptome sequencing reveals that PG0893 is the only highly upregulated gene when exposed to nitrite. Binding of HcpR to DNA appears to be heme dependent. We constructed a homology model of HcpR based on an orthologue, Dnr, a heme binding sensor. The homology model is a homo-dimer composed of three domains – an N-terminal heme-binding domain, a dimerization helix, and a C-terminal DNA-binding domain. Analytical ultracentrifugation confirms the dimeric oligomerization of HcpR. We created an ab initio model using Small Angle X-ray Scattering (SAXS) and derived structural parameters to refine the homology model. Through various techniques, including resonance Raman spectroscopy, we show that HcpR binds heme in a specific manner. The presence of heme bound in the N-terminal domain allows HcpR to bind the diatomic gas molecule Nitric Oxide (NO) as shown by UV-Vis and Raman spectroscopy.
Conclusions: Up-regulation of PG0893 is HcpR dependent. HcpR is heme binding protein that utilizes its N-terminal domain to bind Nitric Oxide.