Porphyromonas gingivalis is a pathogen found in patients with periodontitis that produces toxic cysteine proteases called gingipains. Gingipains are characterized as narrow-spectrum virulence targets. Broad-spectrum antibiotics do not eliminate P. gingivalis but, instead, add to its resistance. Sometimes found in the brains of Alzheimer’s patients, it has been suggested that P. gingivalis, and the gingipains it produces, is involved in cognitive decline. Specifically, lysinegingipan and arginine gingipain A and B are crucial to the pathogenicity of P. gingivalis and are involved in host colonization, suppression of host defenses, nutrient acquisition, and tissue destruction. It has been found that inhibiting gingipain production decreases P. gingivalis brain colonization, reducing neurodegeneration in Alzheimer’s disease. Similarly, it has been suggested that dietary nitrate supplementation may limit P. gingivalis proliferation. The work presented here builds on a clinical trial where participants were given nitrate pills and monitored for changes in oral microbiome community composition. Twenty human oral microbiome RNA samples were subjected to reverse transcriptase quantitative PCR (RT-qPCR) analysis to quantify the abundance of P. gingivalis and the expression levels of genes involved in gingipain production, iron acquisition from host heme, as well as nitrate metabolism. This study designed and validated primers and probes for qPCR for hmuY, kgp, and narG. We found that hmuY, kgp, and narG were amplified of correct size in the positive control samples (100 ng/µL stock P. gingivalis, AlphaDNA), suggesting that the primer targets are accurate. This work will lay foundation for quantifying the absolute abundance of P. gingivalis and the expression levels of hmuY, kgp, and narG in the cDNA samples using qPCR, and, overall, how nitrate supplementation affects these abundances. Additionally, results from the qPCR analysis will give us more precise measurements of how much Porphyromonas sp. are present in the samples which can then be compared to the physiological responses to nitrate supplementation from the study conducted by Freeze et al. (2022). It is hypothesized that reduction in the expression of gingipains and iron acquisition genes, and an increase in the expression of nitrate reductase genes, post nitrate supplementation, will correlate with reduced P. gingivalis abundance
