An etiological relationship between periodontitis, a significant oral health problem, and the anaerobe Porphyromonas gingivalis may be related to the expression of a variety of putative virulence factors. The objective of the experiments described here was to develop a quantitative reverse transcription polymerase chain reaction (QRT-PCR) method to examine P. gingivalis gene expression in human dental plaque from periodontitis subjects. PCR primers and probes for six target genes representing putative virulence factors were chosen and evaluated in vitro for specificity. A potential cross-reactivity level of only 10 copies/107 whole genomic equivalents was occasionally observed with non-P. gingivalis microbes. P. gingivalis cells stressed in vitro by a 5 °C temperature increase showed a rapid rise in the mRNA associated with the molecular chaperons (htpG, dnaK, groEL), SOD (sodA) and gingipain (rgp-1) genes. We examined the stability of bacterial RNA in plaque specimens and found no significant difference in the amount of RNA obtained before or after storage 3 months in a stabilizing buffer (p = 0.786, t-test). Sixty-five percent of plaque samples obtained from two clinical locations contained P. gingivalis; there was a mean level of gene expression (fold increase) for all samples tested for groEL, dnaK, htpG, sodA, PG1431 and rgp-1 of 0.84 ± 2.03 to 7.85 ± 10.0. ANOVA showed that the levels of stress gene transcription for dnaK and htpG were significantly elevated (p < 0.05) at diseased sites; groEL gene transcription approached statistically significant elevation (p = 0.059). We found correlations between probing depth and increased transcription of groEL, htpG and rgp-1 and between attachment loss and htpG. When sorted by disease status, we detected correlations between disease status and elevated expression of dnaK and htpG.
Bibliographical noteFunding Information:
This work was supported in part by an award from the National Institutes of Health/NIDCR DE11117 and by 3M. The authors wish to thank Monique Upton and Chestevia Gibson at 3M for their excellent technical assistance in the development of the RT-PCR methodology and Allison Combs at the University of Michigan for sharing her data on the basal levels of htpG activation in vitro. We also thank Amy Purdy, Thomas Cafarella, James Miller and Stephanie Quam at the University of Minnesota. Genomic DNA from Chlamydia pneumoniae for cross-reactivity testing was a generous gift provided by Grace M.L. Ong, Department of Virology, Royal Hospital Trust, Belfast, Northern Ireland. Preliminary sequence data for P. gingivalis was obtained from The Institute for Genomic Research website at http://www.tigr.org . Sequencing of Porphyromonas gingivalis was accomplished with support from the National Institute of Dental and Craniofacial Research (NIDCR), part of the National Institutes of Health (NIH) in collaboration with The Forsyth Institute. Identification of PG1431 was accomplished through the Oral Pathogen Sequence Databases at the Bioscience Division of Los Alamos National Laboratory.
- Porphyromonas gingivalis
- QRT-PCR analysis