title: Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo.
abstract: 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/10(7) whole genomic equivalents was occasionally observed with non-P. gingivalis microbes. P. gingivalis cells stressed in vitro by a 5 degrees 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.
authors.affiliation: BioMaterials Technology Center, 3M Company, St. Paul, MN, USA. ceshelbu@unich.edu
authors.forename: Charles E
authors.initials: CE
authors.lastname: Shelburne
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Gleason
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Germaine
authors.forename: Larry F
authors.initials: LF
authors.lastname: Wolff
authors.forename: Brian H
authors.initials: BH
authors.lastname: Mullally
authors.forename: Wilson A
authors.initials: WA
authors.lastname: Coulter
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Lopatin
mesh_terms: D018829:Adhesins, Bacterial; D016866:Bacteroidaceae Infections / Q000382:microbiology; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D003773:Dental Plaque / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D010518:Periodontitis / Q000382:microbiology; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; D017931:DNA Primers; D015342:DNA Probes; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D018832:Molecular Chaperones; D012329:RNA, Bacterial; D013482:Superoxide Dismutase; D003546:Cysteine Endopeptidases