Distribution of Porphyromonas gingivalis Biotypes Defined by Alleles of the kgp (Lys-Gingipain) Gene

Abstract

Paired subgingival plaque samples representing the most-diseased and least-diseased sites were collected from 34 adult patients with diagnosed chronic periodontitis. The percentage of Porphyromonas gingivalis relative to the total anaerobic and gram-negative bacterial load at each site was determined by real-time PCR. Based on variations in the noncatalytic C terminus of the Lys-gingipain (Kgp), it was reasoned that DNA sequence variation in the 3-coding region of the kgp gene might determine functional biotypes. Perusal of the available sequence information in GenBank indicated three such forms of the kgp gene corresponding to P. gingivalis strains HG66, 381, and W83. Analysis of patient samples revealed the presence of a fourth genotype (W83v) that showed duplication of a sequence recognized by the W83 reverse primer. The four biotypes, HG66, 381, W83, and W83v, were present in the study group in the ratio 8:11:6:5, respectively. Each subject was colonized by one predominant biotype, and only three patients were colonized by a trace amount of a second biotype.

Lysine Gingipain (kgp) Biovars ofPorphyromonas gingivalisExhibit Differential Distribution on Oral Mucosal Sites

“…Using paired subgingival samples from the most-and least-diseased sites, we showed that the majority of periodontitis patients in our study were colonized by one predominant biovar with only trace amounts of a second detectable in a few subjects. The frequency of occurrence of biovars HG66, 381, W83, and W83v was 8:11:6:5, respectively (12). In support of this observation is a recent study by Inaba and colleagues (4), who reported that fimA genotype II clinical isolates from nine Japanese periodontitis patients all possessed the HG66 biovar, while isolates from two patients corresponded to biovars 381 and W83 or biovars 381 and W83v.…”

“…However, a recent report highlights the role of gingipains and not fimbriae in preferential binding to matrix proteins, a process that could facilitate colonization of the host (9). Previously we reported that P. gingivalis could be categorized by differential PCR into four kgp biovars: HG66, 381, W83, and W83v, a domain variant of the W83 kgp gene (12). Using paired subgingival samples from the most-and least-diseased sites, we showed that the majority of periodontitis patients in our study were colonized by one predominant biovar with only trace amounts of a second detectable in a few subjects.…”

“…Samples were collected in tubes containing 200 l phosphate-buffered saline and snap-frozen with dry ice in preparation for extraction of DNA using a QIAamp DNA minikit (Qiagen, Clifton Hill, Victoria, Australia). P. gingivalis load and total bacterial load were quantified, and identification of kgp biovars was determined as previously described (8,11,12). Briefly, total bacterial and P. gingivalis loads were determined using the universal primer (300 nM) and probe (175 nM) set or the P. gingivalis-specific primers and probe (100 nM), respectively, using the TaqMan PCR core reagents kit (Applied Biosystems) and an ABI-PRISM 7700 sequence detection system (8,12).…”

“…P. gingivalis load and total bacterial load were quantified, and identification of kgp biovars was determined as previously described (8,11,12). Briefly, total bacterial and P. gingivalis loads were determined using the universal primer (300 nM) and probe (175 nM) set or the P. gingivalis-specific primers and probe (100 nM), respectively, using the TaqMan PCR core reagents kit (Applied Biosystems) and an ABI-PRISM 7700 sequence detection system (8,12). The amount of DNA in each sample was measured against P. gingivalis DNA standards in the range 3.6 fg to 3.6 ng and converted to cell numbers based on one cell containing 2.37 fg of DNA.…”

“…P. gingivalis load was expressed as per ml for subgingival plaque and for mucosal or tongue samples as previously used (3). For identification of kgp biovars, differential PCR was carried out with HotStarTaq master mix (Qiagen, Australia) using the HG66 biovar-specific primer set HG66F and HG66R (amplicon size, 1.9 kb), the 381 biovar-specific primer set kgpF and 381R (amplicon size, 3.4 kb), and the W83/W83v biovarspecific primer set kgpF and W83R (amplicon size, 3.6 kb for W83 and 2.2 kb for W83v) (12). Total bacterial load (described above) was used as a reference marker for bacterial biomass recovered to verify the sampling technique (15).…”