Diversity of oral spirochetes in subgingival niches

Abstract

Periodontal disease encompasses a range of inflammatory infections that affect the human gingiva and underlying tissues that support the tooth. Periodontal disease, as typified by periodontitis, has a varied and highly complex polymicrobial etiology. Oral Treponema, as the only genus of spirochetes identified in the oral cavity, is one of the bacterial groups which have been found associated with the occurrence and severity of periodontal infections. Oral treponeme population has been demonstrated as a complex and diversified community. This study mainly investigated and compared the composition of treponeme and related bacterial populations present in the subgingival plaques from periodontitis subjects and periodontitis-free controls. In the first part of the study, the 16S rDNA clone library-based approach was introduced to systematically compare the diversity and composition of treponeme operational taxonomic units (OTUs) present within subgingival plaques collected from ten periodontitis subjects and ten periodontitis-free controls. The results clearly indicated that the subgingival plaque sampled from periodontitis subjects contained a significantly higher diversity and clonal abundance of oral treponeme OTUs than corresponding samples taken from the periodontitis-free controls. Community-based comparisons demonstrated that the periodontitis and control subjects contained quite distinct populations of treponeme OTUs in their corresponding periodontal niches. Most notably, the difference of treponeme populations with the diversity and clonal abundance increase in phylogroup 2 and decrease in phylogroup 6 was observed in periodontitis subjects compared to controls. Since the Spirochaetes/Synergistetes specific primer was used for 16S rDNA clone library construction, the Synergistetes sequences were also analyzed to investigate and compare the diversity and composition of members of the Synergistetes phylum in the subgingival plaques from periodontitis and control subjects. In agreement with previous studies, the results found Synergistetes clones were significantly more prevalent and more diverse in periodontitis subjects, and the presence of certain Synergistetes OTUs appeared associated with periodontitis. The major surface protein (Msp) was identified and investigated in several Treponema species, particularly phylogroup 1 and phylogroup 2 species, as a key virulence factor. In the second part of the study, the consensus primers, which cover the msp gene sequences present in a diverse set of phylogroup 1 and phylogroup 2 oral treponemes, were designed to systematically characterize the msp genotypes present in clinical treponeme communities in subgingival plaques collected from another ten periodontitis and ten periodontitis-free subjects. The results showed a great diverse of msp genotypes were identified in periodontal environment, particularly in periodontitis subgingival plaques, with majority of them have never been reported before. One of these genotypes, OTK-like NP5_2 appears to be associated with periodontitis. In the last part of the study, DNA aptamers were selected in vitro against the recombinant Msp fragment of T. denticola ATCC 35405 (rMsp405V). The preliminary study successfully selected aptamers with high binding affinity with the target protein as well as rMspNP5_2 which is the newly identified Msp homologue mentioned above. The characteristics of selected aptamers in terms of binding affinity (Kd) and predicted secondary structures were also reported.