Diversity, distributions and virulence factors of established and emerging periodontal pathogens

Abstract

Periodontal diseases are a range of infectious-inflammatory diseases affecting gingivae and tooth supporting structures. They are caused by dysbiotic polymicrobial biofilm communities established within periodontal niches. An estimated 70-80 ‘species-level’ phylotypes of treponeme bacteria (genus Treponema) are known to inhabit the oral cavity, most of which are as-yet uncultivated. However, we still lack a fundamental understanding of the phylogeny and disease-associations for the vast majority of these oral treponemes.

Porphyromonas gingivalis is another species that is widely believed to play a key role in periodontal disease pathogenesis. Thiol protease (Tpr) is one of the many proteinases (peptidases) secreted by P. gingivalis. It is postulated to be involved in nutrient acquisition; however its biological roles are not well-established.

The aims of this study are: 1) to investigate the genomic diversity and clinical distributions of phylogroup 1 and 2 oral treponemes strains; 2) to develop single gene, and multilocus gene sequence analysis (MLSA) approaches for the taxonomic identification and discrimination of oral treponemes; 3) to characterize the biophysical and biochemical properties of the P. gingivalis Tpr protease.

A comprehensive set of phylogroup 1 and 2 oral treponeme strains was assembled, and subjected to 16S rRNA gene sequence analysis to determine their taxonomy. Several highly-conserved genes including uridylate kinase (pyrH), recombinase A (recA), flagellar sheath protein (flaA) and major surface protein (msp) genes were sequenced for each strain and analyzed via a variety of phylogenetic and computational approaches. The topology of the maximum likelihood (ML) phylogenetic trees for the individual pyrH, recA and flaA genes were fairly congruent with each other. The concatenated pyrH-flaA-recA ML tree offered a more effective taxonomic resolution. However, the phylogeny of the msp gene had a distinct and more complex pattern.

The recombinant Tpr protein was expressed in Escherichia coli, in various ‘affinitytagged’ forms. The initially-expressed full-length Tpr protein catalyzed its own proteolytic cleavage, to specifically remove a ca. 7kDa portion. Analysis by mass spectrometry revealed Tpr specifically cleaved a 62 amino acid N-terminal fragment, leaving ca. 48kDa mature form. Site directed mutagenesis was performed on amino acid residues located within the active site as well as residues located at the cleavage site. Results indicated that the Cys229Ala and Cys229Ser Tpr mutants had negligible hydrolytic activities. However, mutations at the cleavage site (Gly62Ala, Met63Ala) were well-tolerated. These results suggested that the specificity of Tpr self-cleavage was not based on the sequence of the locus being cut, but affected by other factors, possibly a topological constraint. With the incubation of appropriate concentrations of Ca^(2+) ions (e.g. 5 mM), additional cleavage events became apparent. As detected on SDS-PAGE gels, and subsequently characterized by peptide mass fingerprinting and peptide sequencing via Edman degradation, a total of 5 self-cutting/hydrolysis recognition sites were identified, which were positioned at various points across the full length Tpr protein sequence. These cleavage events were identified to occur between: Ser11-Lys12, Gly62-Met63, Gln163-Lys164, Thr191-Lys192, and Gly278-Ser279. In summary, my results revealed a complex picture for Tpr proteolytic selfprocessing. The biological implications remain to be firmly established.