The effect of Treponema denticola on Porphyromonas gingivalis phenotypes and transcriptome

© 2018 Dr. Lin Xin KinChronic periodontitis is an inflammatory, bacterial biofilm-associated disease resulting in destruction of the tooth’s supporting tissues. The imminent progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Living in a complex oral polymicrobial community, these two bacterial species display close association via physical interaction and metabolic cooperativity in the biosynthesis and cross-feeding of growth substrates. These interspecies interactions result in the coordination of their physiological activities, some of which exhibit complementary and combinatory effects in enhancing their growth and virulence factors. A previous study demonstrated that coculture of T. denticola and P. gingivalis in a continuous system led to upregulation of T. denticola glycine utilisation systems. Likewise, P. gingivalis increased the production of free glycine by proteolytic hydrolysis of peptide-bound glycine during growth in T. denticola conditioned medium (TdCM), suggesting cross-feeding of glycine from P. gingivalis to T. denticola. Free glycine is an important nutrient source for T. denticola, contributing to a dramatic increase in T. denticola growth rate and biomass. Given that P. gingivalis glycine release was stimulated in TdCM, this study aimed to determine and characterise the T. denticola stimulatory factors that had been released into TdCM. TdCM was fractionated by size filtration and reversed-phase high-performance liquid chromatography (RP-HPLC) to determine the most active stimulatory fractions. The free glycine produced by P. gingivalis in different TdCM fractions was then quantified using a glycine enzyme-linked immunosorbent assay (ELISA) kit and liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS). As several RP-HPLC fractions of TdCM appeared to stimulate the release of free glycine by P. gingivalis, this indicated that there was no specific signalling molecule that stimulated P. gingivalis to produce glycine. Instead, the release of glycine by P. gingivalis was likely due to P. gingivalis proteinases that further digested the peptides that had been partially processed by T. denticola proteases during growth in the medium. Candidate peptidases of P. gingivalis that could be involved in the hydrolysis of glycine-containing peptides into free glycine were selected by bioinformatic predictions of the localisation and specificities of P. gingivalis putative peptidases. Interestingly, inactivation of PG0753 and PG1788 that encode putative PrtQ collagenase and C1 family cysteine peptidase respectively, showed a reduction in the rate of free glycine production relative to P. gingivalis calculated cell number in OB:CM, relative to wild type. These results indicated that these two peptidases might play a role in the release of free glycine by P. gingivalis in the presence of T. denticola stimulatory factors. Other potential multimodal interactions of T. denticola and P. gingivalis were examined by investigating the differential gene expression profiles of P. gingivalis during growth in TdCM, compared to oral bacterial growth medium (OBGM). There were a total of 132 genes that showed differential expression, which included transcripts related to metabolic pathways, signal transduction systems, transcription regulatory systems, nucleic acid interacting protein encoding genes, transporters and hypothetical protein encoding genes. This work generated more testable hypotheses of the molecular effects of T. denticola on P. gingivalis gene expression and as well as potential mechanisms that might contribute to P. gingivalis and T. denticola physiological interactions