Virulence induction of Streptococcus milleri group by periodontal tissue proteins

The Streptococcus milleri group (SMG; S. constellatus, S. intermedius and S. anginosus) are commensal bacteria of humans which are also associated with purulent dental, liver and brain abscesses. The pathogenic mechanisms leading to infection are unclear, but the interaction of these bacteria with connective tissue components is hypothesised to contribute to the induction of virulence factors leading to disease. Amongst the many putative virulence factors of these bacteria are a range of extracellular enzymes and toxins with the ability to degrade host tissues. Objectives: To identify the influence of extracellular matrix proteins on the expression of virulence factors by SMGs. Methods: Rat periodontal ligament cells were grown to confluence and extracellular matrix proteins (PDL-ECM) synthesised were harvested from the media following 2-3 days of further culture in DMEM, 0.2% FBS. 8 strains of SMG representing both disease and commensal isolates from oral and extra-oral sites, were cultured to mid-log phase in the presence/absence of 250mg/ml PDL-ECM. Bacterial supernatants were collected and protein profiles examined by SDS-PAGE. The API-ZYM system was used to determine the production of 19 hydrolytic enzymes for each bacterial isolate. The expression of the intermedilysin gene by S. intermedius was also determined using RT-PCR. Results: SDS-PAGE analysis revealed an extra band (55kDa) for each isolate cultured in the presence of PDL-ECM components. SMG isolates cultured in the presence of the PDL-ECM also exhibited higher levels of b-galactosidase, a-glucosidase, and N-acetyl-b-glucosaminidase. For the majority (75%) of S. intermedius isolates, intermedilysin mRNA expression was significantly increased for isolates grown in the presence of PDL-ECM. This was particularly pronounced for commensal S. intermedius strains. Conclusion: The interaction of SMG with PDL-ECM appears to contribute to the induction of virulence factors. The results therefore demonstrate the importance of bacterial matrix interaction as a pathogenic mechanism leading to establishment of disease.