Activity of Intermedilysin expressed by Streptococcus intermedius against oral keratinocytes

Streptococcus intermedius is an oral bacterium that expresses a human-specific cytotoxin, Intermedilysin (ILY). S. intermedius strains expressing high levels of ILY have been shown to be associated with deep-seated abscesses. Objectives: The aim of this research is to determine the susceptibility of oral epithelial cells to damage by ILY at lethal and sublethal concentrations and to measure target cell responses. Methods: Human oral keratinocyte cells (TR146) were treated with purified ILY at sub-lethal and lethal concentrations (0.0125µg/ml-1µg/ml) for 1h or 2h time periods. Cell viability and cell damage were assessed using 3-(4,5-dimethyl-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and trypan blue uptake whereas recovery of cells was determined using the AlamarBlue™ fluorescent dye system. Cytokines (IL-1α and IL-8) were measured in supernatants taken from TR146 cells using ELISA. Results: A dose dependent decrease in cell viability was observed with increasing ILY concentration (0.0125µg/ml-1µg/ml) which was accompanied by an increase in both LDH release and trypan blue uptake. IL-1α release increased with increasing ILY concentration and exposure time. Conversely, the highest levels of IL-8 release (500pg/ml) occurred with the lower concentration of ILY (0.0125µg/ml). Cells monitored up to 24h post ILY treatment with lower concentrations (≤ 0.05µg/ml) were able to recover in our system. Conclusions: These results demonstrate that ILY is able to cause irrevocable damage to TR146 cells at the higher concentrations used (≥ 0.05 µg/ml). However, at lower concentrations ILY is able to cause an up regulation of the neutrophil recruiting cytokine IL-8 whilst causing minimal changes to all other parameters measured. These results have implications for the pathogenesis of deep seated abscesses by S. intermedius.