Effects of Cytotoxin (Intermedilysin)-producing Streptococcus intermedius on Neutrophil Function

Objectives: Flow cytometric investigation into the effects of cytotoxin (intermedilysin: ILY) producing pyogenic bacterium Streptococcus intermedius strain UNS38 and ily^- gene knockout mutant UNS38B3 on neutrophil function and viability. Methods: Mid-exponential phase bacteria were washed, resuspended in Hanks Balanced Salt Solution (HBSS) and cell numbers determined. 100µl fresh whole blood and bacteria were mixed (bacteria : neutrophil ratios of 1:1, 10:1, 20:1 or 50:1) in 1 ml total vol. and incubated at 37^oC. Neutrophil activation was determined from increased forward light scatter at 488nm. Metabolic burst was measured by increased fluorescence due to 2,7-dichlorofluorescein diacetate (DCFDA). Loss of integrity of the neutrophil cell membrane was measured by propidium iodide (PI) influx. Bacterial uptake by neutrophils was determined by measurement of neutrophil associated fluorescence from live/dead stained bacteria. Reduction in neutrophil numbers was measured using a cell counting kit. Results: Neutrophil activation and metabolic burst were observed to increase in a dose-dependent manner with either the ILY⁺ parent or the ILY^- mutant although consistently to a greater degree with the former. Significant increase (p<0.01) in PI uptake was observed for the ILY⁺ parent strain compared with the ILY^- mutant and a 30% reduction in neutrophil numbers occurred after 20 mins exposure to the ILY⁺ parent strain compared to 0% reduction with the ILY^- mutant. No significant difference was observed in the uptake of either ILY⁺ or ILY^- S. intermedius cells by neutrophils. Conclusions: Cell-associated ILY is a potentially important virulence factor causing reduction in neutrophil numbers probably through loss of membrane integrity and lysis. At sublethal levels ILY modulates neutrophil function by increasing activation and respiratory burst with ILY-producing bacteria being phagocytosed. Further work is required to fully elucidate the role of ILY in the virulence of S. intermedius in abscesses.