Abstract
Streptolysin O (SLO) is a cholesterol-dependent cytolysin produced by the important human pathogen, group A Streptococcus (Streptococcus pyogenes or GAS). In addition to its cytolytic activity, SLO mediates the translocation of GAS NAD-glycohydrolase (NADase) into human epithelial cells in vitro. Production of both NADase and SLO is associated with augmented host cell injury beyond that produced by SLO alone, but the mechanism of enhanced cytotoxicity is not known. We have now shown that expression of NADase together with SLO dramatically enhanced the lytic activity of GAS culture supernatants for erythrocytes but had no effect on SLO-mediated poration of synthetic cholesterol-rich liposomes. This result revealed a previously unknown contribution of NADase to the cytolytic activity associated with GAS production of SLO. Purified recombinant SLO bound NADase in vitro, supporting a specific, physical interaction of the two proteins. Exposure of human keratinocytes to wild-type GAS, but not to a NADase-deficient mutant strain, resulted in profound depletion of cellular NAD+ and ATP. Furthermore, expression of recombinant GAS NADase in yeast, in the absence of SLO, induced growth arrest, depletion of NAD+ and ATP, and cell death. These findings have provided evidence that the augmentation of SLO-mediated cytotoxicity by NADase is a consequence of depletion of host cell energy stores through the enzymatic action of NADase. Together, the results have provided mechanistic insight into the cytotoxic effects of a unique bipartite bacterial toxin.
Highlights
NAD-glycohydrolases are enzymes that catalyze the hydrolysis of the nicotinamide-ribose bond of NADϩ to yield nicotinamide and adenosine diphosphoribose
By performing more detailed testing, we found that the NADase mutant strain 771ngaϪ produces similar amounts of Streptolysin O (SLO) hemolytic activity as wild-type strain 771 during early exponential phase but substantially less than strain 771 during later phases of growth (Fig. 1A)
These results raised the possibility that the attenuated cytotoxic effects of 771ngaϪ could be attributable, at least in part, to reduced SLO cytolytic activity in this mutant strain
Summary
Bacterial Strains and Culture Conditions—M-type 3 GAS strain 950771 (referred to hereafter as 771) and its isogenic mutants 771ngaϪ and 771sloϪ have been described [20]. GAS was grown at 37 °C in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (Difco). Escherichia coli DH5␣ or TOP10 (Invitrogen) was grown in Luria-Bertani medium [21]. Antibiotics were added at the following concentrations: ampicillin, 50 g/ml; kanamycin, 50 g/ml
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