The Natural Variants of the Streptococcus NAD+-glycohydrolase SPN Modulate Divergent Host Cell Signaling Pathways and Death

Streptococcus pyogenes has an arsenal of virulence factors that promote its ability to cause a broad range of diseases and fight host defenses. One of these factors is the S. pyogenes NAD +-glycohydrolase (SPN), which is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death. Every strain examined to date has SPN and recent studies suggest that SPN is continuing to evolve diverging into two variants that correlate with tissue tropism, one of which lacks the signature NADase activity. SPN's roles in both cytotoxicity and niche selection are unknown. To gain insight into the forces driving the adaptation of SPN, a detailed comparison of representative glycohydrolase activity-proficient and -deficient variants was conducted.;Out of a total 454 amino acids, the glycohydrolase activity-deficient variants differed at only 9 highly conserved positions. A mutational approach to exchange these residues between variants revealed that reciprocal changes at 3 specific residues were required to both abolish activity of the proficient version and restore full activity to the deficient variant. Remarkably, all versions of SPN were equally cytotoxic to cultured epithelial cells. However, differences in glycohydrolase activity had a significant influence in cell signaling resulting in cellular necrosis and the subsequent inflammatory response.;Necrosis requires SLO, which activates JNK and PARP. However, in combination with the NADase active SPN cells underwent a glycolytic cell death through the depletion of NAD+ and ATP. As a consequence of active SPN, these cells were unable to accumulate PAR despite PARP activation. These effects were associated with the release of HMGB-1 from the nucleus and low levels of Il-8 and TNF alpha production. In contrast, the NADase inactive SPN accumulated PAR and underwent a JNK mediated cell death. These events likely contribute to the increased Il-8 and TNF alpha production seen in these infections however HMGB-1 is not released as in the NADase inactive infections.;Taken together, these data indicate that the glycohydrolase activity of SPN is not the only contribution the toxin has to the pathogenesis of S. pyogenes and that both versions of SPN play an important role during infection.