The NADase-Negative Variant of the Streptococcus pyogenes Toxin NAD⁺ Glycohydrolase Induces JNK1-Mediated Programmed Cellular Necrosis.

Abstract

ABSTRACTVirulence factors are often multifunctional and contribute to pathogenesis through synergistic mechanisms. For the human pathogen Streptococcus pyogenes, two factors that act synergistically are the S. pyogenes NAD+ glycohydrolase (SPN) and streptolysin O (SLO). Through distinct mechanisms, SLO forms pores in host cell membranes and translocates SPN into the host cell cytosol. Two natural variants of SPN exist, one that exhibits NADase activity and one that lacks this function, and both versions are translocated and act in concert with SLO to cause an accelerated death response in epithelial cells. While NADase+ SPN is known to trigger a metabolic form of necrosis through the depletion of NAD+, the mechanism by which NADase− SPN induces cell death was unknown. In the studies described here, we examined the pathway of NADase− cell death through analysis of activation patterns of mitogen-activated protein kinases (MAPKs). S. pyogenes infection resulted in activation of members of three MAPK subfamilies (p38, ERK, and JNK). However, only JNK was activated in an SLO-specific manner. NADase− SPN induced necrosis in HeLa epithelial cells associated with depolarization of mitochondrial membranes, activation of NF-κB, and the generation of reactive oxygen species. Remarkably, RNA interference (RNAi) silencing of JNK protected cells from NADase−-SPN-mediated necrosis, suggesting that NADase− SPN triggers a form of programmed necrosis dependent on JNK signaling. Taken together, these data demonstrate that SPN acts with SLO to elicit necrosis through two different mechanisms depending on its NADase activity, i.e., metabolic (NADase+) or programmed (NADase−), leading to distinct inflammatory profiles.