Group A Streptococcus (GAS, Streptococcus pyogenes) is an ongoing threat to human health as the agent of streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. In animal models of infection, macrophages have been shown to contribute to host defense against GAS infection. However, as GAS can resist killing by macrophages in vitro and induce macrophage cell death, it has been suggested that GAS intracellular survival in macrophages may enable persistent infection. Using isogenic mutants, we now show that the GAS pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase) mediate GAS intracellular survival and cytotoxicity for macrophages. Unexpectedly, the two toxins did not inhibit fusion of GAS-containing phagosomes with lysosomes but rather prevented phagolysosome acidification. SLO served two essential functions, poration of the phagolysosomal membrane and translocation of NADase into the macrophage cytosol, both of which were necessary for maximal GAS intracellular survival. Whereas NADase delivery to epithelial cells is mediated by SLO secreted from GAS bound to the cell surface, in macrophages, the source of SLO and NADase is GAS contained within phagolysosomes. We found that transfer of NADase from the phagolysosome to the macrophage cytosol occurs not by simple diffusion through SLO pores but rather by a specific translocation mechanism that requires the N-terminal translocation domain of NADase. These results illuminate the mechanisms through which SLO and NADase enable GAS to defeat macrophage-mediated killing and provide new insight into the virulence of a major human pathogen.

Group A Streptococcus (GAS) is a common human pathogen and the etiologic agent of a large number of diseases ranging from mild, self-limiting infections to invasive life-threatening conditions. Two prominent virulence factors of this bacterium are the genetically and functionally linked pore-forming toxin streptolysin O (SLO) and its cotoxin NAD+-glycohydrolase (NADase). Overexpression of these toxins has been linked to increased bacterial virulence and is correlated with invasive GAS disease. NADase can be translocated into host cells by a SLO-dependent mechanism, and cytosolic NADase has been assigned multiple properties such as protection of intracellularly located GAS bacteria and induction of host cell death through energy depletion. Here, we used a set of isogenic GAS mutants and a macrophage infection model and report that streptococcal NADase inhibits the innate immune response by decreasing inflammasome-dependent interleukin 1β (IL-1β) release from infected macrophages. Regulation of IL-1β was independent of phagocytosis and ensued also under conditions not allowing SLO-dependent translocation of NADase into the host cell cytosol. Thus, our data indicate that NADase not only acts intracellularly but also has an immune regulatory function in the extracellular niche.

The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence.