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.
IMPORTANCE: Macrophages constitute an important
element of the innate immune response to mucosal pathogens. They ingest and kill microbes by phagocytosis and secrete inflammatory
cytokines to recruit and activate other effector cells. Group A Streptococcus (GAS, Streptococcus pyogenes), an important cause of
pharyngitis and invasive
infections, has been shown to resist killing by macrophages. We find that GAS resistance to macrophage killing depends on the GAS pore-forming toxin
streptolysin O (SLO) and its cotoxin
NAD-glycohydrolase (
NADase). GAS bacteria are internalized by macrophage phagocytosis but resist killing by secreting SLO, which damages the phagolysosome membrane, prevents phagolysosome acidification, and translocates
NADase from the phagolysosome into the macrophage cytosol.
NADase augments SLO-mediated cytotoxicity by depleting cellular energy stores. These findings may explain the nearly universal production of SLO by GAS clinical isolates and the association of
NADase with the global spread of a GAS clone implicated in invasive
infections.