Streptolysin O and NAD-Glycohydrolase Prevent Phagolysosome Acidification and Promote Group A Streptococcus Survival in Macrophages

Abstract

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.

Contribution of Secreted NADase and Streptolysin O to the Pathogenesis of Epidemic Serotype M1 Streptococcus pyogenes Infections

“…Studies in the recent decade demonstrate SPN and SLO play important roles in circumventing the host immune system via i) inhibiting group A Streptococcus internalization by host cells, 3 ii) promoting apoptosis of macrophages, 29 iii) preventing the bactericidal effects of neutrophils, 7,8 iv) disrupting maturation of the phagosome, and v) promoting intracellular bacterial survival. 5,6,30 Recent advances in S. pyogenes population genomics and pathogenesis further underlined the important roles of SPN and SLO in virulence. We previously showed that polymorphisms in the nga upstream region result in enhanced production of SPN and SLO, which is strongly associated with increased transmission and virulence of epidemic M1 and M89 clones.…”

“…2e4 SLO is a cholesteroldependent cytolysin that forms pores in host cell membranes. SPN and SLO inhibit phagocytosis, 3 inhibit maturation of autophagosomes, 5,6 impair neutrophil oxidative burst, 7 and prevent the killing of S. pyogenes by a variety of host immune cells. 3,5e8 SPN and SLO are interconnected in many ways.…”

Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

“…In addition, Mga in M59 GAS influences the expression of an operon encoding streptolysin O (SLO) and NAD-glycohydrolase (NADase) and its intracellular inhibitor immunity factor for Streptococcus pyogenes NADase (IFS). Both SLO and NADase are proven virulence factors and have been implicated in intracellular survival and GAS resistance to macrophage-mediated cytotoxicity (48)(49)(50)(51)(52). Beyond the core regulon, genes regulated by Mga in the two serotypes diverge significantly and point toward differing metabolic capabilities of the two serotypes.…”

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes

“…S. pyogenes expresses a large arsenal of virulence factors (1, 2), many of which are well studied. These include antiphagocytic hyaluronic acid capsule (3), the cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO) (4,5), and a potent extracellular cysteine protease, SpeB (6,7). These virulence factors are regulated by the CovR/CovS two-component regulatory system (8)(9)(10).…”