DNase Sda1 Allows Invasive M1T1 Group A Streptococcus to Prevent TLR9-Dependent Recognition

Abstract

Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

Author Summary

Group A Streptococcus (GAS) ranks among the top ten human pathogens causing fatal disease. GAS possesses an arsenal of virulence factors that circumvent the primary mammalian defence strategies, the innate immune system. Toll-like receptors (TLRs), allow the host to detect pathogens by recognizing structures or patterns abundant in pathogens but lacking in the mammalian host, including unmethylated CpG-rich bacterial DNA recognized by TLR9. Here we show that GAS DNA but not host DNA triggers TNF-α and interferon type 1 cytokine secretion by monocytic cells, and that this secretion is dependent on the presence of functional TLR9. The highly virulent M1T1 GAS clone expresses the virulence factor DNase Sda1. Sda1-mediated bacterial DNA degradation was shown to prevent TLR9-dependent cytokine release in monocytes, which then fail to effectively phagocytose and kill bacteria. In a mouse necrotizing fasciitis model, the streptococcal DNase Sda1 suppressed TLR9-dependent INF-α and TNF-α induction. Inhibition of TLR9 recognition by a bacterial DNase thus illustrates a novel mechanism of microbial innate immune evasion.