The endopeptidase PepO regulates the SpeB cysteine protease and is essential for the virulence of invasive M1T1 Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus, GAS) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1-serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that 5448ΔpepO is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.Importance Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and non-invasive diseases in western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone, as well as being essential for virulence.