A Puzzling Promiscuous Protease: Role of the Streptococcal SpeB Protein in Host Virulence and Polymicrobial Dynamics

Streptococcus pyogenes, or Group A Streptococcus (GAS) is both a pathogen and asymptomatic colonizer of human hosts, and produces a large number of surface-expressed and secreted factors. The GAS-secreted cysteine protease SpeB has been well studied for its effects on the human host, but its role is highly dependent on infection context. For our work, we utilized the GAS strain AP53CovS+, which is skin-trophic and non-invasive, and generated a speB deletion mutant. We first examined the effect of SpeB production using this non-invasive GAS strain in a model of host skin infection, hypothesizing that the proteolytic activity of SpeB would cause host cell damage and loss of viability. Our results indicated no difference in human keratinocyte cytotoxicity between our wild-type and speB deletion mutant infections, even in conditions optimized for SpeB production, and cytokine arrays to identify SpeB-dependent changes in inflammatory processes require further investigation. We assessed survival in a humanized mouse model after subcutaneous infection with GAS. Similar to the in vitro cytotoxicity studies, there was no difference in mouse survival between wt and ΔspeB infections. Overall, our findings do not support a role for SpeB in the virulence of AP53CovS+ GAS in models of skin and subcutaneous infection. Despite its broad proteolytic activity, studies on how SpeB is utilized in polymicrobial environments are lacking. We used both recombinant and endogenous forms of SpeB protease to evaluate antimicrobial and anti-biofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies similar niches to GAS. We demonstrate that SpeB exhibits potent biofilm disruption activity at multiple stages of S. aureus biofilm formation. We hypothesized that the surface expressed adhesin SdrC in S. aureus was cleaved by SpeB, which contributed to the observed biofilm disruption. Indeed, we found that SpeB cleaved recombinant SdrC in vitro and in the context of the full S. aureus biofilm. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and competition with other microorganisms in its niche.