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

by Katelyn E. Carothers Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an exclusively human bacterial pathogen and colonizer. It is the causative agent of diseases in humans ranging from mild, including pharyngitis and impetigo, to severe, such as necrotizing fasciitis and rheumatic fever. GAS is highly successful due to the production of many surface-expressed and secreted factors, including the cysteine protease SpeB. While this protease has long been recognized for its broad proteolytic activity against both host and streptococcal substrates, the relationship between SpeB and GAS pathogenesis has not been fully elucidated. Many speB deletion studies using different GAS strain backgrounds, sites of infection, and model organisms show different infection outcomes, demonstrating that the relationship of SpeB with virulence is highly dependent on infection context. For our work here, we utilized the GAS strain AP53CovS+, which is skin-trophic and non-invasive, and generated a speB deletion mutant in this background. We first examined the effect of SpeB Katelyn E. Carothers production using this non-invasive GAS strain in a model of host skin infection, hypothesizing that the broad proteolytic activity of SpeB would cause host cell damage resulting in loss of viability. Our results indicated no significant difference in human keratinocyte cytotoxicity between our wild-type and speB deletion mutant infections, even in conditions optimized for SpeB production. Cytokine arrays to identify specific SpeB-dependent inflammatory processes showed initial increases in IL-6 during wild-type infection conditions over ΔspeB, though this result was not corroborated by specific ELISA testing and requires further investigation. Finally, 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. Although GAS-secreted cysteine protease SpeB has been investigated for its effects on the human host, studies on how this factor is utilized in polymicrobial environments are lacking. For our next set of studies, we utilized both recombinant and endogenous SpeB protease to evaluate antimicrobial and anti-biofilm properties against the human pathogen and nasal and skin colonizer Staphylococcus aureus, which occupies similar niches to GAS and causes severe and highly antibiotic resistant infections in humans. 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 Katelyn E. Carothers disruption. Indeed, we found that SpeB cleaved SdrC in recombinant form and from S. aureus biofilms. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and polymicrobial dynamics.