Targeting the streptococcal virulence factor SpyCEP as a novel therapeutic approach

Abstract

The Streptococcus pyogenes cell envelope protease (SpyCEP) is a crucial virulence factor involved in the immunomodulation of the innate immune response to S. pyogenes infection. SpyCEP rapidly and efficiently cleaves the entire family of ELR+ CXC chemokines, resulting in a chronic paucity of infiltrating neutrophils to the site of S. pyogenes infection. As such, expression of SpyCEP is highly associated with driving invasive disease and poor clinical outcome in streptococcal associated diseases, such as necrotizing fasciitis. The aim of this thesis was to determine the need for inhibition of SpyCEP activity, and to then identify potential inhibitors and characterise their activity, both in vitro and in vivo, as an adjunctive treatment in invasive streptococcal infection. This required further interrogation of the role of SpyCEP in streptococcal disease and exploration of novel aspects of SpyCEP function. The work has provided the first quantification of SpyCEP production in vivo, in murine models of S. pyogenes infection and in human samples from clinical S. pyogenes infection. Bioactive SpyCEP was detected and quantified in patient samples, and it was demonstrated that enzyme activity is independent of bacterial viability. Biochemical and structural tools were leveraged to analyse multiple recombinant SpyCEP constructs. The work provided the first evidence that the C-terminal domain of SpyCEP is necessary for CXCL8 substrate binding and attributed novel proteolytic activity to the C-terminus alone, independent of the N-terminus. A novel kinetic assay was employed to quantify and compare activity of different SpyCEP constructs and demonstrated the C-terminal activity to be 1500-fold less efficient than full length SpyCEP.