Poster Presentation 20th Lancefield International Symposium on Streptococci and Streptococcal Diseases 2017

Context-specific effects of the chemokine cleaving enzyme SpyCEP on neutrophil clearance of S. pyogenes (#199)

Max Pearson 1 , Jennifer Goldblatt 1 , Mark Reglinski 1 , James E Pease 1 , Shiranee Sriskandan 1
  1. Imperial College London, London, LONDON, United Kingdom

Background The Streptococcus pyogenes cell envelope protease (SpyCEP) cleaves the chemokine CXCL8 (IL-8) and all ELR+ CXC chemokines. Reduced neutrophil recruitment is linked with increased bacterial burden and tissue necrosis. SpyCEP over-expression can be associated with an absence of neutrophils or with neutrophil death at sites of infection; we set out to examine the effects of SpyCEP on neutrophils and S. pyogenes clearance.

Methods Recombinant S. pyogenes strains lacking the SpyCEP cell wall anchoring domain were used to express soluble SpyCEP.  SpyCEP was purified by affinity chromatography using an anti-SpyCEP antibody loaded sepharose column, and by His-tag modification and nickel affinity chromatography. Activity of SpyCEP and inhibitors was assessed by IL-8 cleavage assay. The gene encoding SpyCEP, cepA, was disrupted by allelic replacement in contemporary emm 1 clinical isolates; a parent strain, an isogenic covR mutant, and a covS mutant. The impact of SpyCEP on bacterial and neutrophil survival was assessed.

Results The yield of purified SpyCEP was approximately 200 µg /litre and activity was 0.1ng CXCL8/h/ng SpyCEP. SpyCEP had complex effects on human neutrophils dependent on context; necrosis was observed only for purified SpyCEP.  Although uptake of Lactococcus lactis by neutrophils was impaired by SpyCEP expression, expression of SpyCEP by emm1 S. pyogenes did not affect uptake, except in the setting of covR mutation.

Conclusion A spectrum of compensatory virulence mechanisms is employed by emm 1 S.pyogenes to resist opsonophagocytic killing. The impact of SpyCEP on neutrophil uptake, survival, and bacterial clearance depends on context.