Abstract:
<i>Streptococcus pyogenes</i>, a leading human pathogen, is responsible for a wide range of diseases, including skin and soft tissue infections and severe invasive diseases. <i>S. pyogenes</i> produces a large arsenal of virulence factors, including several immune evasion factors. We have identified an open reading frame (<i>spy0136</i>) in the <i>S. pyogenes</i> SF370 genome encoding a protein of unknown function. Using recombinant Spy0136 in a pull-down assay with human plasma and ELISA, we have identified four complement proteins (C1r, C1s, C3, and C5) as binding partners. Treatment of the complement proteins with PNGase F abrogated binding to C1s, C3, and C5, indicating glycan-dependent interactions. rSpy0136 inhibited complement-mediated hemolysis and interfered with all three complement pathways in a Wieslab complement assay. Furthermore, rSpy0136 inhibited deposition of the C3b opsonin and the membrane attack complex (MAC) on the surface of <i>S. pyogenes</i>. We therefore named the previously unknown protein 'complement evasion factor' (CEF).An <i>S. pyogenes Δspy0136/cef</i> deletion mutant showed decreased virulence in an <i>in-vitro</i> whole blood killing assay and a <i>Galleria mellonella</i> (wax moth) infection model. Furthermore, an <i>L. lactis spy0136/cef</i> gain-of-function mutant showed increased survival during growth in whole human blood. Analysis of serum samples from patients with invasive <i>S. pyogenes</i> revealed Spy0136/CEF sero-conversion indicating expression during disease. In summary, we have identified a novel <i>S. pyogenes</i> immune evasion factor that binds to several complement proteins to interfere with complement function. This is the first example of a <i>S. pyogenes</i> virulence factor binding to several different target proteins via glycan-dependent interactions.