Structural determinants in the Staphylococcus aureus derived phenol-soluble modulin α2 peptide required for neutrophil formyl peptide receptor activation

Abstract

ABSTRACTHighly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), peptides which are formylated N-terminally. Nanomolar concentrations of PSMα2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSMα2 is a biased signaling agonist. A shortened N-terminal PSMα2 variant, consisting of the five N- terminal residues, is selectively recognized by the closely related FPR1, showing that the C- terminal part of PSMα2 confers FPR2 selectivity, while the N-terminal part may interact with the FPR1 binding site. In the present study, a combined pharmacological and genetic approach, involving primary neutrophils and engineered FPR “knock-in” and “knock-out” cells, was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides and the receptor downstream signaling induced by these peptides. In comparison to the full-length PSMα2, we show that the peptide in which the N-terminal part of PSMα2 was replaced by fMIFL (an FPR1- selective peptide agonist) potently activates both FPRs for production of superoxide anions and β- arrestin recruitment. A shortened analogue of PSMα2 (PSMα21-12), lacking the nine C-terminal residues activated both FPR1 and FPR2 to produce ROS, whereas β-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSMα21-12 was sufficient to alter FPR2 signaling to include β-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2 biased signaling. In conclusion, we provide novel structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSMα2, originating from Staphylococcus aureus bacteria.