Group A Streptococcus Enn Proteins Bind Collagen through a New PARF Motif: A Potential Role in Acute Rheumatic Fever

Background: Rheumatic heart disease (RHD), a sequela of acute rheumatic fever (ARF) following Group AStreptococcus (GAS) infection, is driven by autoimmune responses against host tissues. Collagen binding by bacterial proteins is a key proposed mechanism, notably, through the peptide associated with rheumatic fever (PARF) motif found in a subset of M proteins.Methods: We searched for PARF motifs in M and M-like proteins from GAS and Streptococcus dysgalactiaesubsp. equisimilis (SDSE) isolates. Enn proteins were tested for collagen binding by ELISA. Rabbit immunization with Enn-derived peptides were conducted to evaluate collagen reactivity and autoantibody generation.Findings: We identified a new PARF motif in Enn proteins from clusters SG1 and SG2. Functional assays and site-directed mutagenesis demonstrated that only SG1/2 Enn proteins bind human collagen through this motif. SG1/SG2-derived peptides led to anti-collagen antibodies induction in rabbits. Retrospective analysis of GAS strains repertoire suggested that up to 32% could encode PARF-containing Enn proteins. Expanded motif prediction revealed additional potential collagen-binding candidates in both GAS and SDSE. Overall, our data suggests that 15-47% of global GAS isolates may express a PARF-containing protein.Interpretation: These findings expand the repertoire of GAS and SDSE proteins inducing autoimmunity through PARF motifs. The immunopathological potential of Enn proteins warrants consideration in vaccine development targeting GAS, especially in high-burden settings.