Dual-mode ClfA-targeting DARPin biologics protect against diverse methicillin-resistant Staphylococcus aureus strains

Abstract Staphylococcus aureus uses the adhesin clumping factor A (ClfA) to bind fibrinogen and promote invasive infection through two distinct interfaces: an exposed, low-affinity site on the N3 head domain and a buried, high-affinity “dock, lock, and latch” (DLL) trench that is exposed only under shear. This dual-interface architecture allows limited antibody penetration, as antibodies typically block only the exposed site. Here, we establish a dual-mode inhibition strategy that overcomes this constraint by combining a high-affinity ClfA-binding designed ankyrin repeat protein (DARPin) with a fibrinogen v-chain peptide capable of occupying the DLL trench. Using cell-free click display and kinetics-guided affinity maturation, we engineer DARPin–v-peptide fusion biologics that simultaneously block both fibrinogen-binding interfaces. These molecules inhibit ClfA–fibrinogen interactions, prevent methicillin-resistant S. aureus agglutination in human plasma, neutralize major clinical ClfA variants, and confer Fc-independent protection in a lethal murine bacteremia model. This work provides a strategy for targeting antibody-intractable force-activated staphylococcal adhesins. Competing Interest Statement Z.C., K.C. and Y.Z. filed a provisional patent application to protect the DARPin molecules and methods of Fg-γ peptide fusion and tetramerization presented in this study. D.M. is a founder of ImmunArtes LLC., a University of Chicago start-up company that seeks to develop immune therapeutics against S. aureus. The other authors declare no competing interests. Footnotes Subject area categorization modified from Molecular Biology to Microbiology; no changes were made to the manuscript. Abbreviations - ClfA - clumping factor A; - bClfA - biotinylated ClfA - MRSA - Methicillin-resistant S. aureus - DLL - dock, lock and latch - Fg - fibrinogen - MSCRAMM - microbial surface components recognizing adhesive matrix molecule - BLI - biolayer interferometry - p53-TD - p53 tetramerization domain;