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

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 γ-chain peptide capable of occupying the DLL trench. Using cell-free click display and kinetics-guided affinity maturation, we engineer DARPin-γ-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.