De novo design of protein antagonists of bacterial pathogen type III secretion system needle assembly

Abstract The type III secretion system (T3SS), a nanoinjector apparatus utilized by Gram-negative bacterial pathogens to colonize host cells, represents a promising target for antibiotic-independent therapeutics. To date, molecules identified to associate with T3SS components have been largely limited to natural proteins, small molecules, and antibodies. Here, we report the rational design and characterization of de novo proteins that associate with the T3SS needle protein. We generate α-helical mini-proteins targeting polymerization hotspots of PrgI, the Salmonella Typhimurium T3SS needle protein. Twenty-four designs successfully recombinantly expressed, folded into stable α-helical bundles, and exhibited distinct oligomerization states. Binding to PrgI* was confirmed in solution, with equilibrium dissociation constants ranging from 1 to 150 µM. Several designs inhibited polymerization of PrgI* T3SS needle in vitro, and one construct was capable of disassembling preformed T3SS needles. Some designs also associated with homologous needle proteins, Burkholderia BsaL and Shigella MxiH, suggesting a potential for broad-spectrum T3SS needle targeting. Together, these results establish a molecular blueprint for the design of functional T3SS needle binding proteins, enabling future efforts to disrupt conserved secretion systems underlying bacterial pathogenesis.