Bacteria rely on surface proteins to interact with solid surfaces, other microorganisms, and, importantly for pathogens, eukaryotic hosts. Gram-negative bacteria, which have complex diderm cell envelopes, have evolved specialized secretion systems to export proteins from the cytoplasm to the cell surface. My research focuses on understanding molecular mechanisms underlying the broadly distributed Two Partner Secretion (TPS or Type Vb) pathway, which includes many important virulence factors and interbacterial contact-dependent inhibition systems.
TPS systems consist of an Omp85 TpsB transporter that transports a large TpsA exoprotein across the Gram-negative outer membrane. The best studied TPS system is FhaB/FhaC, produced by Bordetella species such as B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory disease in a wide range of mammals. The TpsA protein FhaB mediates adherence to host cells, suppresses the initial inflammatory response, and is required for resistance to killing by phagocytic cells by an unknown mechanism. Examination of the FhaB secretion pathway, its topology on the bacterial surface, and interactions between FhaB and another virulence factor –Bordetella’s adenylyl cyclase toxin (ACT)– have led us to hypothesize that FhaB and FhaC protect the bacteria from phagocytes by acting as a toxin delivery system.
Here, we investigated key aspects of this model. We performed Western blots on WCLs and supernatants collected from wildtype (WT) B. bronchiseptica and mutant strains lacking ACT (∆cyaA) and determined that the loss of ACT is correlated with increased degradation of FhaB, indicating that ACT binding stabilizes FhaB on the bacterial surface. We developed a surface-bound toxin delivery assay by decorating FhaB on recipient ∆cyaA bacteria (that cannot produce their own ACT) with ACT from donor cells and used cyclic-AMP ELISAs to determine that the FhaB-bound toxin is delivered to eukaryotic cells in vitro.