Filamentous hemagglutinin (FHA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and is thought to function as an adhesin that facilitates bacterial attachment to host cells. FHA is a processed form of its precursor, FhaB, and it lacks a large C-terminal region found in FhaB. Our recent results lead us to hypothesize that this C-terminal region functions as a toxin/effector delivery device to modulate mammalian host cell function. The goal of this proposal is to test this hypothesis by examining the topology of FhaB before and after binding to target mammalian cells. Towards this goal, we will leverage new technologies recently developed by us. These methods include a heterologous and functional E. coli FhaB expression system that will allow us to assess FhaB-dependent binding and cellular intoxication and a fluorescent dye-based method that allows tracking of engineered FhaB proteins before and after bacterial binding to target mammalian cells. We will also tag putative toxin domains to determine if they enter target cells, and we will track them using microscopy and biochemical methods to follow their localization and fate. Because additional factors in Bordetella spp. could contribute to mammalian cell intoxication, we will complement our analyses with orthogonal approaches using wild-type and engineered Bordetella. In a second aim, we will assess the effects of FhaB fragments on host cell physiology, extending our preliminary studies that indicate that C- terminal fragments of FhaB induce human lung cells to change shape and lose viability. We will express fragments of FhaB in multiple pathogen-relevant cell types and study their effects on cellular physiology and cell viability. The information generated in this proposal has direct potential application to improving the FHA component of the vaccine by including portions of the FhaB C-terminal toxin delivery system. Moreover, FhaB would be the first example of a single bacterial protein that can deliver toxins into eukaryotic cells, which might be potentially harnessed for therapeutic applications.
The goal of this proposal is to determine if bacterial pathogens, including the causative agent of whooping cough, use cell surface proteins to deliver toxins into target animal/human cells. If correct, this hypothesis will help us understand a new mechanism by which pathogenic bacteria intoxicate their hosts and evade the immune system. This new knowledge might potentially be used to design targeted antimicrobials and vaccines that exploit specific molecular features of the toxin delivery pathway.
