Investigation of the interaction of Bordetella FhaB adhesin with microtubules

Pertussis or whooping cough, despite widespread vaccination, remains a major human health problem. Pertussis is highly infectious and is most dangerous for newborns, where 1/3 of infected infants under one year old are hospitalized and one in a hundred of hospitalized babies die. In the last decades, we have seen a resurgence in pertussis cases, which is believed to stem from decreased efficacy of the vaccine. The current vaccine is composed of cellular components of the causative agent of pertussis, Bordetella pertussis; a major component is the protein Filamentous hemagglutinin protein (FHA). FHA is a virulence factor in Bordetella and is essential for adherence and colonization in human hosts. FHA is the mature form of FhaB, following the loss of a signal peptide and FhaB’s C- terminal region whereby it was proposed that this C-terminal region had no function. Notably, FhaB has striking similarities to CdiA, the toxin carrying component of contact dependent growth inhibition (CDI), whereby the C-terminal toxin domain of which is translocated into target bacterial cells upon contact. Our recent work has shown that extreme C-terminal of FhaB (FhaB-CT) is delivered into human cells, where it acts as a microtuble (MT) binding protein. Thus, FhaB-CT likely has a role in Bordetella virulence. While FhaB is translocated into eukaryotic cells and CdiA is translocated into bacterial cells; these similarities lead us to hypothesize that FhaB is part of an uncharacterized anti-eukaryotic CDI system. This proposal aims to characterize the interaction of FhaB-CT with MTs and identify other Gram-negative bacterial FhaB-CT domains with anti-eukaryotic activities. First, we will determine the structure of the complex of FhaB-CT with MT using cryo-EM. Secondly, we will identify other anti-eukaryotic FhaB-CT domains through in silico analysis. We will test these domains for eukaryotic entry and toxin/adhesion activity. The results of this study will (1) lay the groundwork to further explore Bordetella virulence and the activity of B. pertussis FhaB-CT, and (2) identify other FhaB-CT proteins that target eukaryotic cells and characterize their activity. Not only could these results aid the development of a new pertussis vaccine, but with an improved understanding of the interaction between FhaB- CT and MTs we may also offer a new target for pertussis therapeutic development. Lastly, with the discovery of FhaB-CT activity and a novel anti-eukaryotic CDI system, we have found a new branch of CDI systems with direct correlation to human health that require investigation.