The adenylate cyclase toxin (AC toxin) is necessary for disease caused by Bordetella pertussis, which has reemerged in the United States over the last two decades. AC toxin is expressed by seven of eight species of Bordetella which cause respiratory disease in humans and a range of animals. The toxin molecule is a unique hybrid of an adenylate cyclase enzyme and a binding domain homologous to the repeats-in-toxin (RTX) family of pore-forming bacterial protein toxins. AC toxin intoxicates host cells by binding to the cell membrane and translocating its catalytic domain across the lipid-bilayer, resulting in unregulated generation of intracellular cAMP. Prevention of translocation by an antibody to the catalytic domain or by certain mutations will enhance the other major function of the toxin, formation of oligomeric pores which cause lysis of erythrocytes and contribute to non-apoptotic cell death of macrophages. The magnitude of intoxication correlates with the relative surface expression of the (32 integrin, CD11b/CD18, a receptor expressed most abundantly on neutrophils and macrophages;however, the relationship between AC toxin and CD11b/CD18 is not well defined. Our preliminary data show that CD11b/CD18 does not simply increase sensitivity of cells to intoxication, but, when high concentrations of toxin are applied to cells, actually limits intoxication while possibly enhancing oligomer formation. We will investigate the basic mechanisms of the interaction between AC toxin and CD11 b/CD18. The determinants of toxin binding and function may explain a novel finding: cells of an epithelial monolayer are insensitive to intoxication by AC toxin applied to the apical surface, surprising because the toxin can intoxicate all cell types so far tested, and important because the bacterium first encounters these cells upon infection. These studies will shed light upon the mechanism of pore formation by bacterial toxins, and the pathogenesis of disease caused by all Bordetellae. The proposed project will continue my interest in basic science that began at the age of 19. My mentor, Dr. Erik Hewlett, with over 25 years of experience"""""""" studying Bordetella pertussis, is a preeminent scientist in the field of bacterial toxins. His emphasis on learning through scientific collaboration will complement my plan for structured coursework. The K08 award, in combination with the resources of my mentor and the University of Virginia, will provide me with the support necessary for my full development as a clinician scientist.
AC toxin is expressed by seven of the eight species of Bordetella and is also a member of the RTX family of pore-forming bacterial protein toxins that are produced by other pathogenic bacteria such as uropathogenic Escherichia coli. Here, we will characterize the basic mechanisms of cell binding by AC toxin and show that these mechanisms affect the function of the toxin and determine its role as a virulence factor.
