Cholera, and related diarrheal diseases, are the #3 cause of all deaths worldwide. Both cholera and the less virulent """"""""travelers diarrhea"""""""" result from bacterial infections (by Vibrio cholerae and E. coli, respectively) and colonization of the gut where exotoxins are produced that cause all of the clinically important symptoms of the diseases. Cholera toxin (CT) and E. coli heat labile toxin (LT) are structurally (>80% identity), immunologically, and mechanistically very closely related. The key pathophysiological step in this process, the ADP-ribosylation of Gs, can be performed in vitro using purified components and requires a proteinaceous co-factor, termed the ADP-ribosylation factor or Arf. Arfs are ubiquitous, essential, GTP-binding regulators in eukaryotes of most (perhaps all) steps of vesicular membrane transport and are potent activators of phospholipase D. Entry of the CT/LT toxins occurs through a """"""""reverse secretory"""""""" pathway from endosomes to Golgi to ER and back out to Golgi, where A and B subunits dissociate and the active A subunit presumably enters the cytosol. These surprising observations, that this regulator of membrane traffic, Arf, is required for ADP-ribosylation by the toxins in vitro and that the ADP-ribosylation toxins must transit the length of the secretory pathway before gaining access to the cytosol has prompted this detailed examination of the relationship between Arf and CT/LT. Another reason for doing so comes from the recent observations that CT/LT are very potent mucosal adjuvants, with potential applications against a host of human pathogens, including sexually transmitted viruses, e.g., herpes, papilloma, and HIV. The development of toxin based adjuvants has been limited by the toxicity of the toxins alone. The principal goal is to determine the importance of Arf binding to toxins in intact cells with respect to the activation of adenyl cyclase and to the mode of entry of toxins into cell cytosol. Finding mutants that retain adjuvant properties but reduced or absent cellular toxicities may prove essential to the development of these agents as adjuvants. This proposal has three specific aims: (1) Create and characterize point mutants of LTA that have partial or complete loss of the ability to bind and be activated by human Arf proteins. Results will begin to define the LTA-Arf binding surface. (2) Co-crystallize the Arf- GTP-LTA complex and solve the structure. Preliminary data make this a feasible approach and offer a rapid advance in our understanding of this protein interaction. (3) LT/CT mutants, defective in the ability to interact productively with Arf, will be examined for their ability to activate adenyl cyclase, transit the """"""""reverse secretory"""""""" pathway, localize to appropriate intracellular locales, and intoxicate mammalian cells. These studies will address for the first time the relevance of toxin-Arf interaction in live cells and provide important new mechanistic and structural information on this protein interaction that can either support or refute the potential use of loss-of-Arf-binding mutants of LT or CT as adjuvants, as antigens in vaccine development, or other uses.
| Amor, J C; Horton, J R; Zhu, X et al. (2001) Structures of yeast ARF2 and ARL1: distinct roles for the N terminus in the structure and function of ARF family GTPases. J Biol Chem 276:42477-84 |
