Alanine Scan of the Cry5B Toxin Domain Identifies Variants with Increased Activity Against the Nematodes Caenorhabditis elegans and Ancylostoma ceylanicum
Soil-transmitted helminths infect more than 2 billion people worldwide and only one drug (albendazole) is commonly available for use against intestinal parasite worms under conditions for mass drug administration. Recent studies have shown efficacy issues with this drug, stressing the importance of finding a new treatment option. Three-domain crystal (Cry) proteins produced from the soil bacterium Bacillus thuringiensis have been used for decades as a means to control insects that destroy crops and transmit human diseases, and studies have shown many of these proteins to be safe to humans. Our lab has shown that the crystal protein Cry5B is able to kill both the free-living nematode Caenorhabditis elegans, as well as parasitic roundworms (e.g., Ancylostoma ceylanicum, hookworm). Cry proteins intoxicate invertebrates by acting as pore-forming proteins. We believe that the nematode-Cry5B system has potential to unlock mysteries surrounding Cry proteins and to help improve Cry5B as a therapeutic agent.
Here, I took a large scale systematic alanine-scanning approach and attempted to mutate all 698 amino acids in the toxin domain of Cry5B. I initially tested these mutants on C. elegans to assess for changes in toxicity levels, screening for variants with an increase in activity as compared to the wild type. Both the larger mutant set and the subset that had increased activity in my experiments are promising for future study, although the increased activity of the subset will need to be confirmed in assays with C. elegans and intestinal parasites. In the course of these studies, comparisons were made regarding the utility of CryB expressed in E coli, as purified protein, and as a spore/crystal mixture for activity assays and rescreening. Experiments were also carried out to address which system is best suited to test these variants in narrowing down those of interest and in predicting therapeutic potential in vivo. Overall, the Cry protein variants and approaches to their study described in this thesis hold promise for the development of therapeutics for treating one of the most neglected diseases of our time, parasitic worms.