Chromosomal markerless integration of anthelmintic Cry proteins into the Bacillus thuringiensis genome

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that during sporulation produces insecticidal Crystal (Cry) proteins, which play a major role in insect control today. Some Bt Cry proteins, e.g., Cry5Ba, target nematodes and, when given orally, can cure animals of gastrointestinal nematode (GIN) parasites. To eliminate concerns about treating humans and animals with spores and live bacteria, we developed an asporogenous system for scalable and safe Cry protein delivery called IBaCC (Inactivated Bacteria with Cytosolic Crystal(s)), which results in production of a bioactive crystal and a dead bacterium. However, to date, IBaCC involves expression of Cry proteins from antibiotic-selectable plasmids to ensure maintenance. Here, we develop and validate tools for markerless and stable integration and expression of Cry proteins in Bt. We markerlessly integrate an expression construct for Cry5Ba into either the spo0A or the sigK locus and demonstrate robust Cry5Ba expression. We also integrate our Cry5Ba expression construct into both loci simultaneously, increasing expression further. We demonstrate that an expression construct for a second anthelmintic Cry protein, Cry21Aa, can be integrated either alone or in combination with Cry5Ba in a single Bt strain. We furthermore show that these markerless integrants are stable in the absence of a selectable marker. These integrated strains, processed to IBaCC, demonstrate excellent ex vivo nematicidal bioactivity toward the larval stages of the sheep GIN parasite Haemonchus contortus and adult stages of the human hookworm GIN parasite Ancylostoma ceylanicum . This study demonstrates the successful markerless integration of 1-2 identical or dissimilar Cry proteins into Bt. These Cry integrants, in which genes essential to sporulation are deleted or replaced, provide robust Cry expression, stability, and bioactivity. These studies represent an important advance in Bt genetics and toward a safe, deployable, and cost-effective anthelmintic therapy to treat GIN parasitic infections in humans and animals.