Investigating the specificity of Bacillus thuringiensis Cry41Aa toxin through mutagenesis in domain II loops

Bacillus thuringiensis produces a range of toxins that include both the insecticidal Cry toxins, non-insecticidal, non-haemolytic, and Parasporins. The latter exhibits cytocidal activity to some cancer cell lines. Parasporins 3 or Cry41Aa is cytocidal to human hepatic HepG2 cell lines. It contains the five conserved sequence blocks found in many insecticidal 3-domain Cry toxins and is also believed to possess the same 3-domain structure. In addition, it has an extra loop in its domain II as well as an additional ricin domain at its C-terminus. studies on insecticidal Cry toxins have implicated domain II loops in the specificity of a toxin to target a particular cell. In this study the specificity of Cry41Aa towards HepG2 cell lines was investigated. Bioinformatic tools were used to predict domain II loops of Cry41Aa. A number of mutants were created to investigate its specificity. Loop exchange mutants between loop 3 Cry41Aa and Cry loop 3 of insecticidal were created but did not result in a proteolytically stable protein. Domain II hybrids of Cry41Aa and insecticidal Cry toxins were created but these did not result in a proteolytic stable protein. Finally, residue substitutions with alanine in loop 1,3, and the extra loop resulted in stable activated toxins. loop 1 mutants retained toxicity. The extra loop mutant lost toxicity towards HepG2 cell lines. A number of Loop 3 mutants were made. Recombinant, Y514A and W511F retained toxicity towards HepG2 cells. Recombinant W511A and several recombinants at position 509 including F509A did not exert toxicity as confirmed by cell viability assays. Despite the lack of toxicity, membrane damage assays and western blots on HepG2 incubated with F509A revealed the likely presence of pores and phosphorylation of p38. Cell electrophysiology tools were applied to investigate the effect that nontoxic recombinant F509A on artificial and cell membrane. Cry41Aa induced the formation of stable pores, cell membrane damage and subsequent cell death. F509A induced the formation of unstable pores and did not compromise the integrity of cell membrane. The study Findings indicated that Cry41Aa is likely to have a similar mode of action as insecticidal Cry toxins.