Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with ß-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

A parasporin from Bacillus thuringiensis native to Peninsular India induces apoptosis in cancer cells through intrinsic pathway.

Parasporins, a class of non-insecticidal crystal proteins of Bacillus thuringiensis (Bt) are being explored as promising anticancer agents due to their specific toxicity to cancer cells. The present study has identified 25 Bt isolates harbouring parasporin genes from Western Ghats region, the hotspot of biodiversity in India. Among these, the isolate, KAU 41 (Kerala Agricultural University isolate 41) contained non-hemolytic homogenous crystals showing specific cytotoxicity towards cancer cells. SDS-PAGE analysis of this crystal, isolated by aqueous biphasic separation, revealed a 31 kDa sized peptide. The N-terminal sequence deciphered in BLAST analysis showed homology to a hypothetical Bt protein. Upon proteolysis, a 29 kDa active peptide was generated which exhibited heterogenic cytotoxic spectrum on various cancer cells. HeLa cells were highly susceptible to this peptide with IC 50 1 lg/mL and showed characteristics of apoptosis. RT-qPCR analysis revealed the overexpression of APAF1, caspase 3 and 9 by 14.9, 8 and 7.4 fold, respectively which indicates the activation of intrinsic pathway of apoptosis. However, at higher concentrations of peptide (greater than 3 lg/mL), necrotic death was prominent. The results suggest that the 31 kDa protein from Bt isolate, KAU 41 is a parasporin that may have high therapeutic potential.