Isolation and characterization of parasporal anticancer protein from Bacillus thuringiensis strains

Abstract

Extensive research is ongoing to unravel the mystery of the diversified role of Cry proteins from Bacillus thuringiensis (Bt). Anticancer Cry proteins are named parasporins and have the selectivity to kill tissue-specific cancer cells without harming normal cells. These characteristics of parasporins have opened a new horizon of its anticancer activities. This study sought for investigation into new parasporal anticancer proteins from 98 nonhemolytic and 23 non-insecticidal hemolytic Bt strains from 298 indigenous Bt stock. Six sets of primers were designed from the conserved δ-endotoxin, CryBP1, and Etx-Mtx regions of ps1- ps5 and were explored to detect the presence of respective genes through PCR amplification. A total of 27 PCR amplicons were obtained from 121 Bt strains. Primers targeting the ps1 and ps3 (CryBP1) genes amplified desired fragments from the plasmid of 8 Bt strains. Besides, ps1, ps3 and ps4 primers amplified 19 unintended amplicons of various sizes. 25 PCR amplicons were sequenced from both anticipated and unintended amplifications. These generated no expected parasporin or cry genes from the DNA sequencing data, instead 11 plasmid-mediated genes were found, mostly of drug/metabolite transporters, Tn3 transposases, and DNA/RNA manipulators. Alternatively, extraction and purification of parasporal proteins followed by SDS-PAGE were performed from 98 nonhemolytic Bt strains. Of them, seven strains; MyIa2, FHSc4, KkSc1, RaSd1, MyIb1, MyIa1, and SgSp2 revealed some prominent parasporal protein bands with varied molecular masses (17-78 kDa). Among them, proteinase K-digested putative parasporal proteins from 3 strains, MyIa2, FHSc4, and KkSc1, were revealed as cytotoxic to the HeLa cell line. Phase-contrast microscopy also supported the presence of parasporal inclusion bodies from the same three strains. Interestingly, as proteinase K digested parasporal protein did not produce any visible protein band in the SDS gel suggesting that they belong to non-Cry or novel/ undiscovered categories of parasporal proteins which deserve more critical studies. Consequently, the research was covered to investigating the anticancer Cry protein in 23 hemolytic non-insecticidal Bt strains of which five strains produced putative parasporal protein bands on the SDS-PAGE. Bt strains Soil-46 and 28S were found to produce four putative parasporal proteins of identical molecular mass (101, 86, 76, and 28 kDa), whereas BD59S had three (101, 86, and 76 kDa). Besides, a 103 kDa protein band was found from Dsh4 and 45L strains. Approximately 64, 51, 45, 41, and 32 kDa fragments were generated from the putative parasporal protein extracted from 28S and Soil-46 upon tryptic digestion. BD59S had 64, 51, and 41 kDa protein fragments, while Dsh4 and 45L had 45 kDa fragments upon trypsin digestion. On the other hand, parasporal proteins from all five strains formed a 46 kDa identical protein fragment when digested by proteinase K. Then, untreated, trypsin-treated, and proteinase K-treated protein samples from each strain were tested for cytotoxicity on HeLa cancer cell lines. Untreated protein samples from 28S, Soil-46, 45L, and Dsh4 exhibited modest cytotoxicity on the HeLa cell line, while BD59S did not. The trypsin-digested proteins of BD59S exhibited significant cytotoxicity, whereas 28S, Soil-46, 45L, and Dsh4 showed moderate towards the HeLa cell lines. Proteinase K digested proteins of all five strains showed potent iii cytotoxicity on the HeLa cell line. In contrast, the same protein samples at the same concentration didn’t show any cytotoxicity to the normal cell, Vero cell line. In addition, the solubilized parasporal proteins of these Bt strains showed cytotoxicity to brine shrimp nauplii with an LC50 value of 114.19 μg/mL. Furthermore, they were nonhemolytic to sheep red blood cells and non-insecticidal to the fruit fly larvae (Bactrocera cucurbitae). All of these findings meet the requirements of being parasporins. Besides, as the untreated parasporal protein from 4 strains Soil-46, 28S, 45L, and DSh4 showed little toxicity towards the HeLa cell line hence this attitude of parasporal proteins someway coordinated with S-layer protein. Light microscopy showed the presence of round/ atypical parasporal inclusions, while SEM showed that all inclusions were round in five strains. Morphology analysis from SEM indicates that the parasporal inclusions are consistent with the S-layer protein. Whole genome sequencing (WGS) of four strains (BD59S, 28S, Soil-46, and 45L), three strains (BD59S, Soil-46, and 28S) were found belonging to Bt serovar finitimus, whereas 45L belonging to the serovar konkukian. Besides, bacterial WGS data analysis, the 101, 86 and 76 kDa SDS-PAGE observed protein bands were identified as 91 kDa, 86 kDa S-layer, and a three domain Cry-like 73.54 kDa protein, respectively. The 103 kDa protein band of 45L was identified as 103 or 93 kDa S-layer protein of these strains. As anticancer activity was identified in proteinase K and trypsin digested parasporal protein, hence they were compared with anticancer parasporin. The 91 kDa EA1, 86 kDa Sap, and 73.54 kDa Cry-like protein had 19% and 18% identity with PS3, respectively. Besides, the 93 kDa SLP had 16% with PS3, and the 103 kDa SLP had 19.84% and 14.22% similarities with PS3 and PS5, respectively. Based on the results, parasporal anticancer proteins of five indigenous Bt strains namely Bacillus thuringiensis BD59S, Soil-46, 28S, 45L, and Dsh4 are suggested to be as parasporin-like S-layer proteins. This is the first report of S-layer proteins from B. thuringiensis having anticancer potential in the HeLa cancer cell line while non-cytotoxic to the Vero cell line. The cancer-cell killing nature of parasporal S-layer protein from Bt strains thus opens up further investigations to unveil their anticancer potentials to become a candidate for anticancer protein development.