Parasporin-1, a Novel Cytotoxic Protein to Human Cells from Non-Insecticidal Parasporal Inclusions of Bacillus thuringiensis

Abstract

Pro-parasporin-1 is a parasporal inclusion protein of the non-insecticidal Bacillus thuringiensis strain A1190. Cytotoxic fragments, named parasporin-1, were generated from pro-parasporin-1 by trypsin digestion. Parasporin-1 was purified by a combination of chromatography procedures based on the cytotoxic activity to HeLa cells. Two different fragments of 15-kDa and 56-kDa were detected in the purified parasporin-1 fraction. These fragments were tightly associated with each other and could not be separated by chromatography under conditions that preserve cytotoxic activity, indicating that the active form of parasporin-1 is a heterodimer of the 15- and 56-kDa fragments. Amino acid sequencing and MALDI-TOF mass spectrometric analysis revealed that parasporin-1 is generated from pro-parasporin-1 by trypsin digestion at Arg 93 and Arg 231. Of 12 human cell lines tested, parasporin-1 showed strong cytotoxicity to four cell lines derived from cancer tissues, but low to no cytotoxicity to the other cell lines. The time-courses of cytotoxicity indicated that the mode of action of parasporin-1 to sensitive cells differs from that shown for previously isolated cytotoxic proteins from Bacillus thuringiensis, Cyt proteins, and other bacterial pore-forming toxins. Thus, parasporin-1 is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis, and may be useful as a tool to recognize and destroy specific cancer cells.

In vitro selective cytotoxicity of activated parasporal proteins produced by Bacillus thuringiensis serovars kumamotoensis and tohokuensis against human cancer cell lines

“…Furthermore, the parasporal proteins of these B. thuringiensis strains (J61 and J72) were previously reported (Obeidat et al, 2012) to exhibit no insecticidal activity against diptera and lepidoptera. This is in agreement with previous studies (Mizuki et al, 1999bLee et al, 2000;Yamashita et al, 2000;Ito et al, 2004;Okumura et al, 2004;Katayama et al, 2005;Hayakawa et al, 2007;Jung et al, 2007;Uemori et al, 2008;Yasutake et al, 2008;Nagamatsu et al, 2010) that clearly demonstrated that only non-insecticidal parasporal proteins can exhibit discriminating cytotoxicity against different cancer cell types. Unfortunately, activated parasporal proteins from all reference B. thuringiensis strains screened in this study did not exhibit selective cytotoxicity against target cells.…”

“…Mizuki et al (1999b) reported cytotoxicity against human leukemic T cells and other human cancer cells for parasporal proteins produced by some noninsecticidal B. thuringiensis strains. Moreover, it was found that these non-insecticidal Cry proteins, designated parasporins, are capable of preferentially killing cancer cells upon proteolytic degradation (Mizuki et al, 1999bYamashita et al, 2000;Ito et al, 2004;Okumura et al, 2004;Katayama et al, 2005;Hayakawa et al, 2007;Jung et al, 2007;Uemori et al, 2008;Yasutake et al, 2008;Nagamatsu et al, 2010). Six parasporin families, including 19 parasporin toxins isolated from B. thuringiensis strains, were identified by the Committee of Parasporin Classification and Nomenclature (Okumura et al, 2010;Wong, 2010).…”

Investigation of a NovelBacillus thuringiensisGene Encoding a Parasporal Protein, Parasporin-4, That Preferentially Kills Human Leukemic T Cells

“…Recently, we identified four parasporins from noninsecticidal B. thuringiensis strains. 20) Of these, PS-1, PS-2, and PS-3 correspond respectively to Cry31Aa, 9) Cry46Aa, 10) and Cry41Aa.…”

Novel Bacillus thuringiensis δ-Endotoxin Active against Locusta migratoria manilensis

“…B. thuringiensis is a uniquely specific, safe, and effective biological insecticide that has been used for more than 50 years (3,30). The increased use of B. thuringiensis in biological control and its potential application in medicine have encouraged researchers to find novel strains with different toxic spectra or high specific activity and new functional genes of B. thuringiensis (13,22,26,28).There are two known classes of ␦-endotoxins in B. thuringiensis: the insect-specific insecticidal crystal (Cry) proteins and the Diptera-specific cytolytic (Cyt) proteins. The Cry proteins are encoded by cry genes, include more than 480 different genes belonging to 124 subgroups of 60 groups, and show variable degrees of sequence homology (see the B. thuringiensis [Bt] toxin nomenclature website at http://www.lifesci.sussex.ac .uk/home/Neil_Crickmore/Bt/).…”