Crystal Protein of a Novel Bacillus thuringiensis Strain Inducing Cell Cycle Arrest and Apoptotic Cell Death in Human Leukemic Cells

V Beena,V Ramnath,K P Sreekumar,K Karthiayini,P T Philomina,D Girija

doi:10.1038/s41598-019-45928-z

Abstract

Parasporal inclusions of a native non haemolytic Bacillus thuringiensis strain KAU 59 was screened for its cytotoxicity against human lymphocytic leukemic cell line jurkat and normal human lymphocytes. The cytotoxicity of proteinase activated and non activated solubilised parasporal inclusions against both cell lines was assessed by Cell Titer 96 Aqueous Non Radioactive Cell Proliferation Assay Kit using MTS. The 50 per cent effective concentration (EC50) values were deduced from log probit analysis at 48 h. Morphological changes associated with cytotoxicity were evaluated and molecular mechanisms of cell death were elucidated by TUNEL assay at 48 h post-inoculation. The fluorescence assisted cell sorting was done in the flow cytometer to assess the stage of cell cycle arrest. Relative quantification of caspase-3 expression in Jurkat cells treated with parasporal inclusion protein of KAU 59 was done by qRTPCR The results indicated that the protein was cytotoxic to jurkat cells at the same time non toxic to normal lymphocytes. Cytotoxicity was evident only after proteolytic activation. Apoptotic cell death was confirmed in the protein treated cells by TUNEL Assay and also up regulated caspase-3 gene expression (P < 0.001). S phase cell cycle arrest was confirmed by and fluorescence associated cell sorting.

Highlights

Researches on the biological activities of Bacillus thuringiensis (Bt) strains with non-insecticidal parasporal inclusions, which are abundant in nature had led to the discovery of a unique group of proteins called ‘Parasporins’
The results of one dose haemolytic assay showed that the protein isolated from the Bt strain KAU 59 was non-haemolytic to human erythrocytes
To check whether the protein possesses all properties of parasporins, cytotoxicity assay was done with non-haemolytic Bt proteins of KAU 59 in intact state

Summary

Introduction

Researches on the biological activities of Bt strains with non-insecticidal parasporal inclusions, which are abundant in nature had led to the discovery of a unique group of proteins called ‘Parasporins’. A proteolytically processed peptide from Bt strain 89-T-34-22 induced, necrosis like cytotoxicity against MOLT-4 cells characterised by mitochondrial swelling and structural breakdown, disorganisation of Golgi complex, cell ballooning and chromatin condensation[15,16] Protein from a Malaysian Bt strain, Bt 18 could cause phosphatidyl serine externalisation, activation of caspase-3 and S-phase cell cycle arrest in leukemic cell lines: CEM-SS, CCKF-SB and CCRF-HSB-226,27 These changes were found to be initiated by binding of the toxin with glyceraldehyde 3 PO4 dehydrogenase (GAPDH) present on the plasma membrane of the target cell[22,28]. Crystal protein from another Bt strain KAU 4129 was causing cell death by inducing intrinsic pathway of apoptosis in HeLa cells as evidenced by increased expression of apoptotic genes, caspase 3, 9 and Apoptotic Proteases Activating Factor I (APAF I) with no effect of caspase 8

Methods

Results

Conclusion