The Replacement of five Consecutive Amino Acids in the Cyt1A Protein of Bacillus thuringiensis Enhances its Cytotoxic Activity against Lung Epithelial Cancer Cells

Abstract

Cyt1A protein is a cytolytic protein encoded by the cyt gene of Bacillus thuringiensis subsp. israelensis (Bti) as part of the parasporal crystal proteins produced during the sporulation. Cyt1A protein is unique compared to the other endotoxins present in these parasporal crystals. Unlike δ-endotoxins, Cyt1A protein does not require receptors to bind to the target cell and activate the toxicity. It has the ability to affect a broad range of cell types and organisms, due to this characteristic. Cyt1A has been recognized to not only target the insect cells directly, but also recruit other endotoxins by acting as receptors. Due to these mode of actions, Cyt1A has been studied for its cytolytic activity against human cancer cell lines, although not extensively. In this study, we report a novel Cyt1A protein produced by a Bti strain QBT229 isolated from Qatar. When tested for its cytotoxicity against lung cancer cells, this local strain showed considerably higher activity compared to that of the reference Bti and other strains tested. The possible reasons for such enhanced activity were explored at the gene and protein levels. It was evidenced that five consecutive amino acid replacements in the β8 sheet of the Cyt1A protein enhanced the cytotoxicity against the lung epithelial cancer cells. Such novel Cyt1A protein with high cytotoxicity against lung cancer cells has been characterized and reported through this study.

Keywords: Bacillus thuringiensis subsp. israelensis; Novel Cyt1A; cytotoxicity; lung epithelial cancer cell line; protein modelling.

Figure 1

Cytotoxic effects of local Bt subsp. israelensis strains against lung epithelial cancer cells (NCI-H1975). Calculated viability of cells plotted against the concentration of endotoxin proteins (treatment).

Figure 2

Electrophoresis of the amplified PCR products obtained for each δ-endotoxin gene being explored, Lanes L represent 1 kb plus ladder (100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 650 bp, 850 bp, 1 kb, 1.65 kb, 2 kb, 5 kb, and 12 kb), Lanes 1, 2, and 3 represent QBT213, QBT220, and QBT229 respectively, Lanes H14 and HD1 represent references Bt subsp. israelensis and Bt subsp. kurstaki respectively: each panel represents PCR amplification results for (A) 800 bp of cry4A/4B and 1293 bp of cry4B; (B) 291 bp of p19 & 704 bp of p20; (C) 304 bp of cry11 & 1320 bp of cyt1C; (D) 1150 bp of cry10 & 471 bp of cyt2; and (E) 701 bp of cyt1A..

Figure 3

Amino acid alignment of Cyt1A protein sequence from QBT229 and the template TRON.3 representing the Cyt1A sequence and model for Cyt1A protein from Bacillus thuringiensis subsp. israelensis; red box represents the region of β8 sheet with amino acid replacements; amino acids highlighted in red have negative charges and the amino acids highlighted in blue have the positive charge; amino acids included in secondary structures like β sheets, α helix, and η are marked in the template.

Figure 4

Protein modeling and structural and chemical homology comparisons between Cyt proteins. The ClustalX protein model of the Cyt protein expressed by (A) Bt subsp. israelensis H14 and (B) Qatari Bt subsp. israelensis QBT229, showing the amino acid replacements and chemical differences in the region; the positive (blue) and negative (red) charges owing to the amino acids in the Cyt protein of (C) Bt subsp. israelensis H14 and (D) Qatari Bt subsp. israelensis QBT229.