Inhibition of Lethal Toxin Expression in Bacillus anthracis Using Antisense Technology

Park, Chul Min; Kim, Mee Jeong; Ahn, Jang Hoon; Kim, Ki Jeong; Kim, Won Yong; Chung, Sang In

Abstract

Bacillus anthracis is the causative agent of anthrax primarily in animals and rarely in humans. B. anthracis producing 'anthrax toxin', however, could be a major agent of biological warfare. Anthrax toxin is produced from the pXO1 plasmid encoding the lethal toxin (LeTx) consisted of the protective antigen (PA) and the lethal factor (LF). In this study, we tested whether specific antisense oligonucleotide could inhibit the gene expression in B. anthracis. The antisense oligonucleotide was forced into bacterial cells either by lipofection or heat shock method. The expression of LeTx in B. anthracis was analyzed by the Western blot analysis and the MTT assay using to Raw 264.7 cells. The LeTx protein was purified and used for the production of specific antibodies. The expression of LeTx could be confirmed only in B. anthracis strains haboring pXO1 plasmid. B. anthracis treated with the antisense oligonucleotide through heat shock method markedly inhibited the production of PA. In the Western blot analysis, the expression of PA was inhibited from 25 micrometer and was completely inhibited at 50 micrometer of the antisense oligonucleotide. In the MTT assay, the cytotoxicity was reduced to 20% at 20 micrometer of the antisense oligonucleotide. Above results suggest that the antisense technology would be applied for the research on gene function in B. anthracis.