Evaluation of the Knocking-down Effect of Shiga Toxins in the Enterohemorrhagic E. coli O157 (EHEC); a Strategy Towards RNA-based Therapeutics

Abstract

Antibiotic discovery was considered as a “Miracle”, until the antimicrobial resistance developed against them became the major clinical concern. Shiga toxin producing E. coli (STEC) is also such kind of microbe against which misuse and overuse of antibiotics has led to the development of resistant bacterial strains. Among STEC, E. coli O157:H7 strain is the most notorious and have reported in many outbreaks. Few novel anti-bacterial strategies have successfully been administrated to treat the bacterial infections while many are under investigation. Use of synthetic non-coding RNA is also such kind of strategy which has the potential to become a trusted way of treating bacterial infections. For regulation of gene, synthetic sRNAs need to bind with mRNA of the target gene via Watson-crick complementary model and need promoter, mRNA binding region, Hfq protein and terminator for their proper functioning. In this study, a synthetic non-coding RNA cassette was designed against the shiga toxin 2 gene (stx-2) and was incorporated into E. coli O157:H7 with the help of pAB.001 plasmid (already harboring the promoter, Hfq and terminator regions). Effects caused by synthetic sRNA on shiga toxin production, in target bacteria, were analyzed by real time RT-PCR and MTT assay. Computational web tools based predictions indicate that synthetic sRNA against stx-2 showed maximum repression capability. Synthetic sRNA designed against stx-2 gene also showed significant reduction at the mRNA and protein levels after analysis in the wet lab. A comparative analysis of the effects caused by similar synthetic sRNAs designed against stx-1 was performed and it was observed that stx-1 gene showed no significant change at the mRNA level compared to stx-2 which showed a significant reduction. However a clear reduction in the shiga toxin level of both genes was observed at the protein level. ii Difference in silencing effect at mRNA level was observed due to different production rate of target mRNAs.