Silencing of the shiga toxin Stx-1 in enterohemorrhagic Escherichia coli O157 (EHEC) via synthetic non-coding RNA: a novel antibacterial approach

Abstract

The emergence of resistance in bacteria against antibiotics and the dwindling number of new antibiotics pose a double threat to our continued protection from bacterial pathogens. Advancement in the field of functional genomics has led to many evidences that small regulatory RNA plays a pivotal role in gene regulation in all living organisms. In bacteria, sRNA are basically described as small non-coding transcripts (50 –300 nucleotides) that recruits RNA chaperone proteins, majorly Hfq, and affects mRNA stability and expression level. These natural sRNAs have inspired the design of synthetic trans-acting RNA molecules to efficiently control gene expression in microorganisms. Reversion of antibiotic resistance using synthetic RNAs can be a potential tool in combating antibiotic resistance in pathogenic bacteria. Enterohemorrhagic E. coli (EHEC), a cause of worldwide outbreak is a food borne pathogen that causes mild to bloody diarrhea, hemorrhagic colitis and often complicated with hemolytic uremic syndrome (HUS). Its strong pathogenicity is due to virulence factors such as shiga toxin 1 (Stx 1) and shiga toxin 2 (Stx-2). Our study involves designing a synthetic sRNA against the E. coli shiga toxin 1 gene (sxt-1) followed by assessing the repression efficiency through computational prediction tools. After successfully expressing the anti-shiga sRNA via plasmid pAB.001 which harbors the Hfq downstream to the sRNA transcript, the construct was transferred in STEC O157 cells via conjugation. The effect of anti-shiga sRNA on the mRNA level of the target gene was determined by quantitative real time RT-PCR, while toxin level was verified via MTT cytotoxic assay. Toxin filtrates from wild type E. coli O157 strain was compared to anti-Shiga sRNA containing E. coli O157 strain for cytotoxicity. The assay results clearly indicated that anti-shiga sRNA toxin filtrate had greater number of viable cells as compared to WT.O157 strain toxin. Lower cell viability indicates reduced levels of shiga toxin in the supernatant of E. coli O157 strain containing anti-shiga sRNA. Synthetic sRNA could be a potential tool in treating complicated EHEC infections. Interrupting expression of resistance genes using synthetic sRNA, we can not only restore the antibiotic susceptibility of these bacteria extending the lifespan of existing antibiotics but can also efficiently eradicate their infections.