Reduction of bacterial viability by antisense technology targeting a vital gene

CAROL DAVIES SALA; ALFONSO J. C. SOLER BISTUÉ; ANGELES ZORREGUIETA; MARCELO E. TOLMASKY

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

Sociedad Argentina de Microbiología General

External Guide Sequence (EGS) is an antisense technology that has been successfully used to silence genes of numerous pathogenic Gram negative bacteria. EGS targeting of resistance or essential genes led to reversal of resistant phenotypes or cell viability, respectively. This technology consists of the utilization of endogenous RNase P and an EGS, a short oligonucleotide -or analogue- that elicits RNase P- mediated cleavage of an RNA target. FtsZ is a bacterial tubuline-like protein necessary for normal cell division. A decrease in ftsZ expression is enough to interfere with normal formation of the divisome and consequently with cell division. Hence, an EGS targeting ftsZ mRNA could be a novel antimicrobial agent. Using computational modeling of the ftsZ mRNA secondary structure we designed several EGSs complementary to specific regions of the molecule and tested their ability to elicit its RNase- P mediated cleavage in vitro. The most promising EGS, EGS4, was selected to test its effectiveness in vivo. For this we transformed E. coli BL21(DE3)pLysS with a recombinant plasmid, pEGS4, harboring the EGS4 sequence downstream of the T7 promoter and followed by a hammerhead ribozyme sequence. To determine bacterial viability, cultures in mid log were induced with IPTG, cultured for 60 ? 90 minutes, and serially diluted and plated for colony forming units counting. Controls were carried out using plasmid pEGS4S, which codes for an EGS with a sequence complementary to EGS4. The experiments were repeated 3 times and the results were expressed as mean log10(CFU/ml) ± SD. Statistical significance was analyzed by an unpaired two-tailed t-test. P < 0.01 was considered statistically significant. Our results show that there was a significant reduction in the CFU/ml in the cultures of cells producing EGS4 compared to those expressing EGS4sense (p < 0.0001), confirming that this EGS has a detrimental effect, most probably due to the inhibition of cell division. Our results show that EGS technology could become a new kind of antimicrobial agents or could be used in combination with existing antibiotics to potentiate their effect.