New antimicrobial strategies: inhibition of ftsZ vital gene by EGS technology

DAVIES SALA, C.; SOLER BISTUÉ AJ; ZORREGUIETA, A; TOLMASKY, M E

Congreso; VII Congreso Argentino de Microbiología General,; 2011

Sociedad Argentina de Microbiología General (SAMIGE)

FtsZ is a highly conserved bacterial tubuline-like protein whose correct dosage is necessary for proper cell division. A relatively small decrease in <i>ftsZ</i> expression is enough to interfere with septation and concomitantly with cell division. Therefore, this gene could be used as target of novel antimicrobials. External Guide Sequence (EGS) technology consists of inducing degradation of the target RNA by RNAse P in the presence of the EGS, an appropriate complementary oligonucleotide or analog.  We have recently showed that EGSs composed of a mix of deoxynucleotide and locked nucleic acid (LNA) residues efficiently induced RNase P cleavage of a target mRNA in vivo. In this work we define EGSs that induce RNase P-mediated decay of <i>ftsZ</i> mRNA.  These EGSs could be the foundation for a new kind of antimicrobial agents or could be used in combination with existing antibiotics and other EGSs that inhibit expression of antibiotic resistance genes. We designed EGSs complementary to four <i>ftsZ</i> mRNA regions available for interaction as determined by <i>in silico</i> secondary structure modeling.  Our initial analyses were carried out using oligoribonucleotide EGSs (RNA-EGSs).  The RNA-EGSs were used in electrophoresis mobility shift assays (EMSA) to determine their ability to bind the mRNA target.  Selected RNA-EGSs were then tested to assess their efficiency in eliciting RNase P-mediated cleavage of <i>ftsZ</i> mRNA in vitro. For this, radioactive 5´-end labeled mRNA was incubated with the appropriate ratio of the catalytic M1 RNA and the cofactor protein C5. RNA-EGSs targeting two of the selected regions (RNA-EGS2 and RNA-EGS4) showed high affinity of binding the <i>ftsZ</i> mRNA and elicited RNAse P-mediated cleavage in vitro.  Since RNA-EGS4 showed the highest cleavage inducing efficiency, a nuclease resistant isosequential EGS, LNA1-EGS4, consisting of a mix of deoxynucleotide and LNA residues was designed and tested. The results of these assays indicated that LNA1-EGS4 mediates cleavage of the target mRNA with an efficiency similar to that of the RNA-EGS4. Our results indicate that there are at least two regions of the <i>ftsZ</i> mRNA accessible for productive interaction with EGSs composed by either RNA or nuclease resistant analogs.  Future experiments will determine if these later compounds also interfere with expression of <i>ftsZ</i> in vivo.