Inhibition of cell division induced by External Guide Sequence targeting ftsZ

DAVIES SALA, C.; SOLER BISTUE, A J C; KORPRAPUN, L; ZORREGUIETA, A; TOLMASKY, M E

San Franciso

Congreso; International Conference on Antimicrobial Agentes and Chemotherapy (ICAAC); 2012

Background: External guide sequences (EGSs) are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a tRNA-like complex. FtsZ, a critical component of the divisome, is the most conserved bacterial cell division gene, and it has been validated as potential target for new antimicrobial agents. Methods: The secondary structure of ftsZ mRNa was predicted using the mfold software. EGS activity was assayed in vitro by incubation of 5′-end-radiolabeled ftsZ mRNA, 30 µM EGS, 60 nM M1 RNA, and 3 µM C5 protein followed by denaturing polyacrylamide gel electrophoresis. In vivo assessment of EGS activity was done by inducing its expression with 1 mM isopropyl-beta-thio galactopyranoside (IPTG) from a recombinant clone in E. coli BL21(DE3)(pLysS) followed by microscopy. Results: Four regions potentially accessible for interaction with complementary EGSs were identified on the basis of the ftsZ mRNA structure. EGSs targeting these regions were designed and tested to determine their binding efficiency as well as the ability to induce RNAse P-mediated digestion of the mRNA.  Although several EGSs showed high binding affinity, only one of them, EGS4, elicited cleavage of 90% of the substrate mRNA.  To test the activity of EGS4 in vivo, E. coli BL21(DE3)(pLysS) was transformed with pEGS4, a recombinant clone harboring an insert that includes a T7 promoter followed by the EGS4 coding region, the ACCA sequence, and a sequence required to generate a hammerhead ribozyme to generate the correct 3′ terminus of the EGS4 by cis cleavage. Microscopy analysis of cells from to E. coli BL21(DE3)(pLysS)(pEGS4) cultures incubated for 60 minutes after IPTG addition showed significant filamentation. Conclusions:  EGS technology can be a viable strategy to generate antimicrobials that interfere with bacterial cell growth by inhibition of cell division through reduction of expression of FtsZ. Our in vitro binding and mRNA degradation assays show that, in agreement with our previous findings, there is not good correlation between binding affinity of EGSs to a target RNA and RNase P-mediated cleavage in vitro mediated by the same EGSs.