IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Inhibition of cell division induced by External Guide Sequence targeting ftsZ
Autor/es:
CAROL DAVIES SALA; ALFONSO J. C. SOLER BISTUÉ; LEEANN KORPRAPUN; ANGELES ZORREGUIETA; MARCELO E. TOLMASKY
Lugar:
San Francisco
Reunión:
Congreso; 52nd International Conference on Antimicrobial Agents and Chemotherapy (ICAAC); 2012
Institución organizadora:
American Society for Microbiology
Resumen:
Background: External guide sequences (EGSs) are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA. FtsZ, a critical component of the divisome, is the most conserved bacterial cell division gene, and is a potential target for new antimicrobials. Methods: The secondary structure of ftsZ mRNA was predicted using mfold. EGS activity was assayed by incubating 0.25 μM 5′-end-radiolabeled ftsZ mRNA, 5 μM EGS, 0.25μM M1 RNA, and 7 μM C5 protein followed by denaturing PAGE. In vivo assessment of EGS activity was done by inducing its expression 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 hammerhead ribozyme sequence required to generate the correct 3′ terminus by cis cleavage. Microscopy analysis of cells from E. coli(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 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.