Artículo de Acceso Abierto. Puede ser descargado en su versión final
Consulte el artículo en la página del editor
Consulte la política de Acceso Abierto del editor


EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ. © 2012 Sala et al.


Documento: Artículo
Título:Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting ftsZ
Autor:Sala, C.D.; Soler-Bistué, A.J.C.; Korprapun, L.; Zorreguieta, A.; Tolmasky, M.E.
Filiación:Fundación Instituto Leloir-Instituto de Investigaciones Bioquímicas Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Center for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, United States
Unité Plasticité du Génome Bacté rien, Département Génomes et Génétique, Institut Pasteur, Paris, France
Palabras clave:messenger RNA; ribonuclease P; transfer RNA; article; bacterial cell; binding affinity; binding site; complex formation; controlled study; Escherichia coli; experimental design; external guide sequence technology; ftsZ gene; gene; gene targeting; gene technology; growth inhibition; in vitro study; mitosis inhibition; nonhuman; promoter region; protein secondary structure; recombinant plasmid; RNA binding; RNA cleavage; RNA structure; sequence analysis; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Cell Division; Cytoskeletal Proteins; Drug Design; Electrophoretic Mobility Shift Assay; Escherichia coli; Microscopy, Confocal; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides, Antisense; Promoter Regions, Genetic; Ribonuclease P; RNA Cleavage; Terminator Regions, Genetic
Título revista:PLoS ONE
Título revista abreviado:PLoS ONE
CAS:ribonuclease P, 71427-00-4; transfer RNA, 9014-25-9; Anti-Bacterial Agents; Bacterial Proteins; Cytoskeletal Proteins; FtsZ protein, Bacteria; Oligoribonucleotides, Antisense; Ribonuclease P,


  • Goehring, N.W., Beckwith, J., Diverse paths to midcell: assembly of the bacterial cell division machinery (2005) Curr Biol, 15, pp. R514-R526
  • Vicente, M., Rico, A.I., The order of the ring: assembly of Escherichia coli cell division components (2006) Mol Microbiol, 61, pp. 5-8
  • Vicente, M., Rico, A.I., Martinez-Arteaga, R., Mingorance, J., Septum enlightenment: assembly of bacterial division proteins (2006) J Bacteriol, 188, pp. 19-27
  • Margolin, W., FtsZ and the division of prokaryotic cells and organelles (2005) Nat Rev Mol Cell Biol, 6, pp. 862-871
  • Corbin, B.D., Wang, Y., Beuria, T.K., Margolin, W., Interaction between cell division proteins FtsE and FtsZ (2007) J Bacteriol, 189, pp. 3026-3035
  • Mingorance, J., Rivas, G., Velez, M., Gomez-Puertas, P., Vicente, M., Strong FtsZ is with the force: mechanisms to constrict bacteria (2010) Trends Microbiol, 18, pp. 348-356
  • de Boer, P.A., Advances in understanding E. coli cell fission (2010) Curr Opin Microbiol, 13, pp. 730-737
  • Goley, E.D., Yeh, Y.C., Hong, S.H., Fero, M.J., Abeliuk, E., Assembly of the Caulobacter cell division machine (2011) Mol Microbiol, 80, pp. 1680-1698
  • Erickson, H.P., Anderson, D.E., Osawa, M., FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one (2010) Microbiol Mol Biol Rev, 74, pp. 504-528
  • Vivancos, A.P., Guell, M., Dohm, J.C., Serrano, L., Himmelbauer, H., Strand-specific deep sequencing of the transcriptome (2010) Genome Research, 20, pp. 989-999
  • Wang, J., Galgoci, A., Kodali, S., Herath, K.B., Jayasuriya, H., Discovery of a small molecule that inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics (2003) J Biol Chem, 278, pp. 44424-44428
  • Goh, S., Boberek, J.M., Nakashima, N., Stach, J., Good, L., Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli (2009) PLoS One, 4, pp. e6061
  • Haydon, D.J., Stokes, N.R., Ure, R., Galbraith, G., Bennett, J.M., An inhibitor of FtsZ with potent and selective anti-staphylococcal activity (2008) Science, 321, pp. 1673-1675
  • Huang, Q., Tonge, P.J., Slayden, R.A., Kirikae, T., Ojima, I., FtsZ: a novel target for tuberculosis drug discovery (2007) Curr Top Med Chem, 7, pp. 527-543
  • Sass, P., Josten, M., Famulla, K., Schiffer, G., Sahl, H.G., Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ (2011) Proc Natl Acad Sci U S A, 108, pp. 17474-17479
  • Jaiswal, R., Beuria, T.K., Mohan, R., Mahajan, S.K., Panda, D., Totarol inhibits bacterial cytokinesis by perturbing the assembly dynamics of FtsZ (2007) Biochemistry, 46, pp. 4211-4220
  • Schaffner-Barbero, C., Martin-Fontecha, M., Chacon, P., Andreu, J.M., Targeting the assembly of bacterial cell division protein FtsZ with small molecules (2012) ACS Chem Biol, 7, pp. 269-277
  • Haydon, D.J., Bennett, J.M., Brown, D., Collins, I., Galbraith, G., Creating an antibacterial with in vivo efficacy: synthesis and characterization of potent inhibitors of the bacterial cell division protein FtsZ with improved pharmaceutical properties (2010) J Med Chem, 53, pp. 3927-3936
  • Altman, S., Ribonuclease P (2011) Philos Trans R Soc Lond B Biol Sci, 366, pp. 2936-2941
  • Lundblad, E.W., Altman, S., Inhibition of gene expression by RNase P (2010) Nature Biotechnol, 27, pp. 212-221
  • Gopalan, V., Vioque, A., Altman, S., RNase P: variations and uses (2002) J Biol Chem, 277, pp. 6759-6762
  • Xiao, G., Lundblad, E.W., Izadjoo, M., Altman, S., Inhibition of expression in Escherichia coli of a virulence regulator MglB of Francisella tularensis using external guide sequence technology (2008) PLoS One, 3, pp. e3719
  • Ko, J.H., Izadjoo, M., Altman, S., Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P (2008) RNA, 14, pp. 1656-1662
  • McKinney, J.S., Zhang, H., Kubori, T., Galan, J.E., Altman, S., Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences (2004) Nucleic Acids Res, 32, pp. 848-854
  • McKinney, J., Guerrier-Takada, C., Wesolowski, D., Altman, S., Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes (2001) Proc Natl Acad Sci U S A, 98, pp. 6605-6610
  • Guerrier-Takada, C., Salavati, R., Altman, S., Phenotypic conversion of drug-resistant bacteria to drug sensitivity (1997) Proc Natl Acad Sci U S A, 94, pp. 8468-8472
  • Soler Bistue, A.J., Martin, F.A., Vozza, N., Ha, H., Joaquin, J.C., Inhibition of aac(6′)-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria (2009) Proc Natl Acad Sci U S A, 106, pp. 13230-132235
  • Soler Bistue, A.J., Ha, H., Sarno, R., Don, M., Zorreguieta, A., External guide sequences targeting the aac(6′)-Ib mRNA induce inhibition of amikacin resistance (2007) Antimicrob Agents Chemother, 51, pp. 1918-1925
  • Shen, N., Ko, J.H., Xiao, G., Wesolowski, D., Shan, G., Inactivation of expression of several genes in a variety of bacterial species by EGS technology (2009) Proc Natl Acad Sci U S A, 106, pp. 8163-8168
  • Guerrier-Takada, C., Li, Y., Altman, S., Artificial regulation of gene expression in Escherichia coli by RNase P (1995) Proc Natl Acad Sci U S A, 92, pp. 11115-11119
  • Studier, F.W., Rosenberg, A.H., Dunn, J.J., Dubendorff, J.W., Use of T7 RNA polymerase to direct expression of cloned genes (1990) Methods Enzymol, 185, pp. 60-89
  • Sambrook, J., Russell, D., (2001) Molecular cloning: A laboratory manual, , Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  • Sarno, R., Ha, H., Weinsetel, N., Tolmasky, M.E., Inhibition of aminoglycoside 6′-N-acetyltransferase type Ib-mediated amikacin resistance by antisense oligodeoxynucleotides (2003) Antimicrob Agents Chemother, 47, pp. 3296-3304
  • Zuker, M., Mathews, D.H., Turner, D.H., Algorithms and thermodynamics for RNA secondary structure prediction: A practical guide (1999) RNA biochemistry and Biotechnology: Kluwer Academic Publishers, pp. 11-43. , Barciszewski J, Clark B, editors
  • Zuker, M., Mfold web server for nucleic acid folding and hybridization prediction (2003) Nucleic Acids Res, 31, pp. 3406-3415
  • Li, Y., Guerrier-Takada, C., Altman, S., Targeted cleavage of mRNA in vitro by RNase P from Escherichia coli (1992) Proc Natl Acad Sci U S A, 89, pp. 3185-3189
  • Ayala, J., Garrido, T., de Pedro, M., Vicente, M., Molecular biology of bacterial septation (1994) Bacterial Cell Wall, pp. 73-101. , Ghuysen J, Hakenbeck R, editors, Amsterdam: Elsevier Science
  • Flardh, K., Garrido, T., Vicente, M., Contribution of individual promoters in the ddlB-ftsZ region to the transcription of the essential cell-division gene ftsZ in Escherichia coli (1997) Molecular Microbiol, 24, pp. 927-936
  • Selinger, D.W., Saxena, R.M., Cheung, K.J., Church, G.M., Rosenow, C., Global RNA half-life analysis in Escherichia coli reveals positional patterns of transcript degradation (2003) Genome Research, 13, pp. 216-223
  • Cam, K., Rome, G., Krisch, H.M., Bouche, J.P., RNase E processing of essential cell division genes mRNA in Escherichia coli (1996) Nucleic Acids Res, 24, pp. 3065-3070
  • Benders, G.A., Powell, B.C., Hutchison 3rd, C.A., Transcriptional analysis of the conserved ftsZ gene cluster in Mycoplasma genitalium and Mycoplasma pneumoniae (2005) J Bacteriol, 187, pp. 4542-4551
  • Goldblum, K., Apririon, D., Inactivation of the ribonucleic acid-processing enzyme ribonuclease E blocks cell division (1981) J Bacteriol, 146, pp. 128-132
  • de la Fuente, A., Palacios, P., Vicente, M., Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene (2001) Biochimie, 83, pp. 109-115
  • Tamura, M., Lee, K., Miller, C.A., Moore, C.J., Shirako, Y., RNase E maintenance of proper FtsZ/FtsA ratio required for nonfilamentous growth of Escherichia coli cells but not for colony-forming ability (2006) J Bacteriol, 188, pp. 5145-5152
  • The 10×′20 Initiative: pursuing a global commitment to develop 10 new antibacterial drugs by 2020 (2010) Clin Infect Dis, 50, pp. 1081-1083. , Infectious Diseases Society of America
  • Rice, L.B., Progress and challenges in implementing the research on ESKAPE pathogens (2010) Infect Control Hospital Epidemiol, 31, pp. 7-10
  • Boberek, J.M., Stach, J., Good, L., Genetic evidence for inhibition of bacterial division protein FtsZ by berberine (2010) PLoS One, 5, pp. e13745
  • Czaplewski, L.G., Collins, I., Boyd, E.A., Brown, D., East, S.P., Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell division protein FtsZ (2009) Bioorg Med Chem Lett, 19, pp. 524-527
  • Meng, J., Wang, H., Hou, Z., Chen, T., Fu, J., Novel anion liposome-encapsulated antisense oligonucleotide restores susceptibility of methicillin-resistant Staphylococcus aureus and rescues mice from lethal sepsis by targeting mecA (2009) Antimicrob Agents Chemother, 53, pp. 2871-2878
  • Mellbye, B.L., Weller, D.D., Hassinger, J.N., Reeves, M.D., Lovejoy, C.E., Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo (2010) J Antimicrob Chemother, 65, pp. 98-106
  • Eriksson, M., Nielsen, P.E., Good, L., Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli (2002) J Biol Chem, 277, pp. 7144-7147
  • Traglia, G., Davies Sala, C., Fuxman Bass, J., Soler Bistué, A., Zorreguieta, A., Internalization of locked nucleic acids/DNA hybrid oligomers into Escherichia coli (2012) BioResearch Open Access
  • Stein, C.A., Hansen, J.B., Lai, J., Wu, S., Voskresenskiy, A., Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents (2010) Nucleic Acids Res, 38, pp. e3
  • Torres, A.G., Threlfall, R.N., Gait, M.J., Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2′-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents (2011) Artif DNA PNA XNA, 2, pp. 71-78
  • Lehto, T., Abes, R., Oskolkov, N., Suhorutsenko, J., Copolovici, D.M., Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy (2010) J Control Release, 141, pp. 42-51
  • Mae, M., El Andaloussi, S., Lundin, P., Oskolkov, N., Johansson, H.J., A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy (2009) J Control Release, 134, pp. 221-227
  • Takara, K., Hatakeyama, H., Ohga, N., Hida, K., Harashima, H., Design of a dual-ligand system using a specific ligand and cell penetrating peptide, resulting in a synergistic effect on selectivity and cellular uptake (2010) Int J Pharm, 396, pp. 143-148
  • Anko, M., Majhenc, J., Kogej, K., Sillard, R., Langel, U., Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane (2012) Biochim Biophys Acta, 1818, pp. 915-924
  • Said Hassane, F., Saleh, A.F., Abes, R., Gait, M.J., Lebleu, B., Cell penetrating peptides: overview and applications to the delivery of oligonucleotides (2010) Cell Mol Life Sci, 67, pp. 715-726
  • Holm, T., Andaloussi, S.E., Langel, U., Comparison of CPP uptake methods (2011) Methods Mol Biol, 683, pp. 207-217


---------- APA ----------
Sala, C.D., Soler-Bistué, A.J.C., Korprapun, L., Zorreguieta, A. & Tolmasky, M.E. (2012) . Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting ftsZ. PLoS ONE, 7(10).
---------- CHICAGO ----------
Sala, C.D., Soler-Bistué, A.J.C., Korprapun, L., Zorreguieta, A., Tolmasky, M.E. "Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting ftsZ" . PLoS ONE 7, no. 10 (2012).
---------- MLA ----------
Sala, C.D., Soler-Bistué, A.J.C., Korprapun, L., Zorreguieta, A., Tolmasky, M.E. "Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting ftsZ" . PLoS ONE, vol. 7, no. 10, 2012.
---------- VANCOUVER ----------
Sala, C.D., Soler-Bistué, A.J.C., Korprapun, L., Zorreguieta, A., Tolmasky, M.E. Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting ftsZ. PLoS ONE. 2012;7(10).