[en] Diversification of the CTX-M beta-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 A and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the beta3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution.
Research center :
d‐BRU - Dental Biomaterials Research Unit - ULiège [BE]
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Ghiglione, Barbara
Rodriguez, Maria Margarita
Herman, Raphaël ; Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Curto, Lucrecia
Dropa, Milena
Bouillenne, Fabrice ; Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Kerff, Frédéric ; Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Gutkind, G. O., Di Conza, J., Power, P., and Radice, M. (2013) β-Lactamase-mediated resistance: a biochemical, epidemiological and genetic overview Curr. Pharm. Des. 19, 164-208 10.2174/138161213804070320
Rossolini, G. M., D'Andrea, M. M., and Mugnaioli, C. (2008) The spread of CTX-M-type extended-spectrum β-lactamases Clin. Microbiol. Infect. 14, 33-41 10.1111/j.1469-0691.2007.01867.x
Canton, R. and Coque, T. M. (2006) The CTX-M β-lactamase pandemic Curr. Opin. Microbiol. 9, 466-475 10.1016/j.mib.2006.08.011
Decousser, J. W., Poirel, L., and Nordmann, P. (2001) Characterization of a chromosomally encoded extended-spectrum class A β-lactamase from Kluyvera cryocrescens Antimicrob. Agents Chemother. 45, 3595-3598 10.1128/AAC.45.12.3595-3598.2001
Humeniuk, C., Arlet, G., Gautier, V., Grimont, P., Labia, R., and Philippon, A. (2002) β-Lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types Antimicrob. Agents Chemother. 46, 3045-3049 10.1128/AAC.46.9.3045-3049.2002
Poirel, L., Kampfer, P., and Nordmann, P. (2002) Chromosome-encoded Ambler class A β-lactamase of Kluyvera georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum β-lactamases Antimicrob. Agents Chemother. 46, 4038-4040 10.1128/AAC.46.12.4038-4040.2002
Rodriguez, M. M., Power, P., Radice, M., Vay, C., Famiglietti, A., Galleni, M., Ayala, J. A., and Gutkind, G. (2004) Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases Antimicrob. Agents Chemother. 48, 4895-4897 10.1128/AAC.48.12.4895-4897.2004
Bonnet, R. (2004) Growing group of extended-spectrum β-lactamases: the CTX-M enzymes Antimicrob. Agents Chemother. 48, 1-14 10.1128/AAC.48.1.1-14.2004
Bradford, P. A. (2001) Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat Clin. Microbiol. Rev. 14, 933-951 10.1128/CMR.14.4.933-951.2001
Power, P., Di Conza, J., Rodriguez, M. M., Ghiglione, B., Ayala, J. A., Casellas, J. M., Radice, M., and Gutkind, G. (2007) Biochemical characterization of PER-2 and genetic environment of blaPER-2 Antimicrob. Agents Chemother. 51, 2359-2365 10.1128/AAC.01395-06
Chen, Y., Delmas, J., Sirot, J., Shoichet, B., and Bonnet, R. (2005) Atomic resolution structures of CTX-M β-lactamases: extended spectrum activities from increased mobility and decreased stability J. Mol. Biol. 348, 349-362 10.1016/j.jmb.2005.02.010
Ibuka, A. S., Ishii, Y., Galleni, M., Ishiguro, M., Yamaguchi, K., Frere, J. M., Matsuzawa, H., and Sakai, H. (2003) Crystal structure of extended-spectrum β-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion Biochemistry 42, 10634-10643 10.1021/bi0342822
Cartelle, M., del Mar Tomas, M., Molina, F., Moure, R., Villanueva, R., and Bou, G. (2004) High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution Antimicrob. Agents Chemother. 48, 2308-2313 10.1128/AAC.48.6.2308-2313.2004
Bae, I. K., Lee, B. H., Hwang, H. Y., Jeong, S. H., Hong, S. G., Chang, C. L., Kwak, H. S., Kim, H. J., and Youn, H. (2006) A novel ceftazidime-hydrolysing extended-spectrum β-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop J. Antimicrob. Chemother. 58, 315-319 10.1093/jac/dkl252
Bonnet, R., Dutour, C., Sampaio, J. L., Chanal, C., Sirot, D., Labia, R., De Champs, C., and Sirot, J. (2001) Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240 - > Gly Antimicrob. Agents Chemother. 45, 2269-2275 10.1128/AAC.45.8.2269-2275.2001
Bonnet, R., Recule, C., Baraduc, R., Chanal, C., Sirot, D., De Champs, C., and Sirot, J. (2003) Effect of D240G substitution in a novel ESBL CTX-M-27 J. Antimicrob. Chemother. 52, 29-35 10.1093/jac/dkg256
Poirel, L., Gniadkowski, M., and Nordmann, P. (2002) Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum β-lactamase CTX-M-15 and of its structurally related β-lactamase CTX-M-3 J. Antimicrob. Chemother. 50, 1031-1034 10.1093/jac/dkf240
Delmas, J., Chen, Y., Prati, F., Robin, F., Shoichet, B. K., and Bonnet, R. (2008) Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum β-lactamases J. Mol. Biol. 375, 192-201 10.1016/j.jmb.2007.10.026
Antunes, N. T., Frase, H., Toth, M., Mobashery, S., and Vakulenko, S. B. (2011) Resistance to the third-generation cephalosporin ceftazidime by a deacylation-deficient mutant of the TEM β-lactamase by the uncommon covalent-trapping mechanism Biochemistry 50, 6387-6395 10.1021/bi200403e
Goessens, W. H., van der Bij, A. K., van Boxtel, R., Pitout, J. D., van Ulsen, P., Melles, D. C., and Tommassen, J. (2013) Antibiotic trapping by plasmid-encoded CMY-2 β-lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in Escherichia coli Antimicrob. Agents Chemother. 57, 3941-3949 10.1128/AAC.02459-12
Levitt, P. S., Papp-Wallace, K. M., Taracila, M. A., Hujer, A. M., Winkler, M. L., Smith, K. M., Xu, Y., Harris, M. E., and Bonomo, R. A. (2012) Exploring the role of a conserved class A residue in the Omega-Loop of KPC-2 β-lactamase: a mechanism for ceftazidime hydrolysis J. Biol. Chem. 287, 31783-31793 10.1074/jbc.M112.348540
Kimura, S., Ishiguro, M., Ishii, Y., Alba, J., and Yamaguchi, K. (2004) Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis Antimicrob. Agents Chemother. 48, 1454-1460 10.1128/AAC.48.5.1454-1460.2004
Novais, A., Comas, I., Baquero, F., Canton, R., Coque, T. M., Moya, A., Gonzalez-Candelas, F., and Galan, J. C. (2010) Evolutionary trajectories of β-lactamase CTX-M-1 cluster enzymes: predicting antibiotic resistance PLoS Pathog. 6, e1000735 10.1371/journal.ppat.1000735
Bush, K. and Jacoby, G. A. Updated functional classification of β-lactamases, Antimicrob. Agents Chemother. 2010, 54, 969-976. 10.1128/AAC.01009-09
Villegas, M. V., Correa, A., Perez, F., Zuluaga, T., Radice, M., Gutkind, G., Casellas, J. M., Ayala, J., Lolans, K., and Quinn, J. P. (2004) CTX-M-12 β-lactamase in a Klebsiella pneumoniae clinical isolate in Colombia Antimicrob. Agents Chemother. 48, 629-631 10.1128/AAC.48.2.629-631.2004
Clinical and Laboratory Standards Institute. ((2013) Performance Standards for Antimicrobial Susceptibility Testing; twenty-third informational supplement M100-S22, Clinical and Laboratory Standards Institute, Wayne, PA, USA.
Foulds, J. and Chai, T. (1979) Isolation and characterization of isogenic E. coli strains with alterations in the level of one or more major outer membrane proteins Can. J. Microbiol. 25, 423-427 10.1139/m79-065
Hansen, J. B. and Olsen, R. H. (1978) Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5 J. Bacteriol. 135, 227-238
Pridmore, R. D. (1987) New and versatile cloning vectors with kanamycin-resistance marker Gene 56, 309-312 10.1016/0378-1119(87)90149-1
Power, P., Radice, M., Barberis, C., de Mier, C., Mollerach, M., Maltagliatti, M., Vay, C., Famiglietti, A., and Gutkind, G. (1999) Cefotaxime-hydrolysing β-lactamases in Morganella morganii Eur. J. Clin. Microbiol. Infect. Dis. 18, 743-747 10.1007/s100960050391
Segel, I. H. (1975) Enzyme Kinetics, Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems; John Wiley & Sons, Inc., New York.
De Meester, F., Joris, B., Reckinger, G., Bellefroid-Bourguignon, C., Frere, J. M., and Waley, S. G. (1987) Automated analysis of enzyme inactivation phenomena. Application to β-lactamases and DD-peptidases Biochem. Pharmacol. 36, 2393-2403 10.1016/0006-2952(87)90609-5
Papp-Wallace, K. M., Bethel, C. R., Distler, A. M., Kasuboski, C., Taracila, M., and Bonomo, R. A. (2010) Inhibitor resistance in the KPC-2 β-lactamase, a preeminent property of this class A β-lactamase Antimicrob. Agents Chemother. 54, 890-897 10.1128/AAC.00693-09
Power, P., Galleni, M., Ayala, J. A., and Gutkind, G. (2006) Biochemical and molecular characterization of three new variants of AmpC β-lactamases from Morganella morganii Antimicrob. Agents Chemother. 50, 962-967 10.1128/AAC.50.3.962-967.2006
Kabsch, W. (2010) Integration, scaling, space-group assignment and post-refinement Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 133-144 10.1107/S0907444909047374
Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240-255 10.1107/S0907444996012255
Painter, J. and Merritt, E. A. (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 439-450 10.1107/S0907444906005270
Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126-2132 10.1107/S0907444904019158
The PyMOL Molecular Graphics System, 1.5.0.4 ed., Schrödinger LLC, New York.
Notredame, C., Higgins, D. G., and Heringa, J. (2000) T-Coffee: A novel method for fast and accurate multiple sequence alignment J. Mol. Biol. 302, 205-217 10.1006/jmbi.2000.4042
Gouet, P., Robert, X., and Courcelle, E. (2003) ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins Nucleic Acids Res. 31, 3320-3323 10.1093/nar/gkg556
Shimamura, T., Ibuka, A., Fushinobu, S., Wakagi, T., Ishiguro, M., Ishii, Y., and Matsuzawa, H. (2002) Acyl-intermediate structures of the extended-spectrum class A β-lactamase, Toho-1, in complex with cefotaxime, cephalothin, and benzylpenicillin J. Biol. Chem. 277, 46601-46608 10.1074/jbc.M207884200
Padayatti, P. S., Helfand, M. S., Totir, M. A., Carey, M. P., Carey, P. R., Bonomo, R. A., and van den Akker, F. (2005) High resolution crystal structures of the trans-enamine intermediates formed by sulbactam and clavulanic acid and E166A SHV-1 β-lactamase J. Biol. Chem. 280, 34900-34907 10.1074/jbc.M505333200
Krieger, E., Darden, T., Nabuurs, S. B., Finkelstein, A., and Vriend, G. (2004) Making optimal use of empirical energy functions: force-field parameterization in crystal space Proteins: Struct., Funct., Genet. 57, 678-683 10.1002/prot.20251
Krieger, E., Joo, K., Lee, J., Raman, S., Thompson, J., Tyka, M., Baker, D., and Karplus, K. (2009) Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8 Proteins: Struct., Funct., Genet. 77, 114-122 10.1002/prot.22570
Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., and Pedersen, L. G. (1995) A smooth particle mesh Ewald method J. Chem. Phys. 103, 8577-8593 10.1063/1.470117
Schmid, F. (1989) Spectral methods of characterizing protein conformation and conformational changes., In Protein Structure: A Practical Approach (Creighton, T. E., Ed.) IRL, New York.
Bae, I. K., Lee, Y. N., Hwang, H. Y., Jeong, S. H., Lee, S. J., Kwak, H. S., Song, W., Kim, H. J., and Youn, H. (2006) Emergence of CTX-M-12 extended-spectrum β-lactamase-producing Escherichia coli in Korea J. Antimicrob. Chemother. 58, 1257-1259 10.1093/jac/dkl397
Ambler, R. P., Coulson, A. F., Frere, J. M., Ghuysen, J. M., Joris, B., Forsman, M., Levesque, R. C., Tiraby, G., and Waley, S. G. (1991) A standard numbering scheme for the class A β-lactamases Biochem. J. 276, 269-270
Wilson, A. J. C. (1949) The probability distribution of X-ray intensities Acta Crystallogr. 2, 318-321 10.1107/S0365110X49000813
Ghuysen, J. M. (1991) Serine β-lactamases and penicillin-binding proteins Annu. Rev. Microbiol. 45, 37-67 10.1146/annurev.mi.45.100191.000345
Ibuka, A., Taguchi, A., Ishiguro, M., Fushinobu, S., Ishii, Y., Kamitori, S., Okuyama, K., Yamaguchi, K., Konno, M., and Matsuzawa, H. (1999) Crystal structure of the E166A mutant of extended-spectrum β-lactamase Toho-1 at 1.8 Å resolution J. Mol. Biol. 285, 2079-2087 10.1006/jmbi.1998.2432
Delmas, J., Leyssene, D., Dubois, D., Birck, C., Vazeille, E., Robin, F., and Bonnet, R. (2010) Structural insights into substrate recognition and product expulsion in CTX-M enzymes J. Mol. Biol. 400, 108-120 10.1016/j.jmb.2010.04.062
Pages, J. M., James, C. E., and Winterhalter, M. (2008) The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria Nat. Rev. Microbiol. 6, 893-903 10.1038/nrmicro1994
Mahendran, K. R., Kreir, M., Weingart, H., Fertig, N., and Winterhalter, M. (2010) Permeation of antibiotics through Escherichia coli OmpF and OmpC porins: screening for influx on a single-molecule level J. Biomol. Screening 15, 302-307 10.1177/1087057109357791
