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• W2005883611 abstract "The development of high level β-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant Streptococcus pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime is 12 mg·liter-1). Mutations T371A, which is adjacent to the catalytic nucleophile Ser370, and TSQF(574–577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with β-lactams were probed in vitro, and their effect on resistance was measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared with that of PBP1a from the β-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370. The development of high level β-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant Streptococcus pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime is 12 mg·liter-1). Mutations T371A, which is adjacent to the catalytic nucleophile Ser370, and TSQF(574–577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with β-lactams were probed in vitro, and their effect on resistance was measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared with that of PBP1a from the β-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370. Clinical isolates of Streptococcus pneumoniae are now often resistant to multiple drugs. In France, where the problem is particularly significant, ∼50% of the isolates have decreased susceptibility to β-lactams, most often combined with resistance to other classes of antibiotics such as erythromycin, tetracyclines, or sulfonamides (1Maugein J. Croizé J. Ros A. Bourdon S. Brun M. Cattier B. Chanal C. Chabanon G. Chardon H. Chomarat M. Coignard B. Demachy M.-C. Donnio P.-Y. Dupont P. Fosse T. Gravet A. Grignon B. Laurans G. Péchinot A. Ploy M.-C. Roussel-Delvallez M. Thoreux P.-H. Varon E. Vergnaud M. Vernet-Garnier V. Weber M. Bulletin Epidémiologique Hebdomadaire. 2006; 1: 6-8Google Scholar). β-Lactams interfere with the formation of peptidoglycan, an essential component of the cell wall, by inhibiting the transpeptidation reaction that cross-links peptidoglycan stem peptides. This reaction, catalyzed by penicillin-binding proteins (PBPs), 2The abbreviations used are: PBPpenicillin-binding proteinTPtranspeptidaseGTglycosyltransferaseCTXcefotaximepenGpenicillin GMES2-morpholinoethanesulfonic acidr.m.s.root-mean-squareGSTglutathione S-transferasePEGpolyethyleneglycol. 2The abbreviations used are: PBPpenicillin-binding proteinTPtranspeptidaseGTglycosyltransferaseCTXcefotaximepenGpenicillin GMES2-morpholinoethanesulfonic acidr.m.s.root-mean-squareGSTglutathione S-transferasePEGpolyethyleneglycol. is essential for the stability of the cell wall. The formation of long-lived covalent adducts by β-lactam antibiotics within the PBP active site results in growth arrest or cell death (2Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (624) Google Scholar). Studies of the mechanisms of pneumococcal resistance to β-lactams have been performed for the past two decades through the employment of techniques ranging from epidemiological to structural analyses, and have converged on a common fact: S. pneumoniae resists β-lactams by introducing amino acid substitutions into key PBPs, which have then a decreased affinity for these antibiotics (3Hakenbeck R. Grebe T. Zahner D. Stock J.B. Mol Microbiol. 1999; 33: 673-678Crossref PubMed Scopus (122) Google Scholar). The altered enzymes retain nonetheless their physiological activities. Of the six pneumococcal PBPs, three are modified in most resistant strains: PBP2b, PBP2x, and PBP1a (4Dowson C.G. Hutchison A. Brannigan J.A. George R.C. Hansman D. Linares J. Tomasz A. Smith J.M. Spratt B.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8842-8846Crossref PubMed Scopus (314) Google Scholar, 5Laible G. Spratt B.G. Hakenbeck R. Mol. Microbiol. 1991; 5: 1993-2002Crossref PubMed Scopus (211) Google Scholar, 6Martin C. Sibold C. Hakenbeck R. EMBO J. 1992; 11: 3831-3836Crossref PubMed Scopus (79) Google Scholar). These low-affinity PBPs are encoded by mosaic genes that combine sequence fragments from various origins, including closely related species such as Streptococcus mitis and Streptococcus oralis (7Dowson C.G. Coffey T.J. Kell C. Whiley R.A. Mol. Microbiol. 1993; 9: 635-643Crossref PubMed Scopus (206) Google Scholar, 8Sibold C. Henrichsen J. Konig A. Martin C. Chalkley L. Hakenbeck R. Mol. Microbiol. 1994; 12: 1013-1023Crossref PubMed Scopus (124) Google Scholar). Although the geographic extent of resistance has been attributed to the spread of a small number of initial clones (9McGee L. McDougal L. Zhou J. Spratt B.G. Tenover F.C. George R. Hakenbeck R. Hryniewicz W. Lefevre J.C. Tomasz A. Klugman K.P. J. Clin. Microbiol. 2001; 39: 2565-2571Crossref PubMed Scopus (440) Google Scholar), the genetic plasticity is such that several dozen of different sequences have now been documented for the three low-affinity PBPs. Examination of the sequences and crystal structures of PBP2x variants have revealed the existence of at least two distinct mechanisms for reducing the affinity of this enzyme for β-lactams (10Pernot L. Chesnel L. Le Gouellec A. Croize J. Vernet T. Dideberg O. Dessen A. J. Biol. Chem. 2004; 279: 16463-16470Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The transpeptidase (TP) domains of low-affinity PBPs typically contain between 20 and 40 amino acid substitutions (when compared with PBPs from the β-lactam-susceptible R6 strain). Many of the substitutions are likely irrelevant to the resistance mechanism, as they have been imported through homologous recombination with mutations that truly decrease the affinity for β-lactams. We have shown recently that out of 41 substitutions in PBP2x from the highly resistant strain 5204, only 6 contribute significantly to lowering the affinity for β-lactams (11Carapito R. Chesnel L. Vernet T. Zapun A. J. Biol. Chem. 2006; 281: 1771-1777Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). penicillin-binding protein transpeptidase glycosyltransferase cefotaxime penicillin G 2-morpholinoethanesulfonic acid root-mean-square glutathione S-transferase polyethyleneglycol. penicillin-binding protein transpeptidase glycosyltransferase cefotaxime penicillin G 2-morpholinoethanesulfonic acid root-mean-square glutathione S-transferase polyethyleneglycol. PBP2b and PBP2x are often referred to as primary determinants of β-lactam resistance, because cultivation of pneumococci in the presence of low amounts of piperacillin or cefotaxime selects for mutations in PBP2b or PBP2x, respectively (12Grebe T. Hakenbeck R. Antimicrob. Agents Chemother. 1996; 40: 829-834Crossref PubMed Google Scholar). However, the levels of resistance conferred by low-affinity PBP2b and/or PBP2x variants are limited by the inhibition of the other unmodified PBPs. High levels of resistance are achieved only upon further alteration of PBP1a (13Barcus V.A. Ghanekar K. Yeo M. Coffey T.J. Dowson C.G. FEMS Microbiol. Lett. 1995; 126: 299-303Crossref PubMed Google Scholar, 14Reichmann P. Konig A. Marton A. Hakenbeck R. Microb. Drug Resist. 1996; 2: 177-181Crossref PubMed Scopus (52) Google Scholar, 15Smith A.M. Klugman K.P. Antimicrob. Agents Chemother. 1998; 42: 1329-1333Crossref PubMed Google Scholar, 16du Plessis M. Bingen E. Klugman K.P. Antimicrob. Agents Chemother. 2002; 46: 2349-2357Crossref PubMed Scopus (67) Google Scholar). Low-affinity PBP1a is therefore the most troublesome PBP from a clinical point of view. PBP1a is a bifunctional, class A enzyme, in that it consists of a short cytoplasmic region, an N-terminal membrane anchor segment, followed by a glycosyltransferase domain (GT), a linker region and a TP domain (17Macheboeuf P. Contreras-Martel C. Job V. Dideberg O. Dessen A. FEMS Microbiol. Rev. 2006; 30: 673-691Crossref PubMed Scopus (298) Google Scholar). The crystal structure of a soluble, proteolyzed form of PBP1a from strain R6 reveals that the TP domain, whose fold is typical of the serine penicilloyl transferase family of enzymes, is flanked by a short C-terminal region and an N-terminal β-strand-rich region, which carries a small peptide of the glycosyltransferase domain and serves as a linker between the two catalytic units (18Contreras-Martel C. Job V. Di Guilmi A.M. Vernet T. Dideberg O. Dessen A. J. Mol. Biol. 2006; 355: 684-696Crossref PubMed Scopus (61) Google Scholar). Among 47 publicly available different sequences representing the TP domains from PBP1a variants (residues 264–654), 7 are very similar to the sequence of PBP1a from strain R6 with 1 to 4 amino acid substitutions. In contrast, 39 sequences are clearly mosaic and are presumably all from strains with reduced susceptibility to β-lactams. They carry between 13 and 60 substitutions over the entire TP domain. Individual reversion of the substitutions in PBP1a from strain 3191 (19Smith A.M. Klugman K.P. Antimicrob. Agents Chemother. 2003; 47: 387-389Crossref PubMed Scopus (29) Google Scholar), which harbors 52 mutations in the TP domain, reduced the level of resistance in only two cases: a L539W substitution that is present in only two sequences, and the TSQF to NTGY substitutions at positions 574–577, which are present in nearly all mosaic sequences (38 out of 39). Here we present the crystal structure of a soluble form of PBP1a from the highly resistant pneumococcal strain 5204 (MICpenG = 6.0 mg·liter-1; MICCTX = 12.0 mg·liter-1) (20Chesnel L. Pernot L. Lemaire D. Champelovier D. Croize J. Dideberg O. Vernet T. Zapun A. J. Biol. Chem. 2003; 278: 44448-44456Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This PBP1a carries 48 substitutions when compared with the R6 strain, including 45 within the TP domain. The reaction between penicillin G and cefotaxime, and four different PBP1a clinical variants, as well as point mutants in positions 574–577, were also studied. The impact of these PBP1a variants on resistance was measured. The results are interpreted within the framework of the crystal structures of PBP1a from strains R6 and 5204. Plasmids and Site-directed Mutagenesis–Pneumococcal strains 4790, 5204, 5245, 5259 (University Hospital, Grenoble, France), and R6 were used as sources of genomic DNA for the amplification of pbp1a genes. The fragments encoding the extracellular region of PBP1a (residues 37–719, henceforth noted PBP1a) were PCR-amplified and introduced as BamHI/XhoI fragments in place of the pbp2x fragment in pGEX-S-pbp2x*-f1 (21Mouz N. Di Guilmi A.M. Gordon E. Hakenbeck R. Dideberg O. Vernet T. J. Biol. Chem. 1999; 274: 19175-19180Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This vector had been previously modified by site-directed mutagenesis to eliminate a second undesired BamHI site (GGATCC into GGGTCC) in the f1 region. Site-directed mutagenesis of pbp1a genes from strains R6 and 5204, to introduce or revert mutations T371A and TSQF(574–577)NTGY, were performed using Kunkel's automated method (22Kunkel T. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (632) Google Scholar, 23Carapito R. Gallet B. Zapun A. Vernet T. Anal. Biochem. 2006; 355: 110-116Crossref PubMed Scopus (11) Google Scholar) following production of single-stranded plasmids. In addition, replacement of residues 490–570 of 4790-PBP1a by the corresponding residues from 5245-PBP1a, was performed by employing a two-step PCR strategy. Three fragments were amplified: the region of 4790-PBP1a N-terminal to the target region, the zone C-terminal to the target region, and the 490–570 region of 5245-PBP1a. The three fragments were combined in a second PCR experiment, and introduced as a BamHI/XhoI fragment into modified pGEX-4T1-f1 (in which a BamHI site had been removed). All clones were verified by double-stranded sequencing (Cogenics Genome Express, Grenoble). Protein Expression and Purification–PBP1a variants were produced by employing the same methodology used for PBP1a from strain R6 (18Contreras-Martel C. Job V. Di Guilmi A.M. Vernet T. Dideberg O. Dessen A. J. Mol. Biol. 2006; 355: 684-696Crossref PubMed Scopus (61) Google Scholar), albeit in this work protein expression was performed in Escherichia coli MC1061 cells grown in autoinduction medium (24Studier F.W. Protein Expr. Purif. 2005; 41: 207-234Crossref PubMed Scopus (3954) Google Scholar) complemented with 100 mg·liter-1 of ampicillin, initially for 3 h at 37 °C and subsequently for 21 h at 20 °C. After similar lysis, centrifugation, and glutathione affinity steps, PBP1a variants were purified on a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated with 10 mm sodium phosphate, pH 7.0, and 0.2 m KCl. Yields were between 4 and 12 mg per liter of culture, depending on the variant. Purity was greater than 95% as judged by Coomassie-stained SDS-PAGE. Crystallization and Data Collection–5204-PBP1a* was prepared for crystallization trials by initial digestion the GST-PBP1a fusion protein with a 1:1000 trypsin:protein ratio for 1 h. The asterisk denotes the proteolyzed form used for crystallization, corresponding mostly to the TP domain. The resulting protein, which consists of two associated peptides (residues 47–70 and 264–653), was subsequently purified from the GST tag with a second glutathione chromatography step, which was followed by anion exchange chromatography (MonoQ HR 5/5) in 50 mm Tris-HCl, pH 8.0, 1 mm EDTA. After elution of the protein with a linear gradient to 0.5 m NaCl, 5204-PBP1a* was loaded onto a gel filtration column (Superdex 200, GE Healthcare) in 20 mm HEPES pH 7.0 and 0.1 m NaCl. Protein-containing fractions were pooled and concentrated to 4 mg/ml prior to crystallization. The homogeneity and identity of the sample were verified by SDS-PAGE and electrospray mass spectrometry using a Q-TOF Micro spectrometer (Micromass, Manchester, UK) with an electro-spray ion source operated with a needle voltage of 3 kV, and sample cone and extraction voltages of 55 and 2 V, respectively. Spectra were recorded in the 500–2100 range of mass-to-charge (m/z) ratios. The sample was infused at 1 μm by diluting the sample in H2O/CH3CN (1/1 v/v), 0.2% formic acid. The data are treated by the Masslynx software (Waters). Measured masses were 2635.3 ± 0.2 Da and 43401 ± 5 Da, corresponding to the tryptic fragments 47–70 (predicted m = 2635 Da) and 264–653 (predicted m = 43395 Da). Crystals appeared in a variety of conditions in pH values ranging from 5.5 to 8.5 with different precipitants (PEG, (NH4)2SO4, NaCl). The best crystals were prepared in 0.1 m MES pH 6.0, 21% PEG 6000, 17 mm BaCl2 at 15 °C. Crystals were cryo-cooled after sequential transfer into crystallization solutions containing increasing amounts of ethylene glycol, until a maximum value of 20%. A dataset was collected to 1.9 Å at the European Synchrotron Radiation Facility (ESRF-Grenoble) ID14-EH3 beamline at 0.931 Å. Data processing was carried out with XDS (25Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3200) Google Scholar). The crystal structure was solved by molecular replacement using AMoRE (26Navaza J. Acta Crystallogr. Sect. D. 2001; 57: 1367-1372Crossref PubMed Scopus (659) Google Scholar) by employing the structure of PBP1a* from strain R6 as a model (PDB code 2C6W) in which active site residues were removed in order to avoid bias, and all residues differing between the two proteins were replaced by alanines. Refinement with REFMAC and CNS (27Collaborative Computing Program Number 4 and Collaborative Computing Project for NMR (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-766Google Scholar, 28Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. T. S. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar), which included energy minimization, temperature factor refinement, and simulated annealing steps, was intercalated with iterative cycles of manual model building in COOT. Water molecules (203) were added to the structure using ARP/warp (29Morris R.J. Perrakis A. Lamzin V.S. Methods Enzymol. 2003; 374: 229-244Crossref PubMed Scopus (469) Google Scholar) in combination with REFMAC. MES and Ba2+ ions were added manually in COOT. Model quality was assessed with PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Data collection and refinement statistics are included in Table 1.TABLE 1Data collection and refinementData collectionSpace groupC2a (Å)122.9b (Å)67.0c (Å)49.1β (°)100.9Resolution limits (Å)19.7-1.9Total number of reflections23,917Number of unique reflections6,200Completeness (%)96.1 (93.2)aThe numbers in parentheses represent the values for the highest resolution shellRsym (%)11.7 (36.8)I/σ(I)11.1 (4.0)RefinementR-factor(%)21.2R-free(%)25.1No. protein atoms2,985No. of water molecules203No. barium ions1Mean B factor (Å2)13.1R.m.s.d from ideal geometryBond lengths (Å)0.006Bond angles (°)1.3a The numbers in parentheses represent the values for the highest resolution shell Open table in a new tab Cavity Calculations–The cavity localization and volumes present in PBP1a* proteins from strains R6 and 5204 were calculated using the SURFNET program (31Laskowski R.A. J. Mol. Graph. 1995; 13: 323-330Crossref PubMed Scopus (809) Google Scholar). The calculations were carried out in the absence of water molecules, and a sphere of radius of 1.5 Å was used. Acylation Efficiencies–The k2/K parameter was measured by following the decrease of the intrinsic fluorescence of the proteins (32Di Guilmi A.M. Mouz N. Andrieu J.P. Hoskins J. Jaskunas S.R. Gagnon J. Dideberg O. Vernet T. J. Bacteriol. 1998; 180: 5652-5659Crossref PubMed Google Scholar), at various concentrations of a large excess of antibiotic, using a SFM-400 stopped-flow apparatus (Bio-Logic). Measurements were performed at 37 °C in a 100 mm sodium phosphate, pH 7.0 buffer with 0.6 μm protein and 50–1000 μm of penicillin G or cefotaxime. The excitation wavelength was 280 nm, and the emission was measured above 305 nm using a cut-off filter. The apparent pseudo-first order rate constant kapp was determined by non-linear fitting of the fluorescence data to equation Fluot = Fluo0·exp(-kapp·t) + a·t + c using the Bio-Kine software (Bio-Logic), where the a·t + c accounts for the slightly drifting fluorescence signal at the end of the reaction. The efficiency of acylation k2/K was determined by least squares linear fitting to the equation kapp = (k2/K)·[antibiotic] with Kaleidagraph (Synergy Software, Reading, PA). The mosaic variants of PBP1a showed no change of intrinsic fluorescence upon binding of penicillin G. Thus, the pseudo first-order rate constant for the acylation of the mosaic protein by penicillin G was measured by direct competition with cefotaxime (33Graves-Woodward K. Pratt R.F. Biochem. J. 1998; 332: 755-761Crossref PubMed Scopus (48) Google Scholar). In these cases, kapp(penG) = kapp(mixture) - kapp(CTX), where kapp(penG) is the apparent rate of the reaction with penicillin G at the concentration of this antibiotic in the mixture, kapp(mixture) is the rate constant measured with a mixture of penicillin G and cefotaxime and kapp(CTX) is the rate constant measured in the absence of penicillin G but with the same concentration of cefotaxime as that present in the mixture. Transformations and Minimal Inhibitory Concentrations–The non-encapsulated S. pneumoniae R6 strain was used as a recipient for genetic transformation with 5204-pbp2x and various pbp1a variants. To prevent correction of the introduced mutations by the Hex mismatch repair system of the bacteria, an altered R6 strain (hexA::spc) (34Claverys J.P. Lacks S.A. Microbiol. Rev. 1986; 50: 133-165Crossref PubMed Google Scholar) was used when point mutations were introduced. Transformation and measurements of minimal inhibitory concentrations by E-tests were performed as described previously (20Chesnel L. Pernot L. Lemaire D. Champelovier D. Croize J. Dideberg O. Vernet T. Zapun A. J. Biol. Chem. 2003; 278: 44448-44456Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Sequence Comparisons–As with other low-affinity PBPs of clinical origin, the mosaicity of PBP1a defies attempts at phylogenetic-like classification. However, it appears that, with a single exception, the mosaic PBP1a sequences all share a common block of substitutions spanning residues 540–612, which includes between 10 and 18 substitutions. This sequence block is characterized by the four consecutive substitutions at positions 574–577 that were found to be important for resistance (19Smith A.M. Klugman K.P. Antimicrob. Agents Chemother. 2003; 47: 387-389Crossref PubMed Scopus (29) Google Scholar). This ubiquitous sequence block is combined in various ways with a variety of other blocks. The only mosaic sequence that does not contain the above-mentioned sequence block is entirely devoid of substitution in its C-terminal part following position 533. In the course of sequencing the PBPs of 23 pneumococcal clinical isolates from the University Hospital in Grenoble, we encountered four distinct groups of mosaic PBP1a sequences, represented by strains 5204, 4790, 5245, and 5259. An alignment of the sequences corresponding to the TP domains of these PBPs is shown in Fig. 1. Sequences from strains 5204, 4790, and 5245 are very similar, and carry 45, 46, and 52 substitutions in the TP domain, respectively, with respect to PBP1a from drug-sensitive strain R6. They are from resistant strains with MICpenG values ≥ 1.5 μg·ml-1, and carry the TSQF(574–577)NTGY substitutions within the 540–612 sequence block, as well as the T371A substitution within the first catalytic motif, next to the active site Ser370. Seven substitutions near the C terminus of the TP domain are absent in 5204-PBP1a and present in PBP1a from strains 4790 and 5245. 4790-PBP1a differs from the two other close sequences by the absence of 9 substitutions, an additional one and a different one in the 495–570 segment. PBP1a from strain 5259 is unusual in that it lacks the T371A substitution, and has no substitution following residue 533, thus lacking the abovementioned mutations in residues 574–577. The TP domain of 5259-PBP1a nevertheless carries 33 mutations, including 24 that are in common with 5204- and 5245-PBP1a. The originating strain 5259 has only a mildly reduced susceptibility to penicillin G (MICpenG = 0.19 μg·ml-1). These four clinical variants of PBP1a, and that from the reference susceptible strain R6, gave us the opportunity to test the impact of their differences on β-lactam resistance. Functional Characterization of PBP1a from Clinical Variants–The recombinant extracellular domains of PBP1a variants from strains R6, 5259, 4790, 5204, and 5245 were produced as GST fusion proteins, and the kinetics of the reaction of each fusion protein with penicillin G and cefotaxime were measured by following the decrease of intrinsic fluorescence that follows binding of the antibiotic. The reaction between PBPs and β-lactams is classically described by Reaction 1, E+IK-1⇄EIk2→EI*k3→E+PREACTION 1 where E is the enzyme, I is the β-lactam, and EI is a non-covalent complex with the dissociation constant K. The covalent acyl-enzyme EI* is formed with the rate constant k2, and finally the enzyme is deacylated with the rate constant k3, to regenerate an active enzyme and release an inactivated compound P. The measured acylation efficiencies, characterized by the second order rate constant k2/K are presented in Table 2.TABLE 2Acylation efficiencies of, and resistance conferred by, PBP1a variants of clinical origins Reactions were performed at 37 °C in 100 mm sodium phosphate, pH 7, and 0.2 m KCl. Rate constants are given with the standard error calculated from two independent experiments with five measurements of kapp each. The susceptible strain R6 (MICCTX < 0.016 μg·ml–1) was cotransformed with 5204-pbp2x.PBP1ak2/KMIC CefotaximePenicillin GCefotaximem–1·s–1μg·ml–1R669000 ± 1500011000 ± 7000.2552598600 ± 6004000 ± 5500.384790820 ± 160700 ± 100.755204430 ± 110480 ± 715245420 ± 90440 ± 401 Open table in a new tab The values of k2/K obtained with R6-PBP1a are consistent with previously published results (32Di Guilmi A.M. Mouz N. Andrieu J.P. Hoskins J. Jaskunas S.R. Gagnon J. Dideberg O. Vernet T. J. Bacteriol. 1998; 180: 5652-5659Crossref PubMed Google Scholar). Mosaic PBP1a proteins have decreased acylation efficiencies by factors of 3 to 25 for cefotaxime, and 8 to 164 for penicillin G. Thus, the alterations in our variants of PBP1a have a greater effect decreasing the reactivity toward penicillin than toward cefotaxime, a cephalosporin. The acylation efficiencies of 5204- and 5245-PBP1a do not differ significantly, indicating that the seven additional substitutions of 5245-PBP1a at the C-terminus of the TP domain are not involved in the low-affinity for β-lactams. The k2/K of 5245-PBP1a for cefotaxime and penicillin G are 1.6- and 2-fold smaller than those of 4790-PBP1a, implying that the differences at 10 positions in the 490–570 region also have a rather limited effect on the reactivity toward β-lactams. The combined effect of the 34 substitutions within the 5259-PBP1a transpeptidase domain, which do not include neither the highly prevalent TSQF(574–577)NTGY substitutions nor the active site T371A mutation, is modest but significant, indicating that mutations other than the aforementioned substitutions can also participate in the decreased affinity of PBP1a for β-lactams. To associate the kinetic observations and the in vivo resistance phenomenon, the four pbp1a genes of clinical origin were introduced into the susceptible recipient strain R6. In order to reveal the effect of mosaic pbp1a genes, and because of the fact that a low affinity PBP1a is incapable of conferring resistance on its own, a fact which is linked to the essential nature and high affinity of R6-PBP2x for cefotaxime, it was necessary to co-transform pbp1a variants together with 5204-pbp2x, which encodes a PBP2x with a very low affinity for cephalosporins (20Chesnel L. Pernot L. Lemaire D. Champelovier D. Croize J. Dideberg O. Vernet T. Zapun A. J. Biol. Chem. 2003; 278: 44448-44456Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The other pneumococcal PBPs are either non-essential, or, as in the case of PBP2b, have a naturally low affinity for cephalosporins. Therefore, resistance to cefotaxime can be conferred by altering only pbp2x and pbp1a. The MIC value for cefotaxime was measured and results are given in Table 2. The PBP1a proteins with the lowest k2/K confer the highest resistance levels. Interestingly, even the modestly decreased efficiency of acylation of 5259-PBP1a increases the resistance level. Role of the T371A and TSQF(574–577)NTGY Mutations–The TSQF(574–577)NTGY block of mutations has been shown to be an important determinant of the level of resistance conferred by PBP1a from strain 3191 (19Smith A.M. Klugman K.P. Antimicrob. Agents Chemother. 2003; 47: 387-389Crossref PubMed Scopus (29) Google Scholar). Another potentially important substitution is T371A, which lies adjacent to Ser370, the active site nucleophile; notably, the same substitution is observed in many mosaic PBP2x variants and has been shown to influence the acylation efficiency of this latter protein (35Mouz N. Gordon E. Di Guilmi A.M. Petit I. Petillot Y. Dupont Y. Hakenbeck R. Vernet T. Dideberg O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13403-13406Crossref PubMed Scopus (75) Google Scholar). To evaluate the impact of these mutations on the reactivity of PBP1a toward β-lactams, a set of six variants were constructed, where the T371A and TSQF(574–577)NTGY mutations were introduced individually or together into R6-PBP1a. The reverse substitutions were also introduced into 5204-PBP1a. The recombinant extracellular domains fused to GST were produced, and their efficiencies of acylation by cefotaxime and penicillin G were measured (Table 3).TABLE 3Acylation efficiencies of, and resistance conferred by, PBP1a point-mutants Reactions were at 37 °C in 100 mm sodium phosphate, pH 7, and 0.2 m KCl. Rate constants are given with the standard error calculated from two independent experiments with five measurements of kapp each. The susceptible strain R6 (MICCTX < 0.016 μg·ml–1) was cotransformed with 5204-pbp2x.PBP1ak2/KMIC CefotaximePenicillin GCefotaximem–1·s–1μg·ml–1R6Wild type69000 ± 1500011000 ± 7000.25T371A2640 ± 1504600 ± 210NTaNT, no transformants were isolated with a resistance to cefotaxime greater than that conferred by 5204-pbp2x aloneTSQF(574–577)NTGY1420 ± 2102000 ± 40NTT371A/TSQF(574–577)NTGY1330 ± 1451140 ± 300.385204Wild type430 ± 110480 ± 71A371T2080 ± 550850 ± 300.5NTGY(574–577)TSQF750 ± 901210 ±" @default.
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• W2005883611 title "Common Alterations in PBP1a from Resistant Streptococcus pneumoniae Decrease Its Reactivity toward β-Lactams" @default.
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