Penicillin-binding Protein 2X Research Articles

Plasmid-mediated ArmA and RmtB 16S rRNA methylases in Escherichia coli isolated from chickens

1. Kimura K, Suzuki S, Wachino J et al. First molecular characterization of group B streptococci with reduced penicillin susceptibility. Antimicrob Agents Chemother 2008; 52: 2890–7. 2. Nagano N, Nagano Y, Kimura K et al. Genetic heterogeneity in pbp genes among clinically isolated group B streptococci with reduced penicillin susceptibility. Antimicrob Agents Chemother 2008; 52: 4258–67. 3. Jones N, Bohnsack JF, Takahashi S et al. Multilocus sequence typing system for group B Streptococcus. J Clin Microbiol 2003; 41: 2530–6. 4. Bisharat N, Jones N, Marchaim D et al. Population structure of group B Streptococcus from a low-incidence region for invasive neonatal disease. Microbiology 2005; 151: 1875–81. 5. Maurer P, Koch B, Zerfass I et al. Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodeling an essential enzyme into a resistance determinant. J Mol Biol 2008; 376: 1403–16.

Discovery of New Inhibitors of Resistant Streptococcus pneumoniae Penicillin Binding Protein (PBP) 2x by Structure-Based Virtual Screening

Penicillin binding proteins (PBPs) are involved in the biosynthesis of the peptidoglycan layer constitutive of the bacterial envelope. They have been targeted for more than half a century by extensively derived molecular scaffolds of penicillins and cephalosporins. Streptococcus pneumoniae resists the antibiotic pressure by inducing highly mutated PBPs that can no longer bind the beta-lactam containing agents. To find inhibitors of PBP2x from Streptococcus pneumoniae (spPBP2x) with novel chemical scaffold so as to circumvent the resistance problems, a hierarchical virtual screening procedure was performed on the NCI database containing approximately 260000 compounds. The calculations involved ligand-based pharmacophore mapping studies and molecular docking simulations in a homology model of spPBP2x from the highly resistant strain 5204. A total of 160 hits were found, and 55 were available for experimental tests. Three compounds harboring two novel chemical scaffolds were identified as inhibitors of the resistant strain 5204-spPBP2x at the micromolar range.

The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

BackgroundThe serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.ResultsDeletion replacement mutation in stkP conferred hypersensitivity to penicillin G and was epistatic on mutations in PBP2X, PBP2B and PBP1A from the resistant 9V clinical isolate URA1258. Genetic analysis of 55 clinical isolates identified 11 StkP alleles differing from the reference R6 allele. None relevant mutation in the kinase or the PASTA domains were found to account for susceptibility of the isolates. Rather the minimal inhibitory concentration (MIC) values of the strains appeared to be determined by their PBP alleles.ConclusionThe results of genetic dissection analysis in lab strain Cp1015 reveal that StkP is involved in the bacterial response to penicillin and is epistatic on mutations PBP 2B, 2X and 1A. However analysis of the clinical isolates did not allow us to find the StkP alleles putatively involved in determining the virulence or the resistance level of a given strain, suggesting a strong conservation of StkP in clinical isolates.

Comparative Genome Analysis of High-Level Penicillin Resistance inStreptococcus pneumoniae

Streptococcus pneumoniae strains with very high levels of penicillin resistance (minimum inhibitory concentration [MIC] >or=8 microg/ml) emerged in the 1990 s. Previous studies have traced the changes in penicillin binding proteins (PBP) that result in decreased penicillin susceptibility, and the role of several PBP genes in high-level resistance. In the present study, we investigated the changes that occurred at the two highest levels of penicillin resistance using NimbleGen's Comparative Genome Sequencing (CGS) technology. DNA from a highly resistant (Pen MIC 16 microg/ml) pneumococcus was used to serially transform the R6 strain to high-level resistance. Four distinct levels of penicillin resistance above the susceptible R6 strain (MIC 0.016 microg/ml) were identified. Using CGS technology, the entire genome sequences of the two highest levels of resistant transformants were examined for changes associated with the resistance phenotypes. At the third level of resistance, changes in PBPs 1a, 2b, and 2x were found, very similar to previous reports. At the fourth resistance level, two additional changes were observed in the R6 transformants. More changes were observed in PBP2x, as well as in peptidoglycan GlcNAc deacetylase (pdgA), which had a missense mutation in the coding region. Genetic transformation with polymerase chain reaction (PCR) products generated from the high-level resistant parent containing either the additional PBP2x or mutant pdgA gene did not increase the MIC of the third-level transformant. Only when both PCR products were simultaneously transformed into the third-level transformant did colonies emerge that were at the highest level of resistance (16-32 microg/ml), equivalent to the highly resistant parent strain. This is the first instance of the involvement of a variant pdgA gene in penicillin resistance. It is also clear from these experiments and the literature that there are multiple paths to the pneumococcus achieving high-level penicillin resistance.

Genetic heterogeneity in pbp genes among clinically isolated group B Streptococci with reduced penicillin susceptibility.

The recent emergence of group B streptococcal isolates exhibiting increased penicillin MICs at the Funabashi Municipal Medical Center and other hospitals in Japan prompted a comparative analysis of the penicillin-binding proteins (PBPs) from those strains with the PBPs from penicillin-susceptible strains comprising four neonatal invasive strains isolated from 1976 to 1988 and two recent isolates. The PBP sequences of the penicillin-susceptible strains were highly conserved, irrespective of their isolation date. Of six strains with reduced susceptibility to penicillin (penicillin MICs, 0.25 to 0.5 mug/ml), strains R1, R2, R5, and R6 shared a unique set of five amino acid substitutions, including V405A adjacent to the (402)SSN(404) motif in PBP 2X and one in PBP 2B. The remaining two strains, R3 and R4, shared several substitutions, including Q557E adjacent to the (552)KSG(554) motif in PBP 2X, in addition to the substitutions in PBP 2B, which are commonly found among penicillin-insusceptible strains. Strains R7 and R8, which had a penicillin MIC of 1 mug/ml, shared a unique set of eight amino acid substitutions (two in PBP 2X; two in PBP 2B, including G613R adjacent to the (614)KTG(616) motif; three in PBP 1A; and one in PBP 2A), and the Q557E substitution in PBP 2X was common to R3 and R4. The binding of Bocillin FL was reduced or not detected in some PBPs, including PBP 2X of penicillin-insusceptible strains, but no significant reduction in the level of pbp2x transcription was found in such strains. The results of phylogenetic comparative analyses imply the absence of epidemic penicillin-insusceptible strains, and several genetic lineages of penicillin-insusceptible strains have been independently emerging through the accumulation of mutations in their pbp genes, especially in pbp2x.

Molecular characteristics of penicillin-binding protein 2b, 2x, and 1a sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates in Shenyang, China

The aim of this study was to investigate the nature of the amino acid motifs found in penicillin-binding proteins (PBP) 2b, 2x, and 1a of penicillin-nonsusceptible Streptococcus pneumoniae isolates from Shenyang, China, and to obtain information regarding the prevalence of alterations within the motifs or in positions flanking the motifs. For 18 clinical isolates comprising 4 penicillin-susceptible S. pneumoniae, 5 penicillin-intermediate S. pneumoniae, and 9 penicillin-resistant S. pneumoniae. the DNA sequences of PBP2b, PBP2x, and PBP1a transpeptidase domains were determined and then genotyped by multilocus sequence typing. Sequence analysis revealed that most penicillin-nonsusceptible S. pneumoniae isolates (penicillin MIC > or = 1.5 microg/mL and cefotaxime MIC > or = 2 microg/mL) shared identical PBP2b, PBP2x, and PBP1a amino acid profiles. Most penicillin-resistant S. pneumoniae isolates were ST320 (4-16-19-15-6-20-1), the double-locus variant of the Taiwan19F-14 clone. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Shenyang, China.

Crystal Structures of Biapenem and Tebipenem Complexed with Penicillin-Binding Proteins 2X and 1A from Streptococcus pneumoniae

Biapenem is a parenteral carbapenem antibiotic that exhibits wide-ranging antibacterial activity, remarkable chemical stability, and extensive stability against human renal dehydropeptidase-I. Tebipenem is the active form of tebipenem pivoxil, a novel oral carbapenem antibiotic that has a high level of bioavailability in humans, in addition to the above-mentioned features. beta-lactam antibiotics, including carbapenems, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in peptidoglycan biosynthesis. To envisage the binding of carbapenems to PBPs, we determined the crystal structures of the trypsin-digested forms of both PBP 2X and PBP 1A from Streptococcus pneumoniae strain R6, each complexed with biapenem or tebipenem. The structures of the complexes revealed that the carbapenem C-2 side chains form hydrophobic interactions with Trp374 and Thr526 of PBP 2X and with Trp411 and Thr543 of PBP 1A. The Trp and Thr residues are conserved in PBP 2B. These results suggest that interactions between the C-2 side chains of carbapenems and the conserved Trp and Thr residues in PBPs play important roles in the binding of carbapenems to PBPs.

Positive selection in penicillin-binding proteins 1a, 2b, and 2x from Streptococcus pneumoniae and its correlation with amoxicillin resistance development

The efficacy of β-lactam antibiotics in Streptococcus pneumoniae has been compromised because of the development of altered penicillin-binding proteins (PBPs), however, this has been less so for amoxicillin than for penicillin. Recently, there have been a number of important methods developed to detect molecular adaptation in protein coding genes. The purpose of this study is to employ modern molecular selection approaches to predict sites under positive selection pressure in PBPs, derived from a large international S. pneumoniae collection of amoxicillin resistant and susceptible isolates, and encompassing a comparative data set of 354 pbp1a, 335 pbp2b, and 389 pbp2x gene sequences. A correspondence discriminant analysis (CDA) of positively selected pbp sites and amoxicillin MIC (minimum inhibitory concentration) values is then used to detect sites under positive selection pressure that are important in discriminating different amoxicillin MICs. Molecular adaptation was evident throughout PBP2X, with numerous positively selected sites in both the transpeptidase (TP) and C-terminal domains, strongly correlated with discriminating amoxicillin MICs. In the case of PBP1A positive selection was present in the glycosyltransfer (GT), TP and C-terminal domains. Sites within the TP domain tended to be correlated with the discrimination of low from intermediate MICs, whereas sites within the C-terminal tail, with a discrimination of intermediate from fully resistant. Most of the positively selected sites within PBP2B were in the N-terminal domain and were not correlated with amoxicillin MICs, however, several sites taken from the literature for the TP domain were strongly associated with discriminating high from intermediate level amoxicillin resistance. Many of the positively selected sites could be directly associated with functional inferences based on the crystal structures of these proteins. Our results suggest that clinical emphasis on TP domain sequences of these proteins may result in missing information relevant to antibiotic resistance development.

Penicillin-binding Protein 2x of Streptococcus pneumoniae: Three New Mutational Pathways for Remodelling an Essential Enzyme into a Resistance Determinant

Mutations in the transpeptidase domain of penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae that reduce the affinity to beta-lactams are important determinants of resistance to these antibiotics. We have now analyzed in vitro and in vivo properties of PBP2x variants from cefotaxime-resistant laboratory mutants and a clinical isolate. The patterns of two to four resistance-specific mutations present in each of the proteins, all of which are placed between 6.6 and 24 Å around the active site, fall into three categories according to their positions in the three-dimensional structure. The first PBP2x group is characterized by mutations at the end of helix α11 and carries the well-known T550A change and/or one mutation on the surface of the penicillin-binding domain in close contact with the C-terminal domain. All group I proteins display very low acylation efficiencies, ≤ 1700 M − 1 s − 1 , for cefotaxime. The second class represented by PBP2x of the mutant C505 shows acylation efficiencies below 100 M − 1 s − 1 for both cefotaxime and benzylpenicillin and contains the mutation L403F at a critical site close to the active serine. PBP2x of the clinical isolate 669 reveals a third mutational pathway where at least the two mutations Q552E and S389L are important for resistance, and acylation efficiency is reduced for both beta-lactams to around 10,000 M − 1 s − 1 . In each group, at least one mutation is located in close vicinity to the active site and mediates a resistance phenotype in vivo alone, whereas other mutations might exhibit secondary effects only in context with other alterations.

Meta-analysis of antibiotic susceptibility and the genotype of penicillin-binding proteins in Streptococcus pneumoniae

To further understanding of the mechanisms of development of resistance to penicillin in Streptococcus pneumoniae, and the role of penicillin-binding proteins (PBPs) mutations to antibiotics resistance a meta-analysis was performed. Major databases, Pubmed, Current Contents, Biosis previews, Web of Science, were searched for studies that published within 1997 through to 2007, and reported the penicillin MIC and the alteration of PBP 1a, 2b and 2x (genes or proteins) of clinical S. pneumoniae isolates. Papers were reviewed by 2 persons and used standard criteria to enroll them. Meta-analysis was performed using a random-effects model. Overall, 20 studies were included in the meta-analysis. For the included 1771 clinical S. pneumoniae isolates, the susceptibility to penicillin decreased in inverse proportion to the presence of mutated pbp genes. The mutations of the conserved amino acid motifs STMK and SRNVP of PBP 1A, STMK and LKSG of PBP2X, and SSNT of PBP2B are critical for the penicillin resistance. Those motifs can be used as markers for the penicillin susceptibility of S. pneumoniae. These results are useful in helping define the mechanism of penicillin resistance in S. pneumoniae.

Crystal Structure of Cefditoren Complexed with Streptococcus pneumoniae Penicillin-Binding Protein 2X: Structural Basis for Its High Antimicrobial Activity

Cefditoren is the active form of cefditoren pivoxil, an oral cephalosporin antibiotic used for the treatment of respiratory tract infections and otitis media caused by bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, and methicillin-susceptible strains of Staphylococcus aureus. Beta-lactam antibiotics, including cefditoren, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. To envision the binding of cefditoren to PBPs, we determined the crystal structure of a trypsin-digested form of PBP 2X from S. pneumoniae strain R6 complexed with cefditoren. There are two PBP 2X molecules (designated molecules 1 and 2) per asymmetric unit. The structure reveals that the orientation of Trp374 in each molecule changes in a different way upon the formation of the complex, but each forms a hydrophobic pocket. The methylthiazole group of the C-3 side chain of cefditoren fits into this binding pocket, which consists of residues His394, Trp374, and Thr526 in molecule 1 and residues His394, Asp375, and Thr526 in molecule 2. The formation of the complex is also accompanied by an induced-fit conformational change of the enzyme in the pocket to which the C-7 side chain of cefditoren binds. These features likely play a role in the high level of activity of cefditoren against S. pneumoniae.

Promoter and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus gordonii

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

The relative frequency of intraspecific lateral gene transfer of penicillin binding proteins 1a, 2b, and 2x, in amoxicillin resistant Streptococcus pneumoniae

Evidence exists for both interspecific and intraspecific recombination (lateral gene transfer; LGT) involving Streptococcus pneumoniae pbp (penicillin binding protein) loci. LGT of capsular genes, or serotype switching, is also know to occur between S. pneumoniae of different serotype. It is not clear whether intraspecific pbp LGT is relatively common, whether there is a difference in the relative frequency of intraspecific LGT of different pbps, and whether serotype switching is more or less frequent than pbp LGT. The purpose of this study was to use comparative evolutionary biology analysis of 216 international clinical S. pneumoniae isolates, from the Alexander Project collection, to gain insight on these issues, as well as the possible role they might be playing in spreading amoxicillin resistance. All 216 isolates were genotyped using MLST and complete or nearly complete sequences for pbp1a, pbp2b, and pbp2x were determined. Amoxicillin MICs were available for each isolate. pbps were genotyped using phylogenetics and two or more pbp types within a MLST sequence type (ST) or clonal complex were taken as putative cases of pbp LGT; these hypotheses were statistically evaluated using the approximately unbiased (AU) test. Serotypes were determined for 171 of these isolates and the minimum number of switching events necessary to explain the serotype phenotypes for each of the STs and clonal complexes were evaluated. The majority (78%) of the amoxicillin resistant isolates were comprised in 5 clonal complexes. The relative frequency of pbp LGT was greatest for pbp2b and 2x (minimum of 10.2 and 7.8%, respectively, of the isolates consistent with the LGT hypothesis), followed by 1a (3.9%). Serotype switching was more frequent than intraspecific pbp LGT (33% of isolates consistent with serotype switching hypothesis). Although intraspecific LGT of pbps is occurring and has played a role in the spread of amoxicillin resistance in S. pneumoniae, clonal dissemination appears to be more significant.

Proinflammatory activation of Toll-like receptor-2 during exposure of penicillin-resistant Streptococcus pneumoniae to -lactam antibiotics

Disease caused by penicillin-resistant Streptococcus pneumoniae (PRSP) is associated with more suppurative complications than disease caused by penicillin-susceptible S. pneumoniae (PSSP). Exposure of S. pneumoniae to beta-lactam antibiotics enhances the proinflammatory activation of human cells by pneumococci via Toll-like receptor-2 (TLR2). To test the hypothesis that penicillin resistance influences cellular TLR2 activation by beta-lactam-exposed pneumococci, we compared TLR2 induction by PSSP (MIC 0.06 mg/L) and a high-level PRSP clinical isolate (159; MIC 16 mg/L) following exposure to penicillin and cefotaxime. Both organisms were treated with penicillin or cefotaxime at and around the MIC. TLR2 signalling was measured as relative IL-8 promoter activation in transfected HeLa cells. On exposure to penicillin, log-phase PSSP and PRSP induced TLR2-proinflammatory activation at levels significantly higher than unexposed bacteria, and maximal in each case at the MIC. Transformants containing low-affinity penicillin-binding proteins (PBP) 2x, 1a and 2b exhibited stepwise resistance to cefotaxime and penicillin. TLR2 activation following penicillin treatment was dependent on an abnormal cell wall (PBP1a and 2x) and autolysis (PBP2b). High affinity PBP2x was required for this effect to be observed in log-phase pneumococci exposed to cefotaxime at the MIC. Cefotaxime-mediated TLR2 activation was not observed in lag-phase transformants exposed to sub-lethal concentrations. These data show that PRSP have similar TLR2-proinflammatory effects to PSSP when exposed to beta-lactam antibiotics but the antibiotic concentration relative to the MIC is critical. This has implications for treatment of pneumococcal disease when tissue concentrations of antibiotic are close to the MIC.

Molecular characterisation of Canadian paediatric multidrug-resistant Streptococcus pneumoniae from 1998–2004

Multidrug-resistant (MDR) Streptococcus pneumoniae (i.e. resistant to three different antimicrobial classes) is a global concern. The molecular epidemiology of MDR S. pneumoniae has not been characterised in Canadian paediatric isolates. Paediatric MDR S. pneumoniae were obtained from a national surveillance study. Susceptibility testing was performed by the methods of the Clinical and Laboratory Standards Institute. Phenotypic and genotypic relatedness were assessed by serotyping and pulsed-field gel electrophoresis (PFGE). Penicillin resistance was assessed with polymerase chain reaction (PCR) followed by DNA sequencing of penicillin-binding proteins (PBPs) 1A, 2B and 2X. Macrolide resistance was assessed by PCR-based detection of mef(E) and erm(B). PCR and sequencing of the dihydrofolate reductase ( DHFR) gene was performed to assess resistance to trimethoprim/sulphamethoxazole (T/S). Seventy (98.6%) of 71 MDR paediatric isolates were concomitantly resistant to penicillin, erythromycin and T/S. Resistance genes mef(E) (66.2%) or erm(B) (22.5%) or both mef(E) and erm(B) (8.5%) were associated with macrolide resistance, and the prevalence of erm(B) increased significantly ( P = 0.0001) over time. Penicillin resistance was associated with amino acid substitutions in PBPs 1A, 2B and 2X. Resistance to T/S was associated with amino acid substitutions in the DHFR gene; in particular, Ile100 → Leu was detected in all isolates analysed. PFGE revealed three clusters of isolates that were genetically related and associated with specific serotypes (Taiwan 19F, Spain 23F, Spain 14 and France 9V), suggesting clonal expansion as the primary means of paediatric MDR S. pneumoniae dissemination in Canada. The heptavalent pneumococcal vaccine Prevnar ®, currently approved in Canada for use in children ≤2 years of age, provided excellent coverage (90.2%) of paediatric MDR S. pneumoniae. In conclusion, paediatric MDR S. pneumoniae simultaneously resistant to penicillin, erythromycin and T/S are genetically similar and disseminating across Canada. Prevnar ® provides excellent coverage of paediatric MDR S. pneumoniae.

Mutational analysis of class A and class B penicillin-binding proteins in Streptococcus gordonii.

High-molecular-weight (HMW) penicillin-binding proteins (PBPs) are divided into class A and class B PBPs, which are bifunctional transpeptidases/transglycosylases and monofunctional transpeptidases, respectively. We determined the sequences for the HMW PBP genes of Streptococcus gordonii, a gingivo-dental commensal related to Streptococcus pneumoniae. Five HMW PBPs were identified, including three class A (PBPs 1A, 1B, and 2A) and two class B (PBPs 2B and 2X) PBPs, by homology with those of S. pneumoniae and by radiolabeling with [3H]penicillin. Single and double deletions of each of them were achieved by allelic replacement. All could be deleted, except for PBP 2X, which was essential. Morphological alterations occurred after deletion of PBP 1A (lozenge shape), PBP 2A (separation defect and chaining), and PBP 2B (aberrant septation and premature lysis) but not PBP 1B. The muropeptide cross-link patterns remained similar in all strains, indicating that cross-linkage for one missing PBP could be replaced by others. However, PBP 1A mutants presented shorter glycan chains (by 30%) and a relative decrease (25%) in one monomer stem peptide. Growth rate and viability under aeration, hyperosmolarity, and penicillin exposure were affected primarily in PBP 2B-deleted mutants. In contrast, chain-forming PBP 2A-deleted mutants withstood better aeration, probably because they formed clusters that impaired oxygen diffusion. Double deletion could be generated with any PBP combination and resulted in more-altered mutants. Thus, single deletion of four of the five HMW genes had a detectable effect on the bacterial morphology and/or physiology, and only PBP 1B seemed redundant a priori.

Mutations in penicillin-binding protein (PBP) genes and in non-PBP genes during selection of penicillin-resistant Streptococcus gordonii.

Penicillin resistance in Streptococcus spp. involves multiple mutations in both penicillin-binding proteins (PBPs) and non-PBP genes. Here, we studied the development of penicillin resistance in the oral commensal Streptococcus gordonii. Cyclic exposure of bacteria to twofold-increasing penicillin concentrations selected for a progressive 250- to 500-fold MIC increase (from 0.008 to between 2 and 4 microg/ml). The major MIC increase (> or = 35-fold) was related to non-PBP mutations, whereas PBP mutations accounted only for a 4- to 8-fold additional increase. PBP mutations occurred in class B PBPs 2X and 2B, which carry a transpeptidase domain, but not in class A PBP 1A, 1B, or 2A, which carry an additional transglycosylase domain. Therefore, we tested whether inactivation of class A PBPs affected resistance development in spite of the absence of mutations. Deletion of PBP 1A or 2A profoundly slowed down resistance development but only moderately affected resistance in already highly resistant mutants (MIC = 2 to 4 microg/ml). Thus, class A PBPs might facilitate early development of resistance by stabilizing penicillin-altered peptidoglycan via transglycosylation, whereas they might be less indispensable in highly resistant mutants which have reestablished a penicillin-insensitive cell wall-building machinery. The contribution of PBP and non-PBP mutations alone could be individualized in DNA transformation. Both PBP and non-PBP mutations conferred some level of intrinsic resistance, but combining the mutations synergized them to ensure high-level resistance (> or = 2 microg/ml). The results underline the complexity of penicillin resistance development and suggest that inhibition of transglycosylase might be an as yet underestimated way to interfere with early resistance development.

Activities of Ceftobiprole and Other β-Lactams against Streptococcus pneumoniae Clinical Isolates from the United States with Defined Substitutions in Penicillin-Binding Proteins PBP 1a, PBP 2b, and PBP 2x

The activities of ceftobiprole and other beta-lactams were examined with 30 Streptococcus pneumoniae isolates containing multiple pbp1a, pbp2b, and pbp2x mutations. The highest ceftobiprole MIC was 1 microg/ml, while the comparator MICs were 16 to 64 microg/ml. Fifty percent inhibitory concentrations for penicillin-binding protein 2x were 0.5 microg/ml (ceftobiprole) and 4 microg/ml (ceftriaxone) in a penicillin- and ceftriaxone-resistant isolate.

Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes

Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure–function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.

Pneumococcal β-Lactam Resistance Due to a Conformational Change in Penicillin-binding Protein 2x

Streptococcus pneumoniae is a life-threatening human pathogen that is increasingly resistant to a wide array of drugs. Resistance to beta-lactams, the most widely used antibiotics, is correlated with tens of amino acid substitutions in their targets; that is, the penicillin-binding proteins (PBPs), resulting from multiple events of recombination. To discriminate relevant substitutions from those that are incidental to the recombination process, we report the exhaustive characterization of all the mutations in the transpeptidase domain of PBP2x from the highly resistant strain 5204. A semi-automated method combining biochemical and microbiological approaches singled out 6 mutations of 41 (15%) that are essential for high level resistance. The hitherto uncharacterized I371T, R384G, M400T, and N605T together with the previously studied T338M and M339F account for nearly all the loss of affinity of PBP2x for beta-lactams. Most interestingly, I371T and R384G cause the conformational change of a loop that borders the entrance of the active site cavity, hampering antibiotic binding. For the first time all the mutations of a PBP relevant to beta-lactam resistance have been identified, providing new mechanistic insights. Most notable is the relationship between the decreased susceptibility to beta-lactams and the dynamic behavior of a loop.