scholarly journals Role of Penicillin-Binding Protein 5 in Expression of Ampicillin Resistance and Peptidoglycan Structure in Enterococcus faecium

2001 ◽  
Vol 45 (9) ◽  
pp. 2594-2597 ◽  
Author(s):  
Farid Sifaoui ◽  
Michel Arthur ◽  
Louis Rice ◽  
Laurent Gutmann
Keyword(s):  
Enterococcus Faecium ◽  
Binding Protein ◽  
Shuttle Vector ◽  
Gene Copy Number ◽  
Reversed Phase ◽  
Site Directed Mutagenesis ◽  
Gene Copy ◽  
Penicillin Binding Protein ◽  
Intrinsic Resistance ◽  
Link Type

ABSTRACT The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired β-lactam resistance was investigated by constructing isogenic strains of Enterococcus faeciumproducing different PBP 5. The pbp5 genes from threeE. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faeciumD344 highly susceptible to ampicillin due to deletion ofpbp5 (MIC, 0.03 μg/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 μg/ml), D344 (MIC, 24 μg/ml), and H80721 (MIC, 512 μg/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 μg/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.

scholarly journals Penicillin-Binding Protein 5 and Expression of Ampicillin Resistance in Enterococcus faecium

2001 ◽  
Vol 45 (5) ◽  
pp. 1480-1486 ◽  
Author(s):  
Louis B. Rice ◽  
Lenore L. Carias ◽  
Rebecca Hutton-Thomas ◽  
Farid Sifaoui ◽  
Laurent Gutmann ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Binding Protein ◽  
Transmembrane Protein ◽  
Shuttle Vector ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Transcriptional Analysis ◽  
Penicillin Binding Protein ◽  
Ampicillin Resistance ◽  
Upstream Open Reading Frames

ABSTRACT We report a structural and transcriptional analysis of thepbp5 region of Enterococcus faecium C68.pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reportedpsr (penicillin-binding protein synthesis repressor) andftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW(ftsWEfm ) alone; (ii) psr andpbp5; (iii) pbp5 alone; and (iv)ftsWEfm , psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from theftsWEfm promoter through the pbp5gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faeciumD344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF(pCWR558) and D344-SRF(pCWR583) were 64 μg/ml. Quantities of pbp5transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor ofpbp5 transcription. However, quantities of psrtranscript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.

scholarly journals Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Charlene Desbonnet ◽  
Amelia Tait-Kamradt ◽  
Monica Garcia-Solache ◽  
Paul Dunman ◽  
Jeffrey Coleman ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Binding Protein ◽  
Affinity Purification ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Intrinsic Resistance ◽  
Uncharacterized Protein ◽  
Class A ◽  
Class B ◽  
Cephalosporin Resistance

ABSTRACT The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins”) is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium ; these experiments yielded a protein designated Pbp5-associated protein (P 5 AP). Transcription of the P 5 AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA . Transformation of class A Pbp-deficient strains with the plasmid-carried P 5 AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P 5 AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. IMPORTANCE β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a resistant phenotype. The present work identifies a novel, previously uncharacterized, protein that interacts with Pbp5, whose expression increases in conjunction with stimuli that increase resistance to cephalosporins, and that confers increased resistance to cephalosporins when overexpressed. P 5 AP may represent a promising new target, inhibition of which could restore cephalosporin susceptibility to E. faecium .

scholarly journals The VanYD DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein

Microbiology ◽  
2001 ◽  
Vol 147 (9) ◽  
pp. 2571-2578 ◽  
Author(s):  
Peter E Reynolds ◽  
O. Herman Ambur ◽  
Barbara Casadewall ◽  
Patrice Courvalin
Keyword(s):  
Enterococcus Faecium ◽  
Binding Protein ◽  
Penicillin Binding Protein

scholarly journals Bias in Template-to-Product Ratios in Multitemplate PCR

1998 ◽  
Vol 64 (10) ◽  
pp. 3724-3730 ◽  
Author(s):  
Martin F. Polz ◽  
Colleen M. Cavanaugh
Keyword(s):  
Bacterial Species ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Binding Energies ◽  
Site Directed Mutagenesis ◽  
Universal Primers ◽  
Gene Copy ◽  
Degenerate Primers ◽  
Relative Product ◽  
Genomic Dnas

ABSTRACT Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

scholarly journals Contribution of Specific Amino Acid Changes in Penicillin Binding Protein 1 to Amoxicillin Resistance in ClinicalHelicobacter pyloriisolates

2010 ◽  
Vol 55 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Nadia N. Qureshi ◽  
Dimitrios Morikis ◽  
Neal L. Schiller
Keyword(s):  
Amino Acid ◽  
Homology Modeling ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Peptic Ulcers ◽  
Penicillin Binding Protein ◽  
Specific Amino Acid ◽  
Binding Cleft ◽  
H Pylori

ABSTRACTAmoxicillin is commonly used to treatHelicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance inH. pyloriis increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4pbp1fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.

Catalytic Mechanism of theStreptomycesK15dd-Transpeptidase/Penicillin-Binding Protein Probed by Site-Directed Mutagenesis and Structural Analysis†,‡

Biochemistry ◽  
2003 ◽  
Vol 42 (10) ◽  
pp. 2895-2906 ◽  
Author(s):  
Noureddine Rhazi ◽  
Paulette Charlier ◽  
Dominique Dehareng ◽  
Danièle Engher ◽  
Marcel Vermeire ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Catalytic Mechanism ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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