scholarly journals Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

1986 ◽  
Vol 167 (3) ◽  
pp. 975-980 ◽  
Author(s):  
M Matsuhashi ◽  
M D Song ◽  
F Ishino ◽  
M Wachi ◽  
M Doi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Molecular Cloning ◽  
High Resistance ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Beta Lactam Antibiotics

scholarly journals Role of Penicillin-Binding Protein 2 (PBP2) in the Antibiotic Susceptibility and Cell Wall Cross-Linking of Staphylococcus aureus: Evidence for the Cooperative Functioning of PBP2, PBP4, and PBP2A

2005 ◽  
Vol 187 (5) ◽  
pp. 1815-1824 ◽  
Author(s):  
Tomasz A. Łęski ◽  
Alexander Tomasz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Antimicrobial Agents ◽  
Binding Protein ◽  
Resistant Mutant ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Experimental Proof

ABSTRACT Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.

scholarly journals Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay.

1996 ◽  
Vol 40 (9) ◽  
pp. 2075-2079 ◽  
Author(s):  
S Roychoudhury ◽  
R E Kaiser ◽  
D N Brems ◽  
W K Yeh
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Methicillin Resistant ◽  
Chromogenic Assay ◽  
Enzymatic Acylation

We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.

scholarly journals In vitro evaluation of BO-3482, a novel dithiocarbamate carbapenem with activity against methicillin-resistant staphylococci.

1997 ◽  
Vol 41 (10) ◽  
pp. 2282-2285 ◽  
Author(s):  
Y Adachi ◽  
K Nakamura ◽  
Y Kato ◽  
N Hazumi ◽  
T Hashizume ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Clostridium Difficile ◽  
Proteus Mirabilis ◽  
Gram Negative Bacteria ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Gram Negative ◽  
Methicillin Resistant ◽  
Beta Lactam Antibiotics

BO-3482, a dithiocarbamate carbapenem, inhibited clinical isolates of methicillin-resistant staphylococci (MRS) at 6.25 microg/ml (MIC at which 90% of isolates tested are inhibited [MIC90]), while the MIC90 of imipenem was > 100 microg/ml. BO-3482 was generally less active than imipenem against methicillin-susceptible Staphylococcus aureus, streptococci, enterococci, and gram-negative bacteria, although BO-3482 showed better activity (MIC90) than imipenem against Enterococcus faecium, Haemophilus influenzae, Proteus mirabilis, and Clostridium difficile. The affinities (50% inhibitory concentrations) of BO-3482 for penicillin-binding protein (PBP) PBP 2' of MRS and PBP 5 of E. faecium (both PBPs have low affinities for ordinary beta-lactam antibiotics) were 3.8 and 20 microg/ml, respectively, reflecting the greater activity of BO-3482 against MRS than against E. faecium.

scholarly journals Penicillin-binding protein 2x of Streptococcus pneumoniae: enzymic activities and interactions with β-lactams

1993 ◽  
Vol 292 (3) ◽  
pp. 735-741 ◽  
Author(s):  
M Jamin ◽  
C Damblon ◽  
S Millier ◽  
R Hakenbeck ◽  
J M Frère
Keyword(s):  
Streptococcus Pneumoniae ◽  
Extracellular Enzymes ◽  
Binding Protein ◽  
Soluble Form ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Easy Method ◽  
Beta Lactam Antibiotics ◽  
Catalytic Behaviour ◽  
First Time

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains.

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