Kinetic properties of the Bacillus licheniformis penicillin-binding proteins.
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ABSTRACT:
SUBMITTER: Lepage S
PROVIDER: S-EPMC1135798 | biostudies-other | 1995 Jul
REPOSITORIES: biostudies-other
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Project description:The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by D,D-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent D, D-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.
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Project description:Bacterial cell division involves the dynamic assembly of a diverse set of proteins that coordinate the invagination of the cell membrane and synthesis of cell wall material to create the new cell poles of the separated daughter cells. Penicillin-binding protein PBP 2B is a key cell division protein in Bacillus subtilis proposed to have a specific catalytic role in septal wall synthesis. Unexpectedly, we find that a catalytically inactive mutant of PBP 2B supports cell division, but in this background the normally dispensable PBP 3 becomes essential. Phenotypic analysis of pbpC mutants (encoding PBP 3) shows that PBP 2B has a crucial structural role in assembly of the division complex, independent of catalysis, and that its biochemical activity in septum formation can be provided by PBP 3. Bioinformatic analysis revealed a close sequence relationship between PBP 3 and Staphylococcus aureus PBP 2A, which is responsible for methicillin resistance. These findings suggest that mechanisms for rescuing cell division when the biochemical activity of PBP 2B is perturbed evolved prior to the clinical use of β-lactams.
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Project description:The Leptospira interrogans ponA and pbpB genes were isolated and characterized. ponA and pbpB encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two L. interrogans strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced L. interrogans PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins from Escherichia coli. It was demonstrated for strain Verdun that ponA and pbpB are transcribed individually from their own promoter. The ponA and pbpB genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The L. interrogans PBP 1 and PBP 3 proteins were synthesized in E. coli and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.
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Project description:BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs ?-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for ?-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.
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Project description:Reduced affinity of penicillin-binding proteins (PBPs) for binding penicillin has been proposed as a mechanism of beta-lactam antibiotic resistance in staphylococci. Penicillin binding by PBPs of three penicillin-susceptible and two penicillin-resistant strains of Staphylococcus aureus was studied in kinetic assays to determine rate constants, drug concentrations at which PBPs were bound and the relationship between concentrations that bound PBPs and concentrations that inhibited bacterial growth. PBPs 1 and 2 of the resistant strains exhibited slower acylation and more rapid deacylation than susceptible strains. In contrast PBP 4, a naturally low-affinity PBP, was modified such that it exhibited a lower rate of deacylation. The concentrations of penicillin at which modified PBPs were bound correlated with concentrations that inhibited growth of the resistant strains. Acquisition of penicillin resistance in these strains of S. aureus results, at least in part, from structural modifications affecting binding of multiple PBPs and appears to include recruitment of a non-essential PBP, PBP 4.
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Project description:A recent clinical report has linked Streptococcus pyogenes ?-lactam antibiotic resistance to mutation in the penicillin binding protein (PBP) PBP2x. To determine whether this is an isolated case or reflects a broader prevalence of mutations that might confer reduced ?-lactam susceptibility, we investigated the relative frequency of PBP sequence variation within a global database of 9,667 S. pyogenes isolates. We found that mutations in S. pyogenes PBPs (PBP2x, PBP1a, PBP1b, and PBP2a) occur infrequently across this global database, with fewer than 3 amino acid changes differing between >99% of the global population. Only 4 of the 9,667 strains contained mutations near transpeptidase active sites of PBP2x or PBP1a. The reported PBP2x T553K substitution was not identified. These findings are in contrast to those of 2,520 S. pneumococcus sequences where PBP mutations are relatively frequent and are often located in key ?-lactam binding pockets. These data, combined with the general lack of penicillin resistance reported in S. pyogenes worldwide, suggests that extensive, unknown constraints restrict S. pyogenes PBP sequence plasticity. Our findings imply that while heavy antibiotic pressure may select for mutations in the PBPs, there is currently no evidence of such mutations becoming fixed in the S. pyogenes population or that mutations are being sequentially acquired in the PBPs.IMPORTANCE ?-Lactam antibiotics are the first-line therapeutic option for Streptococcus pyogenes infections. Despite the global high prevalence of S. pyogenes infections and widespread use of ?-lactams worldwide, reports of resistance to ?-lactam antibiotics, such as penicillin, have been incredibly rare. Recently, ?-lactam resistance, as defined by clinical breakpoints, was detected in two clinical S. pyogenes isolates with accompanying mutations in the active site of the penicillin binding protein PBP2x, raising concerns that ?-lactam resistance will become more widespread. We screened a global database of S. pyogenes genome sequences to investigate the frequency of PBP mutations, identifying that PBP mutations are uncommon relative to those of Streptococcus pneumoniae These findings support clinical observations that ?-lactam resistance is rare in S. pyogenes and suggest that there are considerable constraints on S. pyogenes PBP sequence variation.
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