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Wei, Yuping, Teresa Havasy, Derrell C. McPherson, and David L. Popham. "Rod Shape Determination by the Bacillus subtilis Class B Penicillin-Binding Proteins Encoded by pbpA and pbpH." Journal of Bacteriology 185, no. 16 (2003): 4717–26. http://dx.doi.org/10.1128/jb.185.16.4717-4726.2003.
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ABSTRACT The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.
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Pereira, S. F. F., A. O. Henriques, M. G. Pinho, H. de Lencastre, and A. Tomasz. "Role of PBP1 in Cell Division of Staphylococcus aureus." Journal of Bacteriology 189, no. 9 (2007): 3525–31. http://dx.doi.org/10.1128/jb.00044-07.
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ABSTRACT We constructed a conditional mutant of pbpA in which transcription of the gene was placed under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter in order to explore the role of PBP1 in growth, cell wall structure, and cell division. A methicillin-resistant strain and an isogenic methicillin-susceptible strain, each carrying the pbpA mutation, were unable to grow in the absence of the inducer. Conditional mutants of pbpA transferred into IPTG-free medium underwent a four- to fivefold increase in cell mass, which was not accompanied by a proportional increase in viable titer. Examination of thin sections of such cells by transmission electron microscopy or fluorescence microscopy of intact cells with Nile red-stained membranes showed a morphologically heterogeneous population of bacteria with abnormally increased sizes, distorted axial ratios, and a deficit in the number of cells with completed septa. Immunofluorescence with an antibody specific for PBP1 localized the protein to sites of cell division. No alteration in the composition of peptidoglycan was detectable in pbpA conditional mutants grown in the presence of a suboptimal concentration of IPTG, which severely restricted the rate of growth, and the essential function of PBP1 could not be replaced by PBP2A present in methicillin-resistant cells. These observations suggest that PBP1 is not a major contributor to the cross-linking of peptidoglycan and that its essential function must be intimately integrated into the mechanism of cell division.
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Murray, Thomas, David L. Popham, Christine B. Pearson, Arthur R. Hand, and Peter Setlow. "Analysis of Outgrowth of Bacillus subtilis Spores Lacking Penicillin-Binding Protein 2a." Journal of Bacteriology 180, no. 24 (1998): 6493–502. http://dx.doi.org/10.1128/jb.180.24.6493-6502.1998.
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ABSTRACT The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect inpbpA spore outgrowth have shown that (i) outgrowingpbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpAspores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpAspores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.
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Wada, Akihito, and Haruo Watanabe. "Penicillin-Binding Protein 1 ofStaphylococcus aureus Is Essential for Growth." Journal of Bacteriology 180, no. 10 (1998): 2759–65. http://dx.doi.org/10.1128/jb.180.10.2759-2765.1998.
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ABSTRACT pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in anEscherichia coli MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.
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Ropy, Alaa, Gabriel Cabot, Irina Sánchez-Diener та ін. "Role of Pseudomonas aeruginosa Low-Molecular-Mass Penicillin-Binding Proteins in AmpC Expression, β-Lactam Resistance, and Peptidoglycan Structure". Antimicrobial Agents and Chemotherapy 59, № 7 (2015): 3925–34. http://dx.doi.org/10.1128/aac.05150-14.
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ABSTRACTThis study aimed to characterize the role ofPseudomonas aeruginosalow-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, andampCregulation. For this purpose, we constructed all single and multiple mutants ofdacB,dacC,pbpG, andampCfrom the wild-typeP. aeruginosaPAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC),ampCexpression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of β-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, onlydacCmutation led to significantly increased pentapeptide levels, showing that PBP5 is the majordd-carboxypeptidase inP. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role asdd-carboxypeptidase only if PBP5 is absent, and theirdd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase inampCexpression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the β-lactam susceptibility profiles of the LMM PBP mutants correlated well with theampCexpression data. However, the inactivation ofampCin these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect ofP. aeruginosaLMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, β-lactam trapping, anddd-carboxypeptidase/β-lactamase activity.
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Dasgupta, Arunava, Pratik Datta, Manikuntala Kundu, and Joyoti Basu. "The serine/threonine kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein required for cell division." Microbiology 152, no. 2 (2006): 493–504. http://dx.doi.org/10.1099/mic.0.28630-0.
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A cluster of genes encoded by ORFs Rv0014c–Rv0018c in Mycobacterium tuberculosis encodes candidate cell division proteins RodA and PBPA, a pair of serine/threonine kinases (STPKs), PknA and PknB, and a phosphatase, PstP. The organization of genes encompassing this region is conserved in a large number of mycobacterial species. This study demonstrates that recombinant PBPA of M. tuberculosis binds benzylpenicillin. Knockout of its counterpart in M. smegmatis resulted in hindered growth and defective cell septation. The phenotype of the knockout (PBPA-KO) could be restored to that of the wild-type upon expression of PBPA of M. tuberculosis. PBPA localized to the division site along with newly synthesized peptidoglycan, between segregated nucleoids. In vivo coexpression of PBPA and PknB, in vitro kinase assays and site-specific mutagenesis substantiated the view that PknB phosphorylates PBPA on T362 and T437. A T437A mutant could not complement PBPA-KO. These studies demonstrate for the first time that PBPA, which belongs to a subclass of class B high-molecular-mass PBPs, plays an important role in cell division and cell shape maintenance. Signal transduction mediated by PknB and PstP likely regulates the positioning of this PBP at the septum, thereby regulating septal peptidoglycan biosynthesis.
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Philippe, Nadège, Ludovic Pelosi, Richard E. Lenski, and Dominique Schneider. "Evolution of Penicillin-Binding Protein 2 Concentration and Cell Shape during a Long-Term Experiment with Escherichia coli." Journal of Bacteriology 191, no. 3 (2008): 909–21. http://dx.doi.org/10.1128/jb.01419-08.
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ABSTRACT Peptidoglycan is the major component of the bacterial cell wall and is involved in osmotic protection and in determining cell shape. Cell shape potentially influences many processes, including nutrient uptake as well as cell survival and growth. Peptidoglycan is a dynamic structure that changes during the growth cycle. Penicillin-binding proteins (PBPs) catalyze the final stages of peptidoglycan synthesis. Although PBPs are biochemically and physiologically well characterized, their broader effects, especially their effects on organismal fitness, are not well understood. In a long-term experiment, 12 populations of Escherichia coli having a common ancestor were allowed to evolve for more than 40,000 generations in a defined environment. We previously identified mutations in the pbpA operon in one-half of these populations; this operon encodes PBP2 and RodA proteins that are involved in cell wall elongation. In this study, we characterized the effects of two of these mutations on competitive fitness and other phenotypes. By constructing and performing competition experiments with strains that are isogenic except for the pbpA alleles, we showed that both mutations that evolved were beneficial in the environment used for the long-term experiment and that these mutations caused parallel phenotypic changes. In particular, they reduced the cellular concentration of PBP2, thereby generating spherical cells with an increased volume. In contrast to their fitness-enhancing effect in the environment where they evolved, both mutations decreased cellular resistance to osmotic stress. Moreover, one mutation reduced fitness during prolonged stationary phase. Therefore, alteration of the PBP2 concentration contributed to physiological trade-offs and ecological specialization during experimental evolution.
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Cayô, Rodrigo, María-Cruz Rodríguez, Paula Espinal, et al. "Analysis of Genes Encoding Penicillin-Binding Proteins in Clinical Isolates of Acinetobacter baumannii." Antimicrobial Agents and Chemotherapy 55, no. 12 (2011): 5907–13. http://dx.doi.org/10.1128/aac.00459-11.
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ABSTRACTThere is limited information on the role of penicillin-binding proteins (PBPs) in the resistance ofAcinetobacter baumanniito β-lactams. This study presents an analysis of the allelic variations of PBP genes inA. baumanniiisolates. Twenty-sixA. baumanniiclinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes ofA. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpAormrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.
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Berti, Andrew D., Erin Theisen, John-Demian Sauer та ін. "Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy". Antimicrobial Agents and Chemotherapy 60, № 1 (2015): 451–58. http://dx.doi.org/10.1128/aac.02071-15.
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ABSTRACTThe activity of daptomycin (DAP) against methicillin-resistantStaphylococcus aureus(MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of thepbpAtranscript, encoding PBP1, was specifically induced by DAP exposure whereas expression ofpbpB,pbpC, andpbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain withpbpAunder an inducible promoter, increasedpbpAtranscription was accompanied by reduced susceptibility to, and killing by, DAPin vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in anin vitropharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure ofS. aureusto DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP–β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response.
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Łęski, Tomasz A., and Alexander Tomasz. "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." Journal of Bacteriology 187, no. 5 (2005): 1815–24. http://dx.doi.org/10.1128/jb.187.5.1815-1824.2005.
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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.
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Spencer, Maria de los Angeles, José Rodrigo Martinez, Lina María Rivas, et al. "1453. PBP2, PBP2a and PBP4 Clone-specific Polymorphisms are not Associated to Ceftaroline (CPT) Susceptibility in Chilean Clinical Isolates of Methicillin-Resistant Staphylococcus aureus (MRSA)." Open Forum Infectious Diseases 7, Supplement_1 (2020): S729. http://dx.doi.org/10.1093/ofid/ofaa439.1634.
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Abstract Background CPT is a last-generation cephalosporin active against MRSA due to its affinity for PBP2a. CPT-resistance (CPT-R) is well-described, with mutations in the active transpeptidase domain of PBP2a associated to high-level resistance. The accumulation of changes in the non-penicillin-binding domain of PBP2a has been linked to elevations of the minimal inhibitory concentration (MIC) to CPT to levels around 2-4ug/mL. PBP4 and PBP2 have also been implicated as potentially relevant mecA-independent mechanisms of CPT-R. We recently reported high rates of CPT-non-susceptibility in clinical MRSA strains from Chile. However, the mutational landscape of PBPs in clinical MRSA isolates from Chile and its relation to CPT susceptibility has not been assessed. Methods We analyzed 180 MRSA isolates collected from 2000-2018 in Santiago, Chile. Identification was confirmed by MALDI-TOF and methicillin resistance with cefoxitin disk-diffusion. CPT susceptibility was performed by BMD following CLSI-2019 guidance. Whole-genome sequencing was performed for all isolates; the mutational profile of PBPs was determined using reference sequences for PBP2 (AGY89563.1), PBP2a (NG_047938.1) and PBP4 (X91786.1). Results All isolates were phenotypically-confirmed MRSA and harbored mecA. The MIC50/MIC90 by BMD was 2/2μg/dL; only 71 (39%) isolates were CPT-susceptible (MIC <1µg/mL). Most isolates belonged to ST5/SCCmecI (70%,126/180), ST105/SCCmecII (10%,18/180) and ST8/SCCmecIV (5%, 9/180). All ST5/SCCmecI isolates carried the mutations in PBP2 (Y156D), PBP2a (M122I and E150K), and PBP4 (T189S, L234H, and T409A); CPT-susceptibility among ST5/SCCmecI was only 22%. On the other hand, all ST105/SCCmecII isolates had mutations in PBP2 (S707L) and PBP4 (T189S, L234H, and T409A) and exhibited a higher CPT-susceptibility rate (67%). All 9 isolates belonging to the ST8/SCCmecIV lineage harbored a non-coding mutation in PBP2a (g-6t) and the previously observed L234H change in PBP4. Importantly, no association between specific polymorphisms and MIC to CPT was found. Table 1. PBPs mutations compared to CPT MICs by MLST and SCCmec Conclusion Changes in the studied PBPs were frequent among MRSA circulating in Chile and were conserved among different genetic backgrounds. However, these changes were not associated with the level of CPT MIC among these isolates. Disclosures Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)
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Zhou, Yanjiao, Aude Antignac, Shang Wei Wu та Alexander Tomasz. "Penicillin-Binding Proteins and Cell Wall Composition in β-Lactam-Sensitive and -Resistant Strains of Staphylococcus sciuri". Journal of Bacteriology 190, № 2 (2007): 508–14. http://dx.doi.org/10.1128/jb.01549-07.
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ABSTRACT A close homologue of the acquired Staphylococcus aureus mecA gene is present as a native gene in Staphylococcus sciuri. We determined the patterns of penicillin-binding proteins (PBPs) and the peptidoglycan compositions of several S. sciuri strains to explore the functions of this mecA homologue, named pbpD, in its native S. sciuri environment. The protein product of pbpD was identified as PBP4 with a molecular mass of 84 kDa, one of the six PBPs present in representatives of each of three subspecies of S. sciuri examined. PBP4 had a low affinity for nafcillin, reacted with a monoclonal antibody raised against S. aureus PBP2A, and was greatly overproduced in oxacillin-resistant clinical isolate S. sciuri SS37 and to a lesser extent in resistant laboratory mutant K1M200. An additional PBP inducible by oxacillin and corresponding to S. aureus PBP2A was identified in another oxacillin-resistant clinical isolate, S. sciuri K3, which harbors an S. aureus copy of mecA. Oxacillin resistance depended on the overtranscribed S. sciuri pbpD gene in strains SS37 and K1M200, while the resistance of strain K3 depended on the S. aureus copy of mecA. Our data provide evidence that both S. aureus mecA and S. sciuri pbpD can function as resistance determinants in either an S. aureus or an S. sciuri background and that the protein products of these genes, S. aureus PBP2A and S. sciuri PBP4, can participate in the biosynthesis of peptidoglycan, the muropeptide composition of which depends on the bacterium “hosting” the resistance gene.
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Mahat Chhetri, Pradhumna, Xiang-Kai Yang, Chih-Tung Yang, and Jhy-Der Chen. "One-Dimensional Mercury Halide Coordination Polymers Based on A Semi-Rigid N-Donor Ligand: Reversible Structural Transformation." Polymers 11, no. 3 (2019): 436. http://dx.doi.org/10.3390/polym11030436.
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Four one-dimensional (1D) mercury(II) halide coordination polymers have been synthesized by using a semi-rigid N-donor ligand, 2,2′-(1,4-phenylene)-bis(N-(pyridin-3-yl)acetamide) (1,4-pbpa). While [Hg(1,4-pbpa)Cl2·CH3OH]n, 1, forms a sinusoidal chain, the complexes [Hg(1,4-pbpa)X2]n (X = Cl, 2; Br, 3; I, 4) are helical. The sinusoidal 1 undergoes reversible structural transformation with helical 2 upon removal and uptake of CH3OH, which was accompanied with the conformation adjustment of the 1,4-pbpa ligand from trans anti-anti to trans syn-anti. Pyridyl ring rotation of the 1,4-pbpa ligand that results in the change of the ligand conformation is proposed for the initiation of the structural transformation.
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Moreira, B., S. Boyle-Vavra, B. L. deJonge, and R. S. Daum. "Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 41, no. 8 (1997): 1788–93. http://dx.doi.org/10.1128/aac.41.8.1788.
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The mechanism of glycopeptide resistance in the genus Staphylococcus is unknown. Since these antimicrobial compounds act by binding the peptidoglycan precursor terminus, the target of transglycosylase and transpeptidase enzymes, it was hypothesized that resistance might be mediated in Staphylococcus aureus by increased production or activity of these enzymes, commonly called penicillin-binding proteins (PBPs). To evaluate this possibility, glycopeptide-resistant mutants were prepared by passage of several clinical isolates of this species in nutrient broth containing successively increasing concentrations of the glycopeptide vancomycin or teicoplanin. Decreased coagulase activity and increased resistance to lysostaphin were uniformly present in the vancomycin-resistant mutants. Peptidoglycan cross-linking increased in one resistant isolate and decreased in two resistant isolates. The amounts of radioactive penicillin that bound to each PBP in susceptible and resistant strains were compared; PBP2 production was also evaluated by Western blotting. Increased penicillin labeling and production of PBP2 were found in all resistant derivatives selected by either vancomycin or teicoplanin. Moreover, the increase in PBP2 penicillin labeling occurred early in a series of vancomycin-selected derivatives and was strongly correlated (r > 0.9) with the increase in vancomycin and teicoplanin MIC. An increase in penicillin labeling also occurred, variably, in PBP1, PBP3, and/or PBP4. These data demonstrate a strong correlation between resistance to glycopeptides and increased PBP activity and/or production in S. aureus. Such an increase could allow PBPs to better compete with glycopeptides for the peptidoglycan precursor.
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Gardete, Susana, Hermínia de Lencastre, and Alexander Tomasz. "A link in transcription between the native pbpB and the acquired mecA gene in a strain of Staphylococcus aureus." Microbiology 152, no. 9 (2006): 2549–58. http://dx.doi.org/10.1099/mic.0.29078-0.
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Conditional mutants of pbpB with an IPTG-inducible promoter were used to compare the effects of interrupted transcription of this gene in a meticillin-sensitive (MSSA) and a meticillin-resistant (MRSA) strain of Staphylococcus aureus. After 3 h growth following the removal of IPTG, multiplication of the MSSA strain stopped abruptly, cells began to lyse, and membrane preparations showed greatly decreased quantities of penicillin-binding protein (PBP) 2. In contrast, the MRSA strain continued to grow for at least 20 h in the IPTG-free medium, but with gradually increasing doubling times, which eventually reached 180 min. The peptidoglycan produced during this period of extremely slow growth showed only minor alterations, but cells with abnormal morphology accumulated in the culture, the abundance of mecA transcript gradually declined, and the cellular amounts of PBP2A were significantly decreased. Adding back the IPTG inducer caused rapid resumption in the transcription of pbpB, followed by an increase in the transcription of mecA. No changes were detected in the transcription of pbpA, C and D, the determinant of 16S rRNA or the housekeeping gene pta. Promoter fusion experiments suggested that the transcription of the resistance gene mecA may respond to some regulatory signal generated in the bacteria during changes in the transcription of pbpB.
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de Sousa Borges, Anabela, Jeanine de Keyzer, Arnold J. M. Driessen, and Dirk-Jan Scheffers. "The Escherichia coli Membrane Protein Insertase YidC Assists in the Biogenesis of Penicillin Binding Proteins." Journal of Bacteriology 197, no. 8 (2015): 1444–50. http://dx.doi.org/10.1128/jb.02556-14.
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ABSTRACTMembrane proteins need to be properly inserted and folded in the membrane in order to perform a range of activities that are essential for the survival of bacteria. The Sec translocon and the YidC insertase are responsible for the insertion of the majority of proteins into the cytoplasmic membrane. YidC can act in combination with the Sec translocon in the insertion and folding of membrane proteins. However, YidC also functions as an insertase independently of the Sec translocon for so-called YidC-only substrates. In addition, YidC can act as a foldase and promote the proper assembly of membrane protein complexes. Here, we investigate the effect ofEscherichia coliYidC depletion on the assembly of penicillin binding proteins (PBPs), which are involved in cell wall synthesis. YidC depletion does not affect the total amount of the specific cell division PBP3 (FtsI) in the membrane, but the amount of active PBP3, as assessed by substrate binding, is reduced 2-fold. A similar reduction in the amount of active PBP2 was observed, while the levels of active PBP1A/1B and PBP5 were essentially similar. PBP1B and PBP3 disappeared from higher-Mwbands upon YidC depletion, indicating that YidC might play a role in PBP complex formation. Taken together, our results suggest that the foldase activity of YidC can extend to the periplasmic domains of membrane proteins.IMPORTANCEThis study addresses the role of the membrane protein insertase YidC in the biogenesis of penicillin binding proteins (PBPs). PBPs are proteins containing one transmembrane segment and a large periplasmic or extracellular domain, which are involved in peptidoglycan synthesis. We observe that in the absence of YidC, two critical PBPs are not correctly folded even though the total amount of protein in the membrane is not affected. Our findings extend the function of YidC as a foldase for membrane protein (complexes) to periplasmic domains of membrane proteins.
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Li, Cheng, Christy Hullings, Wei Wang, and Debra M. Palmer Keenan. "Development and Validation of a Perceived Barriers to Physical Activity Scale for Low-Income Adolescents." Journal of Physical Activity and Health 18, no. 5 (2021): 507–15. http://dx.doi.org/10.1123/jpah.2020-0259.
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Background: Low-income adolescents’ physical activity (PA) levels fall below current recommendations. Perceived barriers to physical activity (PBPA) are likely significant predictors of PA levels; however, valid and reliable measures to assess PA barriers are lacking. This manuscript describes the development of the PBPA Survey for Low-Income Adolescents. Methods: A mixed-method approach was used. Items identified from the literature and revised for clarity and appropriateness (postcognitive interviews) were assessed for test–retest reliability with 74 adolescents using intraclass correlation coefficient. Items demonstrating low intraclass correlation coefficients or floor effects were removed. Both exploratory factor analysis and confirmatory factor analysis analyses (n = 1914 low-income teens) were used to finalize the scale; internal consistency was assessed by Cronbach’s alpha. Concurrent validity was established by correlating the PBPA with the PA questionnaire for adolescents using a Spearman correlation. Results: The exploratory factor analysis yielded a 38-item, 7-factor solution, which was cross-validated by confirmatory factor analysis (comparative-fit index, nonnormed fit index = .90). The scale’s Cronbach’s alpha was .94, with subscales ranging from .70 to .88. The PBPA Survey for Low-Income Adolescents’ concurrent validity was supported by a negative PA questionnaire for adolescents’ correlation values. Conclusion: The PBPA Survey for Low-Income Adolescents can be used to better understand the relationship between PBPA among low-income teens. Further research is warranted to validate the scale with other adolescent subgroups.
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Liao, X., and R. E. Hancock. "Susceptibility to beta-lactam antibiotics of Pseudomonas aeruginosa overproducing penicillin-binding protein 3." Antimicrobial Agents and Chemotherapy 41, no. 5 (1997): 1158–61. http://dx.doi.org/10.1128/aac.41.5.1158.
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By using a broad-host-range vector, pUCP27, the Pseudomonas aeruginosa and Escherichia coli pbpB genes, which encode penicillin-binding protein 3 (PBP3), were separately overexpressed in a P. aeruginosa strain, PAO4089, that is deficient in producing chromosomal beta-lactamase. Susceptibility studies indicated that overproduction of the P. aeruginosa PBP3 in PAO4089 resulted in twofold-increased resistance to aztreonam, fourfold-increased resistance to cefepime and cefsulodin, and eightfold-increased resistance to ceftazidime, whereas overproduction of the P. aeruginosa PBP3 in PAO4089 did not affect susceptibility to PBP1-targeted cephaloridine or PBP2-targeted imipenem. Similar results were obtained with PAO4089 overproducing E. coli PBP3, with the exception that there was no influence on the MICs or minimal bactericidal concentrations of cefsulodin and cefepime, which have very low affinities for E. coli PBP3. These data are consistent with the conclusion that PBP3 has to achieve a certain level of saturation, with beta-lactams targeted to this protein, to result in cell inhibition or death.
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Sayed, Alaa R. M., Nirav R. Shah, Kari B. Basso та ін. "First Penicillin-Binding Protein Occupancy Patterns for 15 β-Lactams and β-Lactamase Inhibitors in Mycobacterium abscessus". Antimicrobial Agents and Chemotherapy 65, № 1 (2020): e01956-20. http://dx.doi.org/10.1128/aac.01956-20.
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ABSTRACTMycobacterium abscessus causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of M. abscessus infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With β-lactam antibiotics being safe, double β-lactam and β-lactam/β-lactamase inhibitor combinations are of interest for improving the treatment of M. abscessus infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different β-lactams in M. abscessus. We determined the preferred PBP targets of 13 β-lactams and 2 β-lactamase inhibitors in two M. abscessus strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the β-lactam concentrations that half-maximally inhibited Bocillin binding (50% inhibitory concentrations [IC50s]). Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016 to 0.5 mg/liter) (cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031 to 1 mg/liter) (ceftriaxone and cefotaxime) or intermediate (0.35 to 16 mg/liter) (ceftazidime and cefoxitin) concentrations. Sulbactam, aztreonam, carumonam, mecillinam, and avibactam (cluster 3) inactivated the same PBPs as cephalosporins but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2 to 16 mg/liter. Carbapenems, ceftriaxone, and cefotaxime were the most promising β-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in M. abscessus provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with β-lactams.
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Paik, Johanna, Iza Kern, Rudi Lurz, and Regine Hakenbeck. "Mutational Analysis of the Streptococcus pneumoniae Bimodular Class A Penicillin-Binding Proteins." Journal of Bacteriology 181, no. 12 (1999): 3852–56. http://dx.doi.org/10.1128/jb.181.12.3852-3856.1999.
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ABSTRACT One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding–transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.
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Kocaoglu, Ozden, та Erin E. Carlson. "Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Escherichia coli Strain DC2". Antimicrobial Agents and Chemotherapy 59, № 5 (2015): 2785–90. http://dx.doi.org/10.1128/aac.04552-14.
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ABSTRACTPenicillin-binding proteins (PBPs) are integral players in bacterial cell division, and their catalytic activities can be monitored with β-lactam-containing chemical probes. Compounds that target a single PBP could provide important information about the specific role(s) of each enzyme, making identification of such molecules important. We evaluated 22 commercially available β-lactams for inhibition of the PBPs in liveEscherichia colistrain DC2. Whole cells were titrated with β-lactam antibiotics and subsequently incubated with a fluorescent penicillin derivative, Bocillin-FL (Boc-FL), to label uninhibited PBPs. Protein visualization was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and fluorescent scanning. The examined β-lactams exhibited diverse PBP selectivities, with amdinocillin (mecillinam) showing selectivity for PBP2, aztreonam, piperacillin, cefuroxime, cefotaxime, and ceftriaxone for PBP3, and amoxicillin and cephalexin for PBP4. The remaining β-lactams did not block any PBPs in the DC2 strain ofE. colior inhibited more than one PBP at all examined concentrations in this Gram-negative organism.
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Moyá, Bartolomé, Laura Zamorano, Carlos Juan, Yigong Ge, and Antonio Oliver. "Affinity of the New Cephalosporin CXA-101 to Penicillin-Binding Proteins of Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 54, no. 9 (2010): 3933–37. http://dx.doi.org/10.1128/aac.00296-10.
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ABSTRACT CXA-101, previously designated FR264205, is a new antipseudomonal cephalosporin. The objective of this study was to determine the penicillin-binding protein (PBP) inhibition profile of CXA-101 compared to that of ceftazidime (PBP3 inhibitor) and imipenem (PBP2 inhibitor). Killing kinetics, the induction of AmpC expression, and associated changes on cell morphology were also investigated. The MICs for CXA-101, ceftazidime, and imipenem were 0.5, 1, and 1 μg/ml, respectively. Killing curves revealed that CXA-101 shows a concentration-independent bactericidal activity, with concentrations of 1× the MIC (0.5 μg/ml) producing a >3-log reduction in bacterial load after 8 h of incubation. Live-dead staining showed that concentrations of CXA-101 as low as 0.5× the MIC stopped bacterial septation and induced an intense filamentation, which is consistent with the documented high affinity of PBP3. CXA-101 was found to be a potent PBP3 inhibitor and showed affinities ≥2-fold higher than those of ceftazidime for all of the essential PBPs (1b, 1c, 2, and 3). Compared to imipenem, in addition to the obvious inverse PBP2/PBP3 affinities, CXA-101 showed a significantly higher affinity for PBP1b but a lower affinity for PBP1c. Furthermore, CXA-101, like ceftazidime and in contrast to imipenem, was found to be a very weak inducer of AmpC expression, consistent with the low PBP4 affinity documented.
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Hanzel, Adrienn. "Újszerű megközelítés: pontozáson alapuló, testmozgást ösztönző módszer – cikkismertetés." Egészségfejlesztés 59, no. 3 (2018): 57–58. http://dx.doi.org/10.24365/ef.v59i3.302.
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Bevezetés: A közelmúltban végzett vizsgálatok arra hívják fel a figyelmet, hogy a középkorú nők két alapvető dolog miatt nem végeznek fizikai aktivitást, egyrészt az idő hiánya miatt, másrészt a motiváció hiánya miatt. Ezért a szerzők úgy gondolták, hogy ki kellene dolgozni egy olyan technikát, amely beleilleszthető a mindennapi életbe. Kutatás célja: Megvizsgálni, hogy az új "pontozás alapú" fizikai aktivitás (PBPA) mérési módszernek milyen hatásai vannak a testösszetételre az inaktív nők körében, akik túlsúlyosak vagy elhízottak, szemben a strukturált testmozgást végzőkkel. Módszerek: 76 túlsúlyos vagy elhízott, középkorú inaktív nőt (41 ± 2 év; átlagos BMI 29,2 ± 3,4 kg/m2) véletlenszerűen osztottak be a 3 csoportba, 24 hetes vizsgálati időszakra. Egyik csoport a "pontozás alapú" fizikai aktivitást végzőkből állt, akiknek hetente 30 pontot kellett összegyűjteni, másik csoport a strukturált testmozgást végzők, akik heti 5-ször 30 perc mérsékelt intenzitású testmozgást végeztek, a harmadik csoport pedig a kontroll volt, akik továbbra is szokásos inaktív életmódot folytattak. A PBPA csoportba tartozó személyeket egy táblázattal látták el, melyben különböző tevékenységek voltak feltüntetve, például: porszívózás, kertészkedés, ablakpucolás, vasalás stb. Mindegyik tevékenységnek volt egy adott pontja, mely pontszám tízpercnyi mozgásformára vonatkozott. A fizikai aktivitás pontokat a metabolikus egyenértékből (MET) adaptálták, mely alapján 30 pont volt egyenértékű a 150 perces tempós gyaloglással. A 0., 4., 12. és 24. héten testösszetétel (DXA: dual energy X-ray absorptiometry) és antropometriai mérések történtek. A saját bevalláson alapuló tevékenységeket heti rendszerességgel rögzítették. A 0. és a 24. héten a táplálkozást önbevallással (táplálkozási napló), a fizikai aktivitást objektív aktivitásmérő műszerrel (accelerométer) adták meg. Eredmények: A 76 résztvevőből 58 fő teljesítette a 24 hetes intervenciós időszakot, innen származtak az elsődleges végpontokra vonatkozó adatok (testösszetétel, antropometria). A fizikai aktivitás objektív mérésére 19 résztvevőnél volt lehetőség, és 41 főnél vettek fel élelmiszerfogyasztásra vonatkozó adatokat. A PBPA csoportba tartozó nők szignifikáns súlycsökkenést értek el a 24. hét végére: -3,3 ± 5,9 kg (-3,4 ± 7,1%, p = 0,004). A derékbőség (-2,8 ± 4,6 cm vs. +2,1 ± 6,6 cm, p = 0,024), valamint a testzsír mennyisége (-2,3 ± 4,6 kg vs. +0,1 ± 2,0 kg, p = 0,075) a PBPA csoportban a 24. hét végére csökkent, szemben a kontroll csoport értékeivel. A hasi lokalizációjú elhízás mind a 12. héten (-6,1 ± 12,6%, p = 0,005), mind a 24. héten (-10,1 ± 18,4%, p = 0,005) csökkent a PBPA csoportban, a viszcerális zsírszövet pedig még nagyobb mértékű csökkenést mutatott (- 5,8 ± 26,0%) szemben a kontroll csoporttal (+7,8 ± 18,3%, p = 0,053) a 24. hét végére. A testösszetétel, a testtömeg és a derékbőség változatlanok voltak a strukturált testmozgást végzőknél. A PBPA csoportban nőtt a könnyű fizikai aktivitás végzése, ugyanakkor trendszerűen csökkent az ülő életmódot folytatók száma. Ráadásul a PBPA csoportban a napi energia-felvétel csökkenésének tendenciája is megfigyelhető volt –445 ± 564 kcal-ával (p = 0,074), és jelentős csökkenést tapasztaltak a napi zsírbevitel mennyiségében is (p = 0,042). Következtetés: Az egyszerű ösztönzésen alapuló, fizikai aktivitást támogató új pontrendszer hatékony stratégiának tűnik a testtömeg és a testzsír csökkentésére a túlsúlyos és elhízott inaktív nőknél, szemben akár a strukturált testmozgást végzőkkel is. Tanulságok a hazai szakemberek számára: A szerzők által ismertetett módszer könnyen beilleszthető a mindennapi rutinba, ezáltal nagyobb rugalmasságot és színesebb, teljesíthetőbb választási lehetőséget biztosít a célpopulációnak. A vizsgálat eredményei az egészségügyben dolgozó szakemberek számára is fontos népegészségügyi üzenetet hordoznak, mely a lakosság tájékoztatásával jelentős mértékben hozzájárulhat az egészség javításához, megőrzéséhez.
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Sieradzki, Krzysztof, and Alexander Tomasz. "Gradual Alterations in Cell Wall Structure and Metabolism in Vancomycin-Resistant Mutants ofStaphylococcus aureus." Journal of Bacteriology 181, no. 24 (1999): 7566–70. http://dx.doi.org/10.1128/jb.181.24.7566-7570.1999.
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ABSTRACT In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 μg/ml; MIC of vancomycin, 1.5 μg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to β-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.
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Rice, Louis B., Lenore L. Carias, Susan Rudin та ін. "Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium". Journal of Bacteriology 191, № 11 (2009): 3649–56. http://dx.doi.org/10.1128/jb.01834-08.
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ABSTRACT Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of β-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the ΔpbpF ΔponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the l,d-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the ΔpbpF ΔponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different β-lactam antibiotics differed as a function of its partner glycosyltransferase.
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Papp-Wallace, Krisztina M., Baui Senkfor, Julian Gatta та ін. "Early Insights into the Interactions of Different β-Lactam Antibiotics and β-Lactamase Inhibitors against Soluble Forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3". Antimicrobial Agents and Chemotherapy 56, № 11 (2012): 5687–92. http://dx.doi.org/10.1128/aac.01027-12.
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ABSTRACTAcinetobacter baumanniiis an increasingly problematic pathogen in United States hospitals. Antibiotics that can treatA. baumanniiare becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of β-lactam antibiotics, to β-lactam–sulbactam susceptibility and β-lactam resistance inA. baumannii. Decreased expression of PBPs as well as loss of binding of β-lactams to PBPs was previously shown to promote β-lactam resistance inA. baumannii. Using anin vitroassay with a reporter β-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC50s) for PBP1a fromA. baumanniiand PBP3 fromAcinetobactersp. ranged from 1 to 5 μM for a series of β-lactams. In contrast, PBP3 demonstrated a narrower range of IC50s against β-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 μM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC50s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective againstA. baumannii. Unraveling the contribution of PBPs to β-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel β-lactams.
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Davies, Todd A., Wenchi Shang, Karen Bush, and Robert K. Flamm. "Affinity of Doripenem and Comparators to Penicillin-Binding Proteins in Escherichia coli and Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 52, no. 4 (2008): 1510–12. http://dx.doi.org/10.1128/aac.01529-07.
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ABSTRACT Doripenem, a parenteral carbapenem, exhibited high affinity for penicillin-binding protein 2 (PBP2) and PBP3 in Pseudomonas aeruginosa and PBP2 in Escherichia coli, the primary PBPs whose inhibition leads to cell death. This PBP affinity profile correlates with the broad-spectrum gram-negative activity observed with doripenem.
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Chen, Longxin, Chaoyang Zhu, Hui Guo, et al. "Epitope-directed antibody selection by site-specific photocrosslinking." Science Advances 6, no. 14 (2020): eaaz7825. http://dx.doi.org/10.1126/sciadv.aaz7825.
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Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human interleukin-1β and complement 5a. In both cases, more than one-third of hits identified bind to the target epitopes, demonstrating the feasibility and versatility of this method.
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Fernandez, Regina, Liliana I. Paz, Roberto R. Rosato, and Adriana E. Rosato. "Ceftaroline Is Active against Heteroresistant Methicillin-Resistant Staphylococcus aureus Clinical Strains despite Associated Mutational Mechanisms and Intermediate Levels of Resistance." Antimicrobial Agents and Chemotherapy 58, no. 10 (2014): 5736–46. http://dx.doi.org/10.1128/aac.03019-14.
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ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) is an important infectious human pathogen responsible for diseases ranging from skin and soft tissue infections to life-threatening endocarditis. β-Lactam resistance in MRSA involves acquisition of penicillin-binding protein 2a (PBP2a), a protein with low affinity for β-lactams that mediates cell wall assembly when the normal staphylococcal PBPs (PBP1 to -4) are blocked by these agents. Many MRSA strains display heterogeneous expression of resistance (HeR) against β-lactam antibiotics. The β-lactam-mediated homoresistant (HoR) phenotype is associated with both expression of themecAgene and activation of the LexA-RecA-mediated SOS response, a regulatory network induced in response to DNA damage. Ceftaroline (CPT) is the only FDA-approved cephalosporin targeting PBP2a. We investigated the mechanistic basis of CPT activity against HeR-MRSA strains, including a set of strains displaying an intermediate level of resistance to CPT. Mechanistically, we found that 1 exposure of HeR-MRSA to subinhibitory concentrations of CPT selected for the HoR derivative activated the SOS response and increased mutagenesis. Importantly, CPT-selected HoR cells remained susceptible to CPT while still being resistant to most β-lactams, and 2-CPT activity in HeR-MRSA resided in an attenuated induction ofmecAexpression in comparison to other β-lactams. In addition, 3-CPT intermediate-resistant strains displayed a significant increase in CPT-inducedmecAexpression accompanied by mutations in PBP2, which together may interfere with the complete repression by CPT of both PBP2a and PBP2a-PBP2 interactions and thus be a determining factor in the low level of CPT resistance in the absence ofmecAgene mutations. The present study provides mechanistic evidence that CPT represents an alternative therapeutic option for the treatment of heteroresistant MRSA strains.
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Asli, Abdelhamid, Eric Brouillette, Kevin M. Krause, Wright W. Nichols, and François Malouin. "Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria." Antimicrobial Agents and Chemotherapy 60, no. 2 (2015): 752–56. http://dx.doi.org/10.1128/aac.02102-15.
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ABSTRACTAvibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated.Staphylococcus aureuswas of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. InEscherichia coli,Pseudomonas aeruginosa,Haemophilus influenzae, andS. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed inStreptococcus pneumoniae(IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling ofS. aureusPBP4 by Bocillin FL. In PBP competition assays withS. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets.
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el Kharroubi, A., P. Jacques, G. Piras, J. Van Beeumen, J. Coyette, and J. M. Ghuysen. "The Enterococcus hirae R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus penicillin-binding protein 2′ are similar." Biochemical Journal 280, no. 2 (1991): 463–69. http://dx.doi.org/10.1042/bj2800463.
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The penicillin-resistant Enterococcus hirae R40 has a typical profile of membrane-bound penicillin-binding proteins (PBPs) except that the 71 kDa PBP5 of low penicillin affinity represents about 50% of all the PBPs present. Water-soluble tryptic-digest peptides were selectively produced from PBP5, their N-terminal regions were sequenced and synthetic oligonucleotides were used as primers to generate a 476 bp DNA fragment by polymerase chain reaction. On the basis of these data, the PBP5-encoding gene was cloned in Escherichia coli by using pBR322 as vector. The gene, included in a 7.1 kb insert, had the information for a 678-amino acid-residue protein. PBP5 shows similarity, in the primary structure, with the high-molecular-mass PBPs of class B. In particular, amino acid alignment of the enterococcal PBP5 and the methicillin-resistant staphylococcal PBP2′ generates scores that are 30, for the N-terminal domains, and 53, for the C-terminal domains, standard deviations above that expected for a run of 20 randomized pairs of proteins having the same amino acid compositions as the two proteins under consideration.
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SALMAN, ABBAS Z., QUSAY A. JAWAD, KHALID S. RIDAH, LINA M. SHAKER, and AHMED A. AL-AMIERY. "SELECTED BIS-THIADIAZOLE: SYNTHESIS AND CORROSION INHIBITION STUDIES ON MILD STEEL IN HCL ENVIRONMENT." Surface Review and Letters 27, no. 12 (2020): 2050014. http://dx.doi.org/10.1142/s0218625x20500146.
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The inhibition effect of synthesized corrosion inhibitor namely 5,5′-(1,4-phenylene)bis([Formula: see text]-phenyl-1,3,4-thiadiazol-2-amine) (PBPA) on the corrosion of mild steel in 1-M hydrochloric acid environment are examined by gravimetric techniques at various temperature (303–343 K). The synthesized inhibitor concentrations are 0.1–0.5[Formula: see text]mM. The inhibition efficiency increased with the increase of the inhibitor concentration. The inhibition efficiency reached 94% at the highest studied concentration of 0.5[Formula: see text]mM for 5[Formula: see text]h of immersion time and 303[Formula: see text]K. Moreover, the inhibition efficiency decreased with the temperature increase. The adsorption of tested inhibitor molecules on the surface of mild steel follows the Langmuir adsorption isotherm. The studied inhibitor molecules showed excellent inhibition since PBPA molecules have nitrogen and sulfur atoms in addition to phenyl and thiadiazol rings which were linked together in conjugation system.
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Dumitrescu, Oana, Priya Choudhury, Sandrine Boisset та ін. "β-Lactams Interfering with PBP1 Induce Panton-Valentine Leukocidin Expression by TriggeringsarAandrotGlobal Regulators of Staphylococcus aureus". Antimicrobial Agents and Chemotherapy 55, № 7 (2011): 3261–71. http://dx.doi.org/10.1128/aac.01401-10.
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ABSTRACTPrevious articles reported that beta-lactam antibiotics increase the expression ofStaphylococcus aureusPanton-Valentine leukocidin (PVL) by activating its transcription. We investigated the mechanisms underlying the inductor effect of beta-lactams on PVL expression by determining targets and regulatory pathways possibly implicated in this process. We measured PVL production in the presence of oxacillin (nonselective), imipenem (penicillin-binding protein 1 [PBP1] selective), cefotaxime (PBP2 selective), cefaclore (PBP3 selective), and cefoxitin (PBP4 selective).In vitro, we observed increased PVL production consistent withluk-PV mRNA levels that were 20 to 25 times higher for community-acquired methicillin-resistantS. aureus(CA-MRSA) cultures treated with PBP1-binding oxacillin and imipenem than for cultures treated with other beta-lactams or no antibiotic at all. This effect was also observedin vivo, with increased PVL mRNA levels in lung tissues from CA-MRSA-infected mice treated with imipenem but not cefoxitin. To confirm the involvement of PBP1 inhibition in this pathway, PBP1 depletion by use of an induciblepbp1antisense RNA showed a dose-dependent relationship between the level ofpbp1antisense RNA and theluk-PV mRNA level. Upon imipenem treatment of exponential-phase cultures, we observed an increasedsarAmRNA level after 30 min of incubation followed by a decreasedrotmRNA level after 1 to 4 h of incubation. Unlike theagrandsaeRSpositive regulators, which were nonessential for PVL induction by beta-lactams, thesarA(positive) androt(negative) PVL regulators were necessary for PVL induction by imipenem. Our results suggest that antibiotics binding to PBP1 increase PVL expression by modulatingsarAandrot, which are essential mediators of the inductor effect of beta-lactams on PVL expression.
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Zawadzka-Skomiał, Joanna, Zdzislaw Markiewicz, Martine Nguyen-Distèche, Bart Devreese, Jean-Marie Frère, and Mohammed Terrak. "Characterization of the Bifunctional Glycosyltransferase/Acyltransferase Penicillin-Binding Protein 4 of Listeria monocytogenes." Journal of Bacteriology 188, no. 5 (2006): 1875–81. http://dx.doi.org/10.1128/jb.188.5.1875-1881.2006.
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ABSTRACT Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M−1 s−1 from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to β-lactam antibiotics but did increase the resistance of the mutant to moenomycin.
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Kocaoglu, Ozden, Ho-Ching T. Tsui, Malcolm E. Winkler та Erin E. Carlson. "Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Streptococcus pneumoniae D39". Antimicrobial Agents and Chemotherapy 59, № 6 (2015): 3548–55. http://dx.doi.org/10.1128/aac.05142-14.
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ABSTRACTSelective fluorescent β-lactam chemical probes enable the visualization of the transpeptidase activity of penicillin-binding proteins (PBPs) at different stages of bacterial cell division. To facilitate the development of new fluorescent probes for PBP imaging, we evaluated 20 commercially available β-lactams for selective PBP inhibition in an unencapsulated derivative of the D39 strain ofStreptococcus pneumoniae. Live cells were treated with β-lactam antibiotics at different concentrations and subsequently incubated with Bocillin FL (Boc-FL; fluorescent penicillin) to saturate uninhibited PBPs. Fluorophore-labeled PBPs were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence scanning. Among 20 compounds tested, carbapenems (doripenem and meropenem) were coselective for PBP1a, PBP2x, and PBP3, while six of the nine penicillin compounds were coselective for PBP2x and PBP3. In contrast, the seven cephalosporin compounds tested display variability in their PBP-binding profiles. Three cephalosporin compounds (cefoxitin, cephalexin, and cefsulodin) and the monobactam aztreonam exhibited selectivity for PBP3, while only cefuroxime (a cephalosporin) was selective for PBP2x. Treatment ofS. pneumoniaecultures with a sublethal concentration of cefuroxime that inhibited 60% of PBP2x activity and less than 20% of the activity of other PBPs resulted in formation of elongated cells. In contrast, treatment ofS. pneumoniaecultures with concentrations of aztreonam and cefoxitin that inhibited up to 70% of PBP3 activity and less than 30% of other PBPs resulted in no discernible morphological changes. Additionally, correlation of the MIC and IC50s for each PBP, with the exception of faropenem, amdinocillin (mecillinam), and 6-APA, suggests that pneumococcal growth inhibition is primarily due to the inhibition of PBP2x.
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Davies, Todd A., Malcolm G. P. Page, Wenchi Shang, Ted Andrew, Malgosia Kania, and Karen Bush. "Binding of Ceftobiprole and Comparators to the Penicillin-Binding Proteins of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae." Antimicrobial Agents and Chemotherapy 51, no. 7 (2007): 2621–24. http://dx.doi.org/10.1128/aac.00029-07.
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ABSTRACT Ceftobiprole exhibited tight binding to PBP2a in methicillin-resistant Staphylococcus aureus, PBP2x in penicillin-resistant Streptococcus pneumoniae, and PBP3 and other essential penicillin-binding proteins in methicillin-susceptible S. aureus, Escherichia coli, and Pseudomonas aeruginosa. Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria.
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Oliveira, Clarice de, Camila Bellaver Alberti, Laura Boeck Silva, and Gabriela Rosa Nodari. "THE PEOPLE’S BOUROUGH PLAN OF ACTION: A COUNTER-PROJECT OF INSURGENT CITIZENSHIP1." REAd. Revista Eletrônica de Administração (Porto Alegre) 25, no. 3 (2019): 247–77. http://dx.doi.org/10.1590/1413-2311.274.97304.
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ABSTRACT Porto Alegre is divided in eight Boroughs of Planning. Each Borough has a representative in the Urban Planning City Council (UPCC). In 2018 social movements organized to conquer this. From this, some of the Boroughs’ councilors felt the need to better inform themselves on the terms and subjects discussed in the UPCC, and to ensure that their local issues would be discussed. For that manner, the People’s Borough Plan of Action (PBPA) project was created by a coalition of social movements, the architect’s association (IAB-RS) and the university to perform counter-hegemonic actions. The project is based on the insurgent planning theory, which understands urban development from the standpoint of the global south as being essentially performed by communities, activists and grassroots strategies. Thus, the project moves across both invited and invented spaces of action in a non-binary relationship, with the aim of providing the grassroots movements of insurgent citizenship with technical assistance to support their claims and desires over the city they live. Regarding the City’s Master Plan revision, the PBAP represents a counter-plan related to the creation of differential spaces. Therefore, they create a moment of realization of the right to the city.
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McPherson, Derrell C., Adam Driks, and David L. Popham. "Two Class A High-Molecular-Weight Penicillin-Binding Proteins of Bacillus subtilis Play Redundant Roles in Sporulation." Journal of Bacteriology 183, no. 20 (2001): 6046–53. http://dx.doi.org/10.1128/jb.183.20.6046-6053.2001.
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ABSTRACT The four class A penicillin-binding proteins (PBPs) ofBacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamedpbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic δ-lactam and l-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.
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Gardete, S., A. M. Ludovice, R. G. Sobral, S. R. Filipe, H. de Lencastre та A. Tomasz. "Role of murE in the Expression of β-Lactam Antibiotic Resistance in Staphylococcus aureus". Journal of Bacteriology 186, № 6 (2004): 1705–13. http://dx.doi.org/10.1128/jb.186.6.1705-1713.2004.
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ABSTRACT It was shown earlier that Tn551 inserted into the C-terminal region of murE of parental methicillin-resistant Staphylococcus aureus strain COL causes a drastic reduction in methicillin resistance, accompanied by accumulation of UDP-MurNAc dipeptide in the cell wall precursor pool and incorporation of these abnormal muropeptides into the peptidoglycan of the mutant. Methicillin resistance was recovered in a suppressor mutant. The murE gene of the same strain was then put under the control of the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter P spac . Bacteria grown in the presence of suboptimal concentrations of IPTG accumulated UDP-MurNAc dipeptide in the cell wall precursor pool. Both growth rates and methicillin resistance levels (but not resistance to other antibiotics) were a function of the IPTG concentration. Northern analysis showed a gradual increase in the transcription of murE and also in the transcription of pbpB and mecA, parallel with the increasing concentrations of IPTG in the medium. A similar increase in the transcription of pbpB and mecA, the structural genes of penicillin-binding protein 2 (PBP2) and PBP2A, was also detected in the suppressor mutant. The expression of these two proteins, which are known to play critical roles in the mechanism of staphylococcal methicillin resistance, appears to be—directly or indirectly—under the control of the murE gene. Our data suggest that the drastic reduction of the methicillin MIC seen in the murE mutant may be caused by the insufficient cellular amounts of these two PBPs.
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Ishida, Kenji, Trinh Viet Hung, Kwangkyoung Liou, Hei Chan Lee, Chang-Hun Shin, and Jae Kyung Sohng. "Characterization of pbpA and pbp2 Encoding Penicillin-binding Proteins Located on the Downstream of Clavulanic Acid Gene Cluster in Streptomyces clavuligerus." Biotechnology Letters 28, no. 6 (2006): 409–17. http://dx.doi.org/10.1007/s10529-005-6071-5.
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Duez, Colette, Marc Vanhove, Xavier Gallet, et al. "Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein fromBacillus subtilis." Journal of Bacteriology 183, no. 5 (2001): 1595–99. http://dx.doi.org/10.1128/jb.183.5.1595-1599.2001.
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ABSTRACT Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits dd-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781–788, 1992), which is rapidly inactivated by many β-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k 2/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M−1s−1 for the Actinomadura sp. strain R39 peptidase, 1,400 M−1 s−1 for B. subtilis PBP4a, and 7,000 M−1 s−1 forEscherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k 2/K = 46,000 M−1 s−1). PBP4a is also much more thermostable than the R39 enzyme.
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Moya, Bartolome, Sachin Bhagwat, Gabriel Cabot, German Bou, Mahesh Patel, and Antonio Oliver. "Effective inhibition of PBPs by cefepime and zidebactam in the presence of VIM-1 drives potent bactericidal activity against MBL-expressing Pseudomonas aeruginosa." Journal of Antimicrobial Chemotherapy 75, no. 6 (2020): 1474–78. http://dx.doi.org/10.1093/jac/dkaa036.
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Abstract Objectives The combination of cefepime and the novel β-lactam enhancer zidebactam (WCK 5222) is under development for the treatment of difficult-to-treat Gram-negative infections. Against MBL-producing pathogens, cefepime and zidebactam induce cell elongation and spheroplast formation, indicating PBP3 and PBP2 dysfunction, respectively, having a potent bactericidal effect as a combination. The objective of the present study was to determine the mechanistic basis of the bactericidal effect of cefepime/zidebactam on MBL-expressing pathogens. Methods Pseudomonal PBP-binding affinities of cefepime, zidebactam and imipenem were assessed at different timepoints and also in the presence of purified VIM-1 using a Bocillin FL competition assay. The antibacterial activity of cefepime/zidebactam against three VIM-expressing Pseudomonas aeruginosa isolates was assessed by time–kill and neutropenic mouse lung/thigh infection studies. Results Amidst cefepime-hydrolysing concentrations of VIM-1, substantial cefepime binding to target PBPs was observed. High-affinity binding of zidebactam to PBP2 remained unaltered in the presence of VIM-1; however, MBL addition significantly affected imipenem PBP2 binding. Furthermore, the rate of cefepime binding to the primary target PBP3 was found to be higher compared with the imipenem PBP2 binding rate. Finally, complementary PBP inhibition by cefepime/zidebactam resulted in enhanced bactericidal activity in time–kill and neutropenic mouse lung/thigh infection studies against VIM-6-, VIM-10- and VIM-11-expressing P. aeruginosa, thus revealing the mechanistic basis of β-lactam enhancer action. Conclusions For the first time ever (to the best of our knowledge), this study demonstrates that in the presence of VIM-1 MBL, β-lactamase-labile cefepime and β-lactamase-stable zidebactam produce effective inhibition of respective target PBPs. For cefepime, this seems to be a result of a faster rate of PBP binding, which helps it overcome β-lactamase-mediated hydrolysis.
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Murray, T., D. L. Popham, and P. Setlow. "Identification and characterization of pbpA encoding Bacillus subtilis penicillin-binding protein 2A." Journal of bacteriology 179, no. 9 (1997): 3021–29. http://dx.doi.org/10.1128/jb.179.9.3021-3029.1997.
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Ferraro, John R., Anita Furlani, and Maria Vittoria Russo. "Vibrational Spectroscopy of Iodine-Doped Substituted Polyacetylenes." Applied Spectroscopy 41, no. 5 (1987): 830–33. http://dx.doi.org/10.1366/0003702874448166.
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Raman and far-infrared (FIR) data for iodine-doped substituted polyacetylenes, such as poly(2-propyn-1-ol)-(POHP) and poly(benzylpropargylamine)-(PBPA) are reported. The data are consistent with an I5−(I2 … I3)− dopant species in the heavily doped polymers. Solution and solid data provided similar vibrational results. Stability of the polymeric radical cation and the polyiodide anion moiety in tetrahydrofuran is attributed to the solvation effects of the ions by the solvent. New data on heavily doped poly(phenyl)acetylene (PPA) are presented.
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Bhardwaj, Pooja, Moutusee Z. Islam, Christi Kim, Uyen Thy Nguyen, and Kelli L. Palmer. "ddcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410." PLOS ONE 16, no. 4 (2021): e0249631. http://dx.doi.org/10.1371/journal.pone.0249631.
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Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that cause life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a synergistic effect of the two antimicrobials was observed. In this study, we used multiple approaches to investigate the mechanism of synergism between chlorhexidine and vancomycin in the VanA-type VRE strain E. faecium 1,231,410. We generated clean deletions of 7 of 11 pbp, transpeptidase, and carboxypeptidase genes in this strain (ponA, pbpF, pbpZ, pbpA, ddcP, ldtfm, and vanY). Deletion of ddcP, encoding a membrane-bound carboxypeptidase, altered the synergism phenotype. Furthermore, using in vitro evolution, we isolated a spontaneous synergy escaper mutant and utilized whole genome sequencing to determine that a mutation in pstB, encoding an ATPase of phosphate-specific transporters, also altered synergism. Finally, addition of excess D-lactate, but not D-alanine, enhanced synergism to reduce vancomycin MIC levels. Overall, our work identified factors that alter chlorhexidine and vancomycin synergism in a model VanA-type VRE strain.
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Arbeloa, Ana, Heidi Segal, Jean-Emmanuel Hugonnet та ін. "Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance in Enterococcus faecalis". Journal of Bacteriology 186, № 5 (2004): 1221–28. http://dx.doi.org/10.1128/jb.186.5.1221-1228.2004.
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ABSTRACT Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 ΔponA ΔpbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.
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Memmi, Guido, Sergio R. Filipe, Mariana G. Pinho, Zhibiao Fu та Ambrose Cheung. "Staphylococcus aureus PBP4 Is Essential for β-Lactam Resistance in Community-Acquired Methicillin-Resistant Strains". Antimicrobial Agents and Chemotherapy 52, № 11 (2008): 3955–66. http://dx.doi.org/10.1128/aac.00049-08.
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ABSTRACT Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (MRSA) (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs by virtue of their genomic background and increased virulence in animal models. Here, we show that in two common CA-MRSA isolates, USA300 and MW2 (USA400), a loss of penicillin binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance, thus demonstrating that mecA, encoding PBP2A, is not the sole determinant of methicillin resistance in CA-MRSA. The loss of PBP4 was also found to severely affect the transcription of PBP2 in cells after challenge with oxacillin, thus leading to a significant decrease in peptidoglycan cross-linking. Autolysis, which is commonly associated with the killing mechanism of penicillin and β-lactams, does not play a role in the reduced resistance phenotype associated with the loss of PBP4. We also showed that cefoxitin, a semisynthetic β-lactam that binds irreversibly to PBP4, is synergistic with oxacillin in killing CA-MRSA strains, including clinical CA-MRSA isolates. Thus, PBP4 represents a major target for drug rediscovery against CA-MRSA, and a combination of cefoxitin and synthetic penicillins may be an effective therapy for CA-MRSA infections.
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Sanbongi, Yumiko, Takashi Ida, Midori Ishikawa, et al. "Complete Sequences of Six Penicillin-Binding Protein Genes from 40 Streptococcus pneumoniae Clinical Isolates Collected in Japan." Antimicrobial Agents and Chemotherapy 48, no. 6 (2004): 2244–50. http://dx.doi.org/10.1128/aac.48.6.2244-2250.2004.
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ABSTRACT All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 μg/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs ≧ 2 μg/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 μg/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were ≧1 μg/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 μg/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to β-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with β-lactam resistance.
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Moyá, Bartolomé, Alejandro Beceiro, Gabriel Cabot та ін. "Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities". Antimicrobial Agents and Chemotherapy 56, № 9 (2012): 4771–78. http://dx.doi.org/10.1128/aac.00680-12.
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ABSTRACTWe investigated the mechanisms leading toPseudomonas aeruginosapan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence ofoprD,ampD, anddacBmutations was explored by PCR followed by sequencing and the expression ofampCand efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressedampCand one or several efflux pumps, particularlymexBand/ormexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n= 1) or diminished expression (n= 1) of PBP1a and a lack of PBP4 expression (n= 4), which correlated with AmpC overexpression driven bydacBmutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in thein vivoemergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.
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Hoskins, JoAnn, Patti Matsushima, Deborah L. Mullen, et al. "Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in Streptococcus pneumoniae." Journal of Bacteriology 181, no. 20 (1999): 6552–55. http://dx.doi.org/10.1128/jb.181.20.6552-6555.1999.
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ABSTRACT The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a inStreptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2amutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, andpbp2a genes are dispensable but that eitherpbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.