scholarly journals Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

2013 ◽  
Vol 57 (7) ◽  
pp. 3137-3146 ◽  
Author(s):  
Jeffrey D. Smith ◽  
Malika Kumarasiri ◽  
Weilie Zhang ◽  
Dusan Hesek ◽  
Mijoon Lee ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Binding Protein ◽  
Main Function ◽  
Penicillin Binding Protein ◽  
Active Site Residues ◽  
Content Type ◽  
A Cell ◽  
Dynamics Simulations

ABSTRACTPenicillin-binding protein 5 (PBP5) is one of the most abundant PBPs inPseudomonas aeruginosa. Although its main function is that of a cell walldd-carboxypeptidase, it possesses sufficient β-lactamase activity to contribute to the ability ofP. aeruginosato resist the antibiotic activity of the β-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance byP. aeruginosa, an important human pathogen, and to the understanding of the evolution of β-lactamase activity from the PBP enzymes. We purified a soluble version ofP. aeruginosaPBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Underin vitroconditions, Pa sPBP5 demonstrates bothdd-carboxypeptidase and expanded-spectrum β-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A β-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 ofEscherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in theE. coliPBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the β-lactam resistance ofP. aeruginosawhile highlighting how broader β-lactamase activity may be encoded in the structural folds shared by the PBP and serine β-lactamase classes.

scholarly journals Structural Insights into Ceftobiprole Inhibition of Pseudomonas aeruginosa Penicillin-Binding Protein 3

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Vijay Kumar ◽  
Christie Tang ◽  
Christopher R. Bethel ◽  
Krisztina M. Papp-Wallace ◽  
Jacob Wyatt ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Binding Protein ◽  
Covalent Bond ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Cephalosporin Antibiotic ◽  
Structural Insights ◽  
Fine Tune ◽  
Advanced Generation

ABSTRACT Ceftobiprole is an advanced-generation broad-spectrum cephalosporin antibiotic with potent and rapid bactericidal activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, as well as susceptible Gram-negative pathogens, including Pseudomonas sp. pathogens. In the case of Pseudomonas aeruginosa, ceftobiprole acts by inhibiting P. aeruginosa penicillin-binding protein 3 (PBP3). Structural studies were pursued to elucidate the molecular details of this PBP inhibition. The crystal structure of the His-tagged PBP3-ceftobiprole complex revealed a covalent bond between the ligand and the catalytic residue S294. Ceftobiprole binding leads to large active site changes near binding sites for the pyrrolidinone and pyrrolidine rings. The S528 to L536 region adopts a conformation previously not observed in PBP3, including partial unwinding of the α11 helix. These molecular insights can lead to a deeper understanding of β-lactam-PBP interactions that result in major changes in protein structure, as well as suggesting how to fine-tune current inhibitors and to develop novel inhibitors of this PBP.

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

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

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

scholarly journals Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

2012 ◽  
Vol 56 (9) ◽  
pp. 4771-4778 ◽  
Author(s):  
Bartolomé Moyá ◽  
Alejandro Beceiro ◽  
Gabriel Cabot ◽  
Carlos Juan ◽  
Laura Zamorano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Binding Protein ◽  
Efflux Pumps ◽  
Binding Affinities ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Reverse Transcription Pcr

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.

scholarly journals In Vitro and In Vivo Activities of β-Lactams in Combination with the Novel β-Lactam Enhancers Zidebactam and WCK 5153 against Multidrug-Resistant Metallo-β-Lactamase-Producing Klebsiella pneumoniae

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Bartolome Moya ◽  
Isabel M. Barcelo ◽  
Gabriel Cabot ◽  
Gabriel Torrens ◽  
Snehal Palwe ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Molecular Targets ◽  
Multidrug Resistant ◽  
The Novel ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Time Kill

ABSTRACT Zidebactam and WCK 5153 are novel bicyclo-acyl hydrazide (BCH) agents that have previously been shown to act as β-lactam enhancer (BLE) antibiotics in Pseudomonas aeruginosa and Acinetobacter baumannii. The objectives of this work were to identify the molecular targets of these BCHs in Klebsiella pneumoniae and to investigate their potential BLE activity for cefepime and aztreonam against metallo-β-lactamase (MBL)-producing strains in vitro and in vivo. Penicillin binding protein (PBP) binding profiles were determined by Bocillin FL assay, and 50% inhibitory concentrations (IC50s) were determined using ImageQuant TL software. MICs and kill kinetics for zidebactam, WCK 5153, and cefepime or aztreonam, alone and in combination, were determined against clinical K. pneumoniae isolates producing MBLs VIM-1 or NDM-1 (plus ESBLs and class C β-lactamases) to assess the in vitro enhancer effect of BCH compounds in conjunction with β-lactams. Additionally, murine systemic and thigh infection studies were conducted to evaluate BLE effects in vivo. Zidebactam and WCK 5153 showed specific, high PBP2 affinity in K. pneumoniae. The MICs of BLEs were >64 μg/ml for all MBL-producing strains. Time-kill studies showed that a combination of these BLEs with either cefepime or aztreonam provided 1 to >3 log10 kill against MBL-producing K. pneumoniae strains. Furthermore, the bactericidal synergy observed for these BLE–β-lactam combinations translated well into in vivo efficacy even in the absence of MBL inhibition by BLEs, a characteristic feature of the β-lactam enhancer mechanism of action. Zidebactam and WCK 5153 are potent PBP2 inhibitors and display in vitro and in vivo BLE effects against multidrug-resistant (MDR) K. pneumoniae clinical isolates producing MBLs.

scholarly journals 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

2015 ◽  
Vol 60 (1) ◽  
pp. 451-458 ◽  
Author(s):  
Andrew D. Berti ◽  
Erin Theisen ◽  
John-Demian Sauer ◽  
Poochit Nonejuie ◽  
Joshua Olson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Protein ◽  
Membrane Damage ◽  
Metabolic Stress ◽  
Inducible Promoter ◽  
Penicillin Binding Protein ◽  
Compensatory Response ◽  
Content Type ◽  
Antimicrobial Efficiency

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.

scholarly journals Clinical Variants of the Native Class D β-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems

2016 ◽  
Vol 60 (10) ◽  
pp. 6155-6164 ◽  
Author(s):  
Emma C. Schroder ◽  
Zachary L. Klamer ◽  
Aysegul Saral ◽  
Kyle A. Sugg ◽  
Cynthia M. June ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Hydrolytic Activity ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Active Site Residues ◽  
Content Type ◽  
Class D ◽  
Clinical Variants ◽  
Dynamics Simulations

ABSTRACTThe threat posed by the chromosomally encoded class D β-lactamase ofAcinetobacter baumannii(OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of β-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of β-lactams against this troublesome pathogen.

scholarly journals VNRX-5133 (Taniborbactam), a Broad-Spectrum Inhibitor of Serine- and Metallo-β-Lactamases, Restores Activity of Cefepime in Enterobacterales and Pseudomonas aeruginosa

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Jodie C. Hamrick ◽  
Jean-Denis Docquier ◽  
Tsuyoshi Uehara ◽  
Cullen L. Myers ◽  
David A. Six ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Activity ◽  
Broad Spectrum ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Clinical Isolates ◽  
Active Site Residues ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Reversible Covalent

ABSTRACT As shifts in the epidemiology of β-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved β-lactam (BL)–β-lactamase inhibitor (BLI) combinations address widespread serine β-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-β-lactamases (KPC, OXA-48) or clinically important metallo-β-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by β-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki) values ranging from 0.019 to 0.081 μM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 μg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.

scholarly journals Interspecies Transfer of the Penicillin-Binding Protein 3-Encoding GeneftsIbetween Haemophilus influenzae and Haemophilus haemolyticus Can Confer Reduced Susceptibility to β-Lactam Antimicrobial Agents

2015 ◽  
Vol 59 (7) ◽  
pp. 4339-4342 ◽  
Author(s):  
Annette Søndergaard ◽  
Elizabeth A. Witherden ◽  
Niels Nørskov-Lauritsen ◽  
Stephen G. Tristram
Keyword(s):  
Homologous Recombination ◽  
Haemophilus Influenzae ◽  
Antimicrobial Agents ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Clinical Strains ◽  
Content Sequencing ◽  
Interspecies Recombination

ABSTRACTMutations inftsI, encoding penicillin-binding protein 3, can cause decreased β-lactam susceptibility inHaemophilus influenzae. Sequencing offtsIfrom clinical strains has indicated interspecies recombination offtsIbetweenH. influenzaeandHaemophilus haemolyticus. This study documented apparently unrestricted homologous recombination offtsIbetweenH. influenzaeandH. haemolyticus in vitro. Transfer offtsIfrom resistant isolates conferred similar but not identical increases in the MICs of susceptible strains ofH. influenzaeandH. haemolyticus.

scholarly journals Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

2015 ◽  
Vol 197 (8) ◽  
pp. 1525-1535 ◽  
Author(s):  
Serena Rinaldo ◽  
Alessandro Paiardini ◽  
Valentina Stelitano ◽  
Paolo Brunotti ◽  
Laura Cervoni ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Metal Binding ◽  
Binding Mode ◽  
Structural Features ◽  
Structural Basis ◽  
Content Type ◽  
Secondary Messenger ◽  
Wide Range

ABSTRACTThe intracellular level of the bacterial secondary messenger cyclic di-3′,5′-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the threePseudomonas aeruginosaHD-GYP proteins, which we have previously characterizedin vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site.IMPORTANCEIn bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 fromPseudomonas aeruginosa, involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs.

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