Characterization of β-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry

Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.     

Cation–π Interactions in β-Lactamases: The Role in Structural Stability

β-lactam group of antibiotics is the most widely used therapeutic molecules for treating bacterial infections. The main mode of bacterial resistance to β-lactams is by β-lactamases. In the present study, we report our results on the role of cation–π interactions in β-lactamases and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while tyrosine is comparatively higher than phenylalanine and tryptophan in the π group. Our results indicate that cation–π interactions might play an important role in the global conformational stability of β-lactamases.     

Rapid determination of carbapenem resistance by low-cost colorimetric methods: Propidium Iodide and alamar blue staining

Journal of Microbiology - Carbapenems are a class of β-lactam antibiotics with a broad antimicrobial activity spectrum. Owing to their sturdy structures resistant to most β-lactamases,...     

Novel and Improved Crystal Structures of H. influenzae,E. coli and P. aeruginosa Penicillin-Binding Protein 3 (PBP3) and N. gonorrhoeae PBP2: Toward a Better Understanding of β-Lactam Target-Mediated Resistance

Even with the emergence of antibiotic resistance, penicillin and the wider family of β-lactams have remained the single most important family of antibiotics. The periplasmic/extra-cytoplasmic targets of penicillin are a family of enzymes with a highly conserved catalytic activity involved in the final stage of bacterial cell wall (peptidoglycan) biosynthesis. Named after their ability to bind penicillin, rather than their catalytic activity, these key targets are called penicillin-binding proteins (PBPs). Resistance is predominantly mediated by reducing the target drug concentration via β-lactamases; however, naturally transformable bacteria have also acquired target-mediated resistance by inter-species recombination. Here we focus on structural based interpretations of amino acid alterations associated with the emergence of resistance within clinical isolates and include new PBP3 structures along with new, and improved, PBP-β-lactam co-structures.     

Evolution of Broad Spectrum β-Lactam Resistance in an Engineered Metallo-β-lactamase

The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are β-lactamases, a group of enzymes inactivating β-lactam antibiotics. Metallo-β-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of β-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several β-lactam antibiotics was constructed and employed as a parental MBL in an experiment to examine how an enzyme can evolve toward increased activity with a variety of β-lactam antibiotics. We designed and synthesized a mutant library in which the substrate activity profile was varied by randomizing six active site amino acid residues. The library was expressed in Salmonella typhimurium, clones with increased resistance against seven different β-lactam antibiotics (penicillin G, ampicillin, cephalothin, cefaclor, cefuroxime, cefoperazone, and cefotaxime) were isolated, and the MBL variants were characterized. For the majority of the mutants, bacterial resistance was significantly increased despite marked reductions in both mRNA and protein levels relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of the mutant enzymes were generalists, conferring increased resistance against most of the examined β-lactams.     

Alarming β-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae

Resistance to β-lactams and other antibiotics in the Enterobacteriaceae is frequently associated with plasmidic resistance determinants that are easily transferred among species. β-Lactamase-mediated resistance is increasingly associated with plasmid-encoded extended-spectrum β-lactamases (ESBLs) and carbapenemases, specifically the CTX-M family of ESBLs, the KPC family of serine carbapenemases, and the VIM, IMP, and NDM-1 metallo-β-lactamases. Although clonal dispersion of resistant isolates was seen initially, more diverse genetic platforms are being observed as variations of mobile elements are transferred worldwide. These enzymes are now appearing in multiple combinations of ESBLs and carbapenemases, thereby conferring resistance to virtually all β-lactam antibiotics.     

Neutron Diffraction Studies of a Class A β-Lactamase Toho-1 E166A/R274N/R276N Triple Mutant

β-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the β-lactam antibiotics is the production of β-lactamase enzymes that inactivate β-lactams by rapidly hydrolyzing the amide group of the β-lactam ring. Specifically, the class A extended-spectrum β-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a β-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.     

A boronic-acid-based probe for fluorescence polarization assays with penicillin binding proteins and β-lactamases

Penicillin binding proteins (PBPs) and β-lactamases are involved in interactions with β-lactam antibiotics connected with both antibacterial activity and mediation of bacterial β-lactam resistance. Current methods for identifying inhibitors of PBPs and β-lactamases can be inefficient and are often not suitable for studying weakly and/or reversibly binding compounds. Therefore, improved ligand binding assays for PBPs and β-lactamases are needed. We report the development of a fluorescence polarization (FP) assay for PBPs and "serine" β-lactamases using a boronic-acid-based, reversibly binding "tracer." The tracer was designed based on a crystal structure of a covalent complex between a boronic acid and PBP1b from Streptococcus pneumoniae. The tracer bound to three different PBPs with modest affinity (K(d)=4-12 μM) and more tightly to the TEM1 serine β-lactamase (K(d)=109 nM). β-Lactams and other boronic acids were able to displace the tracer in competition assays. These results indicate that fluorescent boronic acids are suited to serve as reversibly binding tracers in FP-based assays with PBPs and β-lactamases and potentially with other related enzymes.     

Metallo-β-lactamase-mediated antimicrobial resistance and progress in inhibitor discovery

Resistance to β-lactam antibiotics is rapidly growing, substantially due to the spread of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs), which efficiently catalyse β-lactam hydrolysis. Combinations of a β-lactam antibiotic with an SBL inhibitor have been clinically successful; however, no MBL inhibitors have been developed for clinical use. MBLs are a worrying resistance vector because they catalyse hydrolysis of all β-lactam antibiotic classes, except the monobactams, and they are being disseminated across many bacterial species worldwide. Here we review the classification, structures, substrate profiles, and inhibition mechanisms of MBLs, highlighting current clinical problems due to MBL-mediated resistance and progress in understanding and combating MBL-mediated resistance.     

Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

β-Lactamase-mediated resistance to β-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among β-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the β-lactamase family and probably for most carboxypeptidases.     

Novel metagenome-derived carboxylesterase that hydrolyzes β-lactam antibiotics

It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.     

Structural relationship between the active sites of β-lactam-recognizing and amidase signature enzymes: convergent evolution?

The β-lactam-recognizing enzymes (BLRE) make up a superfamily of largely bacterial proteins that include, principally, the dd-peptidases and β-lactamases. The former enzymes catalyze the final step in bacterial cell wall biosynthesis and are inhibited by β-lactam antibiotics, while the latter enzymes catalyze the hydrolytic destruction of β-lactams and represent a major source of bacterial resistance to these antibiotics. The active site of this superfamily of enzymes includes a Ser1/Ser2(Tyr)/Lys1(His)/Lys2 tetrad in which Ser1 is a nucleophilic catalyst that becomes acylated in the formation of an acyl-enzyme intermediate. An oxyanion hole is also present. The amidase signature (AS) enzymes represent another serine amidohydrolase superfamily with no overall structural resemblance to the BLRE. The active site is characterized by a Ser1/Ser2/Lys1/NH tetrad and an oxyanion hole. We point out that there is a close spatial overlap between the two tetrads and speculate that this has arisen from a process of convergent evolution driven by a mechanistic imperative. Conversion of the backbone NH group of the AS tetrad into Lys2 of the BLRE is rationalized and leads to another mechanistic possibility that may dominate BLRE catalysis. The active site triads of other serine amidohydrolases are also briefly and comparatively discussed.     

C4-alkylthiols with activity against Moraxella catarrhalis and Mycobacterium tuberculosis

Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infectious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New approaches to drug development are especially needed to target organisms that exhibit broad antibiotic resistance due to expression of β-lactamases which is the most common mechanism by which bacteria become resistant to β-lactam antibiotics. We designed and synthesized 20 novel monocyclic β-lactams with alkyl- and aryl-thio moieties at C4, and subsequently tested these for antibacterial activity. These compounds demonstrated intrinsic activity against serine β-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=6) β-lactamase producing Moraxella catarrhalis clinical isolates.     

Complete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.     

The study of the role of mutations M182T and Q39K in the TEM-72 β-lactamase structure by the molecular dynamics method

Synthesis of β-lactamases is one of the common mechanisms of bacterial resistance to β-lactam antibiotics such as penicillins and cephalosporins. The widespread use of antibiotics resulted in appearance of numerous extended-spectrum β-lactamase variants or inhibitor-resistant β-lactamases. In TEM type β-lactamases mutations of 92 residues have been described. Several mutations are functionally important and they determine the extended substrate specificity. However, roles of the most so-called associated mutations, located far from the active site, remain unknown. We have investigated the role of associated mutations in structure of β-lactamase TEM-72, which contains two key mutations (G238S, E240K) and two associated mutations (Q39K, M182T) by means of molecular dynamics simulation. Appearance of the key mutations (in 238 and 240 positions) caused destabilization of the protein globule, characterized by increased mobility of amino acid residues. Associated mutations (Q39K, M182T) exhibited opposite effect on the protein structure. The mutation M182T stabilized, while the mutation Q39K destabilized the protein. It appears that the latter mutation promoted optimization of the conformational mobility of β-lactamase and may influence the enzyme activity.     

A disulfide bridge near the active site of carbapenem-hydrolyzing class A β-lactamases might explain their unusual substrate profile

Bacterial resistance to β-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of β-lactamases, enzymes that efficiently hydrolyze the amide bond of the β-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine β-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new β-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of β-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the “classical” TEM-1 β-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238. Proteins 27:47–58 © 1997 Wiley-Liss, Inc.     

Computational analysis of the interactions of a novel cephalosporin derivative with β-lactamases

Background

One of the main concerns of the modern medicine is the frightening spread of antimicrobial resistance caused mainly by the misuse of antibiotics. The researchers worldwide are actively involved in the search for new classes of antibiotics, and for the modification of known molecules in order to face this threatening problem. We have applied a computational approach to predict the interactions between a new cephalosporin derivative containing an additional β-lactam ring with different substituents, and several serine β-lactamases representative of the different classes of this family of enzymes.

Results

The results of the simulations, performed by using a covalent docking approach, has shown that this compound, although able to bind the selected β-lactamases, has a different predicted binding score for the two β-lactam rings, suggesting that one of them could be more resistant to the attack of these enzymes and stay available to perform its bactericidal activity.

Conclusions

The detailed analysis of the complexes obtained by these simulations suggests possible hints to modulate the affinity of this compound towards these enzymes, in order to develop new derivatives with improved features to escape to degradation.

     

Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum beta-lactamases

Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial β-lactamases. Their widespread use prompted the emergence of the extended-spectrum β-lactamases CTX-Ms, which have become highly prevalent. The C7 β-amino thiazol-oxyimino-amide side chain of ceftazidime has a protective effect against most CTX-M β-lactamases. However, Asp240Gly CTX-M derivatives demonstrate enhanced hydrolytic activity against this compound. In this work, we present the crystallographic structures of Asp240Gly-harboring enzyme CTX-M-16 in complex with ceftazidime-like glycylboronic acid (resolution 1.80 Å) and molecular dynamics simulations of the corresponding acyl-enzyme complex. These experiments revealed breathing motions of CTX-M enzymes and the role of the substitution Asp240Gly in the accommodation of ceftazidime. The substitution Asp240Gly resulted in insertion of the C7β side chain of ceftazidime deep in the catalytic pocket and orchestrated motions of the active serine Ser70, the β3 strand and the omega loop, which favored the key interactions of the residues 237 and 235 with ceftazidime.     

3-mercapto-1,2,4-triazoles and N-acylated thiosemicarbazides as metallo-β-lactamase inhibitors

The production of β-lactamases is an effective strategy by which pathogenic bacteria can develop resistance against β-lactam antibiotics. While inhibitors of serine-β-lactamases are widely used in combination therapy with β-lactam antibiotics, there are no clinically available inhibitors of metallo-β-lactamases (MBLs), and so there is a need for the development of such inhibitors. This work describes the optimisation of a lead inhibitor previously identified by fragment screening of a compound library. We also report that thiosemicarbazide intermediates in the syntheses of these compounds are also moderately potent inhibitors of the IMP-1 MBL from Pseudomonas aeruginosa. The interactions of these inhibitors with the active site of IMP-1 were examined using in silico methods.     

Crystal Structure of Serratia fonticola Sfh-I: Activation of the Nucleophile in Mono-Zinc Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.