TRC-1: Emergence of a clavulanic acid-resistant TEM β-lactamase in a clinical strain

A novel TEM-derived plasmid-encoded beta-lactamase, resistant to inhibition by clavulanic acid, has been identified in a clinical strain of Escherichia coli found in Scotland. The beta-lactamase gene was carried on an 81-kb plasmid that conferred no other resistances. The novel enzyme conferred resistance to the amoxycillin/clavulanic acid combination on the host bacterium. The beta-lactamase has a pI of 5.25 and lies between the PSE-4 and SAR-1 beta-lactamases on an isoelectric focusing gel. This beta-lactamase has a Mr value of 25,000, similar to the TEM-1 enzyme and a comparable substrate profile. Its most significant difference is that it is inhibited by clavulanic acid 100-fold less efficiently than the TEM-1 enzyme. The enzyme was confirmed to be derived from the TEM enzymes by probing the plasmid DNA with an intragenic gene probe for TEM-1. This is the first report of a clinical bacterium carrying a TEM-enzyme that confers resistance to clavulanic acid combinations and we have designated the beta-lactamase as TRC-1.     

Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein

beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.     

TEM-E1: a novel β-lactamase conferring resistance to ceftazidime

A novel beta-lactamase, conferring resistance to ceftazidime, has been identified to be encoded by a 31 kb plasmid (pUK720) in a clinical E. coli strain isolated in Belgium. The beta-lactamase, new designated TEM-E1, has a pI of approximately 5.4 and lies in between the iso-electric focused bands of the beta-lactamases TEM-1 and TEM-7. The TEM-E1 beta-lactamase has a similar molecular weight of 22,000 to the TEM-1 and it is also inhibited by clavulanic acid. However, the TEM-E1 enzyme differs from TEM-1 by its low rates and efficiency of hydrolysis for ceftazidime and cefotaxime, TEM-E1 has similar efficiency of hydrolysis values for ceftazidime and cefotaxime, but only confers resistance to ceftazidime.     

Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase.

TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-1 gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 beta-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin.     

Two variants of transferrable extended-spectrum TEM-β-lactamase successively isolated from a clinical Escherichia coli isolate

In a leukaemic patient presenting a septicaemia treated with ceftazidime and amikacin, two clinical Escherichia coli isolates distinguished by their level of resistance to oxyimino-beta-lactams were isolated at an interval of 24 h. The isolates were identified by biotyping and esterase electrophoretic typing and the two host strains were shown to be identical. However, each of these strains exhibited a different transferrable extended-spectrum beta-lactamase. These enzymes had different pI values (5.25 and 5.58), but were both blaTEM-1 mutants. The enzyme with pI 5.25 was identical to TEM-101 (TEM-12) (serine 162 substitution). The enzyme with pI 5.58 showed an additional amino acid substitution (lysine residue instead of an arginine at position 237) and was denominated TEM-23. These data indicate that point-mutations can be successively cumulated in vivo by blaTEM mutants, leading to expression of beta-lactamases with increased hydrolysis rates.     

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.     

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

Nucleotide sequence of the thermostable direct hemolysin gene (tdh gene) of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio parahaemolyticus

During an outbreak of infection with ampicillin-resistant, TEM-1 beta-lactamase-producing Escherichia coli serotype O15, some strains were noted to differ from the majority in that they showed reduced susceptibility to amoxycillin/clavulanic acid (Augmentin), ureidopenicillins and first generation cephalosporins and produced increased amounts of beta-lactamase. The plasmid from one such isolate was compared with that from an isolate that produced normal amounts of beta-lactamase. Restriction analysis with EcoRI revealed extra fragments in the plasmid from the beta-lactamase hyperproducer and use of DNA-DNA hybridisation with a biotinylated TEM-1 probe showed genetic rearrangement in the beta-lactamase hyperproducer so that the TEM gene appeared to be present in larger amounts and was located on a smaller fragment than for the plasmid from the strain that produced normal amounts of beta-lactamase.     

Inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.     

Dissecting the protein-protein interface between beta-lactamase inhibitory protein and class A beta-lactamases

beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases at a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 beta-lactamase and SME-1 beta-lactamase. Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T. (2003) J. Biol. Chem. 278, 45706-45712). This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm). The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-lactamase were also found to be important for binding Bla1. The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding. This may explain why BLIP binds to SHV-1 beta-lactamase with much weaker affinity than to the other three enzymes. Three regions, including two loops that insert into the active pocket of TEM-1 beta-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants. Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 beta-lactamase.     

Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein

Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co-crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta-lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures.     

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.     

Oligonucleotide probes for the detection of TEM-1 and TEM-2 beta-lactamase genes and their transposons

We describe the use of molecular probes to detect the TEM-type beta-lactamase genes. As a general probe, we prepared a 656 base pair restriction fragment, entirely within the TEM structural gene. This probe was specific for the TEM family, hybridizing only with TEM-1 and TEM-2. The TEM-1 and TEM-2 beta-lactamases differ by only one amino acid. We synthesized two oligonucleotides whose central bases correspond to this difference. The use of these oligonucleotides enables us to discriminate between TEM-1 and TEM-2 genes. Using oligonucleotides homologous to parts of Tn3, we also monitored the presence of TnA-like transposons in bacteria harboring different beta-lactamase genes. Only the TEM-1 and TEM-2 genes were found to be on transposons with terminal sequences identical to those of Tn3. All hybridization experiments were performed with both dot-blot and colony-hybridization techniques, and the suitability of these two methods for epidemiological studies is compared.     

Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.     

Structure of the SHV-1 beta-lactamase

The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established. The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A. The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution. Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively. Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A. Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet. All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1. However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1. A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.     

A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase

A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog. We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene. The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region. Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2). The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G). The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E. coli strain of human origin. Thus, parental bla(TEM) genes encoding beta-lactamases in E. coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.     

Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae

We have determined the nucleotide sequence of the plasmid genes blaT-4 and blaT-5 which encode the broad-substrate-range beta-lactamases TEM-4 and TEM-5, respectively. The TEM-4 enzyme, which confers high-level resistance to cefotaxime (Ctx) and ceftazidime (Caz), differed from the TEM-1 penicillinase by four amino acid substitutions. Two of the mutations are identical to those responsible for the wide substrate range of the TEM-3 beta-lactamase which hydrolyses Ctx and Caz. The amino acid sequence of TEM-5, which confers higher levels of resistance to Caz than to other recently developed cephalosporins, differed from that of TEM-1 by three mutations distinct from those of TEM-4. Analysis of the location of the mutations in the primary and tertiary structures of class A beta-lactamases suggests that interactions between the substituted residues and beta-lactam antibiotics non-hydrolysable by TEM-1 and TEM-2 allow TEM-4 and TEM-5 to hydrolyse efficiently novel broad-spectrum cephalosporins such as Ctx and Caz.     

Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum beta-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae

Extended-broad-spectrum beta-lactamase TEM-9, detected in a clinical isolate of Klebsiella pneumoniae, confers high-level resistance to recent cephalosporins, in particular ceftazidime, and to the monobactam aztreonam. Using oligonucleotide probes, we found that the plasmid gene blaT-9 encoding TEM-9 differs from characterized blaT genes by a new combination of already known mutations. Gene blaT-9 was further studied by direct sequencing of an amplified 1.1-kb DNA fragment which contained the open reading frame and its promoter. Analysis of the nucleotide and of the deduced amino acid sequence confirmed the hybridization results and indicated that TEM-9 differs from TEM-1 by four amino acid substitutions: Phe at position 19 and Met at position 261, which have been found in TEM-4 and are known not to expand the enzyme substrate range; Lys 102, detected in TEM-3 and TEM-4, and Ser 162, present in TEM-5 and TEM-7. Each of the latter substitutions enlarges the substrate spectrum of the enzymes and they are found associated for the first time in TEM-9.     

Experimental prediction of the evolution of cefepime resistance from the CMY-2 AmpC beta-lactamase

Understanding of the evolutionary histories of many genes has not yet allowed us to predict the evolutionary potential of those genes. Intuition suggests that current biochemical activity of gene products should be a good predictor of the potential to evolve related activities; however, we have little evidence to support that intuition. Here we use our in vitro evolution method to evaluate biochemical activity as a predictor of future evolutionary potential. Neither the class C Citrobacter freundii CMY-2 AmpC beta-lactamase nor the class A TEM-1 beta-lactamase confer resistance to the beta-lactam antibiotic cefepime, nor do any of the naturally occurring alleles descended from them. However, the CMY-2 AmpC enzyme and some alleles descended from TEM-1 confer high-level resistance to the structurally similar ceftazidime. On the basis of the comparison of TEM-1 and CMY-2, we asked whether biochemical activity is a good predictor of the evolutionary potential of an enzyme. If it is, then CMY-2 should be more able than the TEMs to evolve the ability to confer higher levels of cefepime resistance. Although we generated CMY-2 evolvants that conferred increased cefepime resistance, we did not recover any CMY-2 evolvants that conferred resistance levels as high as the best cefepime-resistant TEM alleles.