Evolution of Metallo-β-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution.
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ABSTRACT:
SUBMITTER: Meini MR
PROVIDER: S-EPMC4212336 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The class B carbapenem-hydrolyzing beta-lactamase IND-1 has been characterized for Chryseobacterium indologenes strain 001. With internal primers for the bla gene for IND-1 (bla(IND-1)) and an internal bla(IND-1) probe, PCR amplifications failed, while hybridization results were positive when DNA from another C. indologenes isolate, strain CIP101026, was used as a template. Thus, a bla(IND)-related gene was cloned from this C. indologenes reference strain. Sequencing of the insert of a recombinant plasmid conferring resistance to carbapenems revealed an open reading frame with a G + C content of 39.9% and coding for a 243-amino-acid preprotein named IND-2. IND-2 shared 80% amino acid identity with IND-1 and had a similar broad-spectrum resistance profile, including resistance to carbapenems. It was classified in functional subgroup 3a of class B carbapenem-hydrolyzing beta-lactamases. IND-1 and IND-2, despite their genetic diversity, possessed similar kinetic parameters, except that ceftazidime was hydrolyzed less by IND-2. To obtain the entire bla(IND)-related gene sequences of eight other C. indologenes isolates, PCR was performed using internal and external primers, followed by inverse PCR techniques. The likely chromosome-mediated metallo-beta-lactamases of the 10 C. indologenes isolates were divided into several groups and subgroups. IND-1, IND-2, IND-2a, IND-3, and IND-4 shared 77 to 99% amino acid identity.
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Project description:A total of 10 metallo-?-lactamase-producing isolates of six different species, including Brevundimonas diminuta (n = 3), Rhizobium radiobacter (n = 2), Pseudomonas monteilii (n = 1), Pseudomonas aeruginosa (n = 2), Ochrobactrum anthropi (n = 1), and Enterobacter ludwigii (n = 1), were detected in the sewage water of a hospital. The presence of bla(VIM-13) associated with a Tn1721-class 1 integron structure was detected in all but one of the isolates (E. ludwigii, which produced VIM-2), and in two of them (R. radiobacter), this structure was located on a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-?-lactamase genes.
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Project description:An evolution of antibiotic-resistant bacteria has resulted in the need for new antibiotics. β-Lactam based drugs are the most predominantly prescribed antibiotics to combat bacterial infections; however, production of β-lactamases, which catalyze the hydrolysis of the β-lactam bond of this class of antibiotics, by pathogenic bacteria such as Bacillus cereus, are rendering them useless. Some inhibitors of β-lactamases have been found, but there are no inhibitors against a class of β-lactamases known as metallo-β-lactamases, and it has been reported that the number of bacteria that produce metallo-β-lactamases is on the rise. Finding inhibitors of metallo-β-lactamases is thus an urgent necessity. One way to approach the problem is by employing the combinatorial method SELEX. The SELEX method is significant in discovering and producing new classes of inhibitors, as well as providing insight into the development of these inhibitors and paves the way for future aptamer applications that further novel drug discovery.
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Project description:The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-beta-lactamase. Acquisition of a metallo-beta-lactamase gene will invariably mediate broad-spectrum beta-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-beta-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-beta-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-beta-lactamase inhibitor studies securing the longevity of important anti-infectives.
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Project description:The metallo-beta-lactamases fall into two groups: Ambler class B subgroups B1 and B2 and Ambler class B subgroup B3. The two groups are so distantly related that there is no detectable sequence homology between members of the two different groups, but homology is clearly detectable at the protein structure level. The multiple structure alignment program MAPS has been used to align the structures of eight metallo-beta-lactamases and five structurally homologous proteins from the metallo-beta-lactamase superfamily, and that alignment has been used to construct a phylogenetic tree of the metallo-beta-lactamases. The presence of genes from Eubacteria, Archaebacteria, and Eukaryota on that tree is consistent with a very ancient origin of the metallo-beta-lactamase family.
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Project description:Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo-β-lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo-β-lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs.
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Project description:β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20-100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.
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Project description:Metallo-beta-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available beta-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of Erwinia carotovora, an important plant pathogen. Although no detectable beta-lactamase activity could be found in E. carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several beta-lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps, and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime, and cefotaxime and, to a lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. CAR-1 is the first example of a functional naturally occurring MBL in the family Enterobacteriaceae (order Enterobacteriales) and highlights the extraordinary structural and functional diversity exhibited by MBLs.
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Project description:This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-beta-lactamase inhibitors. Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-beta-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed K(i) values of 79, 17, and 3.4 microM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-beta-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-beta-lactamase (50% inhibitory concentration > 1,000 microM). The lack of activity against angiotensin-converting enzyme and serine beta-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 A. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B. fragilis producing CfiA, making these strains sensitive to meropenem (MIC < or = 4 microg/ml). Consequently, this series of metallo-beta-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-beta-lactamases.
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Project description:The urgent demand for effective countermeasures against metallo-β-lactamases (MBLs) necessitates development of novel metallo-β-lactamase inhibitors (MBLIs). This study is dedicated to identifying critical chemical moieties within previously developed MBLIs, and critical MBLs should serve as the target in MBLI evaluations. Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), a systematic literature analysis was conducted, and the NCBI RefSeq genome database was exploited to access the abundance profile and taxonomic distribution of MBLs and their variant types. Through the implementation of two distinct systematic approaches, we elucidated critical chemical moieties of MBLIs, providing pivotal information for rational drug design. We also prioritised MBLs and their variant types, highlighting the imperative need for comprehensive testing to ensure the potency and efficacy of the newly developed MBLIs. This approach contributes valuable information to advance the field of antimicrobial drug discovery.
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