Dataset Information

Characterization of the AmpC ?-Lactamase from Burkholderia multivorans.

ABSTRACT: Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. ?-Lactam antibiotics are recommended as therapy for infections due to Bmultivorans, which possesses two ?-lactamase genes, blapenA and blaAmpC PenA is a carbapenemase with a substrate profile similar to that of the Klebsiella pneumoniae carbapenemase (KPC); in addition, expression of PenA is inducible by ?-lactams in Bmultivorans Here, we characterize AmpC from Bmultivorans ATCC 17616. AmpC possesses only 38 to 46% protein identity with non-Burkholderia AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of Bmultivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (? loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward ?-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (Kiapp > 600 ?M), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible in Bmultivorans ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 ?g/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in Bmultivorans compared to PenA, which is the dominant ?-lactamase in Bmultivorans ATCC 17616.

SUBMITTER: Becka SA

PROVIDER: S-EPMC6153817 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Characterization of the AmpC β-Lactamase from Burkholderia multivorans.

Becka Scott A SA Zeiser Elise T ET Barnes Melissa D MD Taracila Magdalena A MA Nguyen Kevin K Singh Indresh I Sutton Granger G GG LiPuma John J JJ Fouts Derrick E DE Papp-Wallace Krisztina M KM

Antimicrobial agents and chemotherapy 20180924 10

Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due to Bmultivorans, which possesses two β-lactamase genes, blapenA and blaAmpC PenA is a carbapenemase with a substrate profile similar to that of the Klebsiella pneumoniae</i ...[more]

PMID: 30012762

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Project description:Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including β-lactams. β-Lactam resistance in Bcc is conferred largely via PenA-like β-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using β-lactamase inhibitors (β-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on kinact/KI or k2/K values (range: 3.4 × 102 to 2 × 106 M-1 s-1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased Ki-app values and decreased kinact/KI or k2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis-Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k2/K, 3.4 × 102 M-1 s-1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1's Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k2/K for the R220A variant compared to PenA1, acyl-enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favorable interactions compared to PenA1. The former likely impacts the ability of all inhibitors to effectively acylate this variant enzyme. Based on the summation of all evidence herein, the utility of these newer β-lactamase inhibitors (i.e., relebactam, enmetazobactam, avibactam, and vaborbactam) in combination with a β-lactam against B. multivorans producing PenA1 and the R220A variant is promising.

Project description:Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.

Dataset Information

Characterization of the AmpC ?-Lactamase from Burkholderia multivorans.

Publications

Characterization of the AmpC β-Lactamase from Burkholderia multivorans.

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