The extensive and sometimes irresponsible use of beta-lactam antibiotics in clinical and agricultural settings has contributed to the emergence and widespread dissemination of antibiotic-resistant ...bacteria. Bacteria have evolved three strategies to escape the activity of beta-lactam antibiotics: 1) alteration of the target site (e.g. penicillin-binding protein (PBPs), 2) reduction of drug permeation across the bacterial membrane (e.g. efflux pumps) and 3) production of beta-lactamase enzymes. The beta-lactamase enzymes inactivate beta-lactam antibiotics by hydrolyzing the peptide bond of the characteristic four-membered beta-lactam ring rendering the antibiotic ineffective. The inactivation of the antibiotic provides resistance to the bacterium. Currently, there are over 300 beta-lactamase enzymes described for which numerous kinetic, structural, computational and mutagenesis studies have been performed. In this review, we discuss the recent work performed on the four different classes (A, B, C, and D) of beta-lactamases. These investigative advances further expand our knowledge about these complex enzymes, and hopefully, will provide us with additional tools to develop new inhibitors and antibiotics based on structural and rational designs.
beta-Lactam antibiotics are the most commonly used antibacterial agents and growing resistance to these drugs is a concern. Metallo-beta-lactamases are a diverse set of enzymes that catalyze the ...hydrolysis of a broad range of beta-lactam drugs including carbapenems. This diversity is reflected in the observation that the enzyme mechanisms differ based on whether one or two zincs are bound in the active site that, in turn, is dependent on the subclass of beta-lactamase. The dissemination of the genes encoding these enzymes among Gram-negative bacteria has made them an important cause of resistance. In addition, there are currently no clinically available inhibitors to block metallo-beta-lactamase action. This review summarizes the numerous studies that have yielded insights into the structure, function, and mechanism of action of these enzymes. PUBLICATION ABSTRACT
Orotidine 5′‐monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5′‐monophosphate to uridine 5′‐monophosphate during pyrimidine nucleotide biosynthesis. This enzyme is one ...of the most proficient known, exhibiting a rate enhancement of over 17 orders of magnitude over the uncatalyzed rate. An interesting question is whether the high proficiency of ODCase is associated with a highly optimized sequence of active site residues. This question was addressed by randomizing 24 residue positions in and around the active site of the E. coli ODCase (pyrF) by site‐directed mutagenesis. The libraries of mutants were selected for function from a multicopy plasmid or by single‐copy replacement at the pyrF locus on the E. coli chromosome. Stringent sequence requirements for function were found for the mutants expressed from the chromosomal pyrF locus. Six positions were not tolerant of substitutions and several others accepted very limited substitutions. In contrast, all positions could be substituted to some extent when the library mutants were expressed from a multicopy plasmid. For the conserved quartet of charged residues Lys44‐Asp71‐Lys73‐Asp76, a cysteine substitution was found to provide function at positions 71 and 76. A lower pKa for both cysteine mutants supports a mechanism whereby the thiolate group of cysteine substitutes for the negatively charged aspartate side chain. The partial function mutants such as D71C and D76C exhibit reduced catalytic efficiency relative to wild type but nevertheless provide a rate enhancement of 15 orders of magnitude over the uncatalyzed rate indicating the catalytic proficiency of the enzyme is robust and tolerant of mutation.
Carbapenemases confer resistance to nearly all β-lactam antibiotics. The extensive spread of carbapenemase-producing multidrug-resistant bacteria contributes significantly to hospital-acquired ...infections. We have developed a novel protein-based binding assay that identifies KPC β-lactamases from clinical isolates. We used the protein-protein interaction between KPCs and a soluble β-lactamase inhibitory protein (BLIP) variant, BLIP
, which specifically inhibits KPCs but not other β-lactamases. In this assay, BLIP
was allowed to form complexes with KPC-2 in bacterial cell lysates and then extracted using His tag binding resins. We demonstrated the presence of KPC-2 by monitoring the hydrolysis of a colorimetric β-lactam substrate. Also, to further increase the accuracy of the method, a BLIP
-mediated inhibition assay was developed. The binding and inhibition assays were validated by testing 127
clinical isolates with known genome sequences for the presence of KPC. Our assays identified a total of 32 strains as KPC-2 producers, a result in 100% concordance with genome sequencing predictions. To further simplify the assay and decrease the time to obtain results, the BLIP
protein was tested in combination with the widely used Carba-NP assay. For this purpose, the genome-sequenced
strains were tested for the presence of carbapenemases with the Carba-NP test with and without the addition of BLIP
The test accurately identified carbapenemase-producing strains and the addition of BLIP
allowed a further determination that the strains contain KPC carbapenemase. Thus, the BLIP
protein is an effective sensor to specifically detect KPC β-lactamases produced by clinical isolates.
Infections caused by carbapenem-resistant
are associated with high therapeutic failure and mortality rates. Thus, it is critical to rapidly identify clinical isolates expressing KPC β-lactamases to facilitate administration of the correct antibiotic treatment and initiate infection control strategies. To address this problem, we developed a protein-based, KPC-specific binding assay in combination with a cell lysate inhibition assay that provided a 100% identification rate of KPC from clinical isolates of known genomic sequence. In addition, this protein sensor was adapted to the Carba-NP assay to provide a rapid strategy to detect KPC-producing isolates that will facilitate informed treatment of critically ill patients.
In Gram-negative bacteria, TEM-1 β-lactamase provides the major mechanism of plasmid-mediated β-lactam resistance. Natural variants of TEM-1 with increased antibiotic resistance have appeared in ...response to the use of extended-spectrum β-lactam antibiotics (e.g., ceftazidime) and β-lactamase inhibitors (e.g., clavulanic acid). Some of the variant enzymes are more efficient at catalyzing β-lactam hydrolysis, whereas others are more resistant to inhibitors. M182T is a substitution observed in both types of variant TEM-1 β-lactamases. This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity. An engineered core mutation, L76N, which diminishes the periplasmic β-lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation. Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 β-lactamase. Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance.
Efficient mechanism-based design of antibiotics that are not susceptible to β-lactamases is hindered by the lack of comprehensive knowledge on the energetic landscapes for the hydrolysis of various ...β-lactams. Herein, we adopted efficient quantum mechanics/molecular mechanics simulations to explore the acylation reaction catalyzed by CTX-M-44 (Toho-1) β-lactamase. We show that the catalytic pathways for β-lactam hydrolysis are correlated to substrate scaffolds: using Glu166 as the only general base for acylation is viable for ampicillin but prohibitive for cefalexin. The present computational workflow provides quantitative insights to facilitate the optimization of future β-lactam antibiotics.
Antibody responses for 882 of the 1,039 proteins in the proteome of Treponema pallidum were examined. Sera collected from infected rabbits were used to systematically identify 106 antigenic proteins, ...including 22 previously identified antigens and 84 novel antigens. Additionally, sera collected from rabbits throughout the course of infection demonstrated a progression in the breadth and intensity of humoral immunoreactivity against a representative panel of T. pallidum antigens.
Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. ...Increasing rates of new syphilis cases per year have been observed recently.
The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome.
The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.
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