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Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies.

ABSTRACT: Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of k_cat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of T_mB and T_mD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

SUBMITTER: Piccirilli A

PROVIDER: S-EPMC8467308 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structure of New Delhi metallo-β-lactamase 1 (NDM-1).

Project description:Antibiotic resistance in bacterial pathogens poses a serious threat to human health and the metallo-β-lactamase (MBL) enzymes are responsible for much of this resistance. The recently identified New Delhi MBL 1 (NDM-1) is a novel member of this family that is capable of hydrolysing a wide variety of clinically important antibiotics. Here, the crystal structure of NDM-1 from Klebsiella pneumoniae is reported and its structure and active site are discussed in the context of other recently deposited coordinates of NDM-1.

| S-EPMC3212353 | biostudies-literature

Inhibitor discovery of full-length New Delhi metallo-?-lactamase-1 (NDM-1).

Project description:New Delhi metallo-?-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of ?-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of ?-lactam antibiotics have been detected. It could effectively hydrolyze most ?-lactams (k cat/K m ratios between 0.03 to 1.28 µmol?¹.s?¹), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

| S-EPMC3652859 | biostudies-literature

Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

Project description:BackgroundNew Delhi Metallo-b-lactamase (NDM-1) is an enzyme emerging around the world conferring resistance to a wide range of β-lactams agents and whose early detection is extremely important. We proposed to standardize the detection of the blaNDM-1 gene using the LOOP-mediated isothermal amplification technique (LAMP).MethodsIn all, 14 Gram-negative bacterial strains isolated from patients presenting pneumonia associated with mechanical ventilation were used for the blaNDM-1 standardization by LAMP. Klebsiella pneumoniae ATCC BAA-2473 and two clinical strains were used as a positive control. All results were compared to the reaction in polymerase chain reaction (PCR), considered gold standard for this detection.ResultsThere was an excellent correlation between the two techniques employed, since all measured clinical strains were negative in both employed tests and two clinical, and a reference strains were positive.ConclusionsThe lamp technique seems to be an excellent option for the rapid detection of blaNDM-1. The amplification time is much shorter than other molecular techniques, the PCR machine is not necessary, it is easy of implementation and costs is low.

| S-EPMC6816916 | biostudies-literature

Oligopeptides as full-length New Delhi metallo-β-lactamase-1 (NDM-1) inhibitors.

Project description:'Superbug' bacteria producing NDM-1 enzyme causing wide public concern were first detected in a patient who visited India in 2008. It's an effective approach to combining β-lactam antibiotics with NDM-1 inhibitor for treating NDM-1 producing strain infection. In our research, we designed ten oligopeptides, tested IC50 values against NDM-1 enzyme, determined the MIC values of synergistic antibacterial effect and explored the binding model. We found that the oligopeptides 2 (Cys-Phe) and 5 (Cys-Asp) respectively presented IC50 values of 113 μM and 68 μM and also displayed favorable synergistic effects of the inhibitors in combination with ertapenem against genetic engineering-host E. coli BL21 (DE3)/pET30a-NDM-1 and a clinical isolate of P. aeruginosa with blaNDM-1. Flexible docking and partial charge study suggested the interaction between oligopeptide and NDM-1. Three types of action effects, hydrogen bond, electrostatic effect and π-π interaction, contributed to the inhibitory activities.

| S-EPMC5441612 | biostudies-literature

Investigating the position of the hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding.

Project description:In an effort to examine the relative position of a hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis, rapid freeze quench double electron electron resonance (RFQ-DEER) spectroscopy was used. A doubly-labeled mutant of NDM-1, which had one spin label on the invariant loop at position 69 and another label at position 235, was prepared and characterized. The reaction of the doubly spin labeled mutant with chromacef was freeze quenched at 500μs and 10ms. DEER results showed that the average distance between labels decreased by 4Å in the 500μs quenched sample and by 2Å in the 10ms quenched sample, as compared to the distance in the unreacted enzyme, although the peaks corresponding to distance distributions were very broad. DEER spectra with the doubly spin labeled enzyme with two inhibitors showed that the distance between the loop residue at position 69 and the spin label at position 235 does not change upon inhibitor binding. This study suggests that the hairpin loop in NDM-1 moves over the metal ion during the catalysis and then moves back to its original position after hydrolysis, which is consistent with a previous hypothesis based on NMR solution studies on a related metallo-β-lactamase. This study also demonstrates that this loop motion occurs in the millisecond time domain.

| S-EPMC4843777 | biostudies-literature

Inhibitory Potential of Polyclonal Camel Antibodies against New Delhi Metallo-?-lactamase-1 (NDM-1).

Project description:New Delhi Metallo-?-lactamase-1 (NDM-1) is the most prevalent type of metallo-?-lactamase, able to hydrolyze almost all antibiotics of the ?-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-?-lactamase (VIM-1) (subclass B1) and L1 metallo-?-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 ?M. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all ?-lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.

| S-EPMC7584030 | biostudies-literature

Clinical Evolution of New Delhi Metallo-?-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.

Project description:Carbapenem-resistant Enterobacteriaceae (CRE) are rapidly spreading and taking a staggering toll on all health care systems, largely due to the dissemination of genes coding for potent carbapenemases. An important family of carbapenemases are the Zn(II)-dependent ?-lactamases, known as metallo-?-lactamases (MBLs). Among them, the New Delhi metallo-?-lactamase (NDM) has experienced the fastest and widest geographical spread. While other clinically important MBLs are soluble periplasmic enzymes, NDMs are lipoproteins anchored to the outer membrane in Gram-negative bacteria. This unique cellular localization endows NDMs with enhanced stability upon the Zn(II) starvation elicited by the immune system response at the sites of infection. Since the first report of NDM-1, new allelic variants (16 in total) have been identified in clinical isolates differing by a limited number of substitutions. Here, we show that these variants have evolved by accumulating mutations that enhance their stability or the Zn(II) binding affinity in vivo, overriding the most common evolutionary pressure acting on catalytic efficiency. We identified the ubiquitous substitution M154L as responsible for improving the Zn(II) binding capabilities of the NDM variants. These results also reveal that Zn(II) deprivation imposes a strict constraint on the evolution of this MBL, overriding the most common pressures acting on catalytic performance, and shed light on possible inhibitory strategies.

| S-EPMC5740384 | biostudies-literature

Rapid Identification of New Delhi Metallo-?-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS.

Project description:There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-?-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90?min. In a blind set, the assay detected 21/24 bla NDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (?0.1 fm/?g) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the bla NDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

| S-EPMC6709477 | biostudies-literature

Identification of a New Delhi metallo-?-lactamase-4 (NDM-4)-producing Escherichia coli in Italy.

Project description:BACKGROUND: During June-July 2012, six imipenem-resistant Escherichia coli isolates were isolated from two patients hospitalized in a ward of one large tertiary-care hospital in Genoa, Italy. Genetic features associated with blaNDM-4 gene were investigated. RESULTS: The isolates exhibited the same PFGE profile and a multidrug-resistant (MDR) phenotype to aminoglycosides, fluoroquinolones, and ?-lactams. The strains produced the NDM-4 carbapenemase and the blaNDM-4 gene was part of the variable region of a class 1 integron. MLST analysis revealed that all isolates belonged to sequence type 405 (ST405). CONCLUSIONS: This is the first report on the emergence of an MDR strain of E.coli producing the NDM-4 MBL in Italy.

| S-EPMC4060580 | biostudies-literature

Novel Variant of New Delhi Metallo-β-lactamase, NDM-20, in <i>Escherichia coli</i>.

Project description:The spread of carbapenem-resistant Enterobacteriaceae (CRE) mediated by New Delhi metallo-β-lactamase (NDM) poses a serious challenge to clinicians and has become a major public health concern. NDM has been evolving into variants that possess different hydrolysis activity toward antibiotics, so as to affect treatment strategy. In addition, very few studies on NDM variants have focused on animal-derived bacterial isolates. Our study reports a novel NDM variant, NDM-20, in an isolate of Escherichia coli CCD1 recovered from the food animal swine in China. The isolate that was assigned to ST1114, exhibited high level resistance to all β-lactams tested, including aztreonam and carbapenems. The gene of blaNDM-20 was located on an IncX3-type plasmid, surrounded by multiple insertion sequences. Sequencing analysis demonstrated that blaNDM-20 contained three point mutations at positions 262 (G→T), 460 (A→C), and 809 (G→A), compared with blaNDM-1, and just one point mutation at position 809 (G→A), relative to blaNDM-5. Functional analysis revealed that the blaNDM-20 transformant, DH5α+pHSG398/NDM-20, exhibited a higher resistance to ertapenem than that of blaNDM-1 transformant DH5α+pHSG398/NDM-1. Kinetic parameter analysis showed that NDM-20 had increased enzymatic activity against some penicillins and cephalosporins but decreased carbapenemase activity relative to NDM-5. The identification of NDM-20 further confirms the evolution and prevalence of NDM variants in bacteria of food-animal origin.

| S-EPMC5826333 | biostudies-literature

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