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. 2020 Sep 28;25(19):4453.
doi: 10.3390/molecules25194453.

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

Rahma Ben Abderrazek  1 , Sarra Chammam  1 , Ayoub Ksouri  1 , Mariagrazia Perilli  2 , Sayda Dhaouadi  1 , Ines Mdini  1 , Zakaria Benlasfar  1 , Gianfranco Amicosante  2 , Balkiss Bouhaouala-Zahar  1   3 , Alessandra Piccirilli  2
Affiliations

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

Rahma Ben Abderrazek et al. Molecules. .

Abstract

New Delhi Metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase, able to hydrolyze almost all antibiotics of the β-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-β-lactamase (VIM-1) (subclass B1) and L1 metallo-β-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 μM. 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 β-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.

Keywords: NDM-1; antibiotic resistance; camel antibodies; metallo-β-lactamases; potential inhibitors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Reactivity of immune serum (1:5000) was tested by ELISA using New Delhi Metallo-β-lactamase-1 (NDM-1) at a concentration of 1 μg/mL. The absorbance values were measured at 450 nm. Error bars represent standard deviation.
Figure 2
Figure 2
Reactivity of immunoglobulin G fractions was tested by ELISA using NDM-1 enzyme at a concentration of 1 μg/mL and various concentrations of IgG subclasses (µg/mL). Preimmune serum was used as a negative control. The signal values were measured at 450 nm. Error bars represent standard deviation.
Figure 3
Figure 3
Analysis of purified IgG fractions on 12% SDS-PAGE under reducing conditions; lane 1, IgG1 with an apparent molecular weight of 50 kDa and 25 kDa. Lanes 2, 3 revealed a single band of 46 and 43 kDa corresponding to the heavy-chain antibodies (IgG2 and IgG3), respectively.
Figure 4
Figure 4
Inhibition effect of IgG1, IgG2, and IgG3 toward NDM-1. (A) Time-dependent inhibition assays of the three antibodies toward NDM-1; (B) residual activity (%) of NDM-1 after incubation (1 min) with increasing concentration of IgG1; (C) residual activity (%) of NDM-1 after incubation (60 min) with increasing concentration of IgG2; (D) residual activity (%) of NDM-1 after incubation (60 min) with increasing concentration of IgG3. Each kinetic value is the mean of three different measurements; the standard deviation (SD) was below 2%.
Figure 5
Figure 5
Inhibition effect of IgG1, IgG2, and IgG3 towardVerona integron-encoded metallo-β-lactamase VIM-1 and L1 metallo-β-lactamase L1. (A) Time-dependent inhibition assays of the three antibodies toward VIM-1 and L1; (B) residual activity (%) of VIM-1 after preincubation (1 min) with increasing concentrations of IgG1, IgG2, and IgG3; (C) time-dependent inhibition assays of the three antibodies toward L1, (D) residual activity (%) of L1 after preincubation with increasing concentrations of IgG1 (1 min), IgG2 (5 min), and IgG3 (1 min). Each kinetic value is the mean of three different measurements; the standard deviation (SD) was below 2%.
Figure 6
Figure 6
Three-dimensional (3D) structural representation of NDM1, L1, and VIM1 metallo-β-lactamases sharing the same catalytic sites. The crystal structures of NDM1, L1, and VIM1 were extracted from the Protein Data Bank (PDB) using codes 3SPE, 1SML, and 5N5G, respectively. (A) Electrostatic potentials are presented on the surface of enzymes using the molecular graphics system PyMOL, with the yellow circle showing a similar conserved cavity forming the catalytic site. (B) Enzyme structures with the same orientation are presented in cartoon mode. Similar structural components (sheets and helices) with conserved shapes are highlighted, and rainbow colors code for different structures, highlighting enzyme compartment similarities. (C) Catalytic sites for the three enzymes are also highlighted in yellow sticks to show the similarity of these functional regions.
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