Variations in the SDN Loop of Class A Beta-Lactamases: A Study of the Molecular Mechanism of BlaC (Mycobacterium tuberculosis) to Alter the Stability and Catalytic Activity Towards Antibiotic Resistance of MBIs

Abstract

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for an immediate search for novel treatment strategies. Recently, BlaC, the principal beta-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. BlaC belongs to Ambler class A, which is generally susceptible to the beta-lactamase inhibitors currently used in clinics: tazobactam, sulbactam, and clavulanate. Alterations at Ser130 in conserved SDN loop confer resistance to mechanism-based inhibitors (MBIs) commonly observed in various clinical isolates. The absence of clinical evidence of S130G conversion in M. tuberculosis draws our attention to build laboratory mutants of S130G and S130A of BlaC. The study involving steady state, inhibition kinetics, and fluorescence microscopy shows the emergence of resistance against MBIs to the mutants expressing S130G and S130A. To understand the molecular reasoning behind the unavailability of such mutation in real life, we have used circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), molecular dynamics (MD) simulation, and stability-based enzyme activity to compare the stability and dynamic behaviors of native and S130G/A mutant form of BlaC. A significant decrease in melting temperature (BlaC TM 60°C, S130A TM 50°C, and S130G TM 45°C), kinetic instability at higher temperature, and comparative dynamic instability correlate the fact that resistance to beta-lactam/beta-lactamase inhibitor combinations will likely not arise from the structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be potentially applied as a part of a successful treatment regimen against M. tuberculosis.

Characterization of a Class A β-Lactamase from Francisella tularensis (Ftu-1) Belonging to a Unique Subclass toward Understanding AMR

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“…Similar experiments were performed with a buffer containing different concentrations of NaCl to see the effect of salt in enzyme catalysis. 33 , 37 …”

“…Similar experiments were performed with a buffer containing different concentrations of NaCl to see the effect of salt in enzyme catalysis. 33,37 Inhibition Kinetics. MBIs (mechanism-based inhibitors) like sulbactam, clavulanic acid, and tazobactam, along with carbapenems like imipenem and meropenem, were used for the potential inhibitory assay against Ftu-1 β-lactamase.…”

The microbiome and the gut-lung axis in tuberculosis: interplay in the course of disease and treatment

“…Furthermore, when oral antibiotics cannot be used, patients may require intravenous antibiotics like carbapenems. However, carbapenems for anti-TB treatment are prescribed in conjunction with clavulanic acid, since MTB has a constitutive betalactamase BlaC, that has a penicillinase, cephalosporinase, and carbapenemase activity that is inhibited by clavulanic acid (Bhattacharya et al, 2021). Moreover, in México and other Latin American countries, clavulanic acid is administered with amoxicillin, which adds another broad-spectrum antibiotic to the treatment (National Center for Preventive Programs and Disease Control, 2020).…”