Analogous mechanisms of resistance to benzothiazinones and dinitrobenzamides in Mycobacterium smegmatis

Abstract

Tuberculosis is still a leading cause of death worldwide. The selection and spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Recently, two different classes of chemical series, the benzothiazinones (BTZ) and the dinitrobenzamide (DNB) derivatives have been found to be highly active against M. tuberculosis, including XDR-TB strains. The target of BTZs is DprE1 protein which works in concert with DprE2 to form the heteromeric decaprenylphosphoryl-β-D-ribose 2'-epimerase, involved in Decaprenyl-Phospho-Arabinose (DPA) biosynthesis. Interestingly, it has been shown that the DNBs block the same pathway thus suggesting that both drugs could share the same target. Moreover, in Mycobacterium smegmatis the overexpression of the NfnB nitroreductase led to the inactivation of the BTZs by reduction of a critical nitro-group to an amino-group. In this work several spontaneous M. smegmatis mutants resistant to DNBs were isolated. Sixteen mutants, showing high levels of DNB resistance, exhibited a mutation in the Cys394 of DprE1. Using fluorescence titration and mass spectrometry it has been possible to monitor the binding between DprE1 and DNBs, achieving direct evidence that MSMEG_6382 is the cellular target of DNBs in mycobacteria. Additionally, M. smegmatis mutants having low levels of resistance to DNBs harbor various mutations in MSMEG_6503 gene encoding the transcriptional repressor of the nitroreductase NfnB. By LC/MS analysis it has been demonstrated that NfnB is responsible for DNB inactivation. Taken together, our data demonstrate that both DNB and BTZ drugs share common resistance mechanisms in M. smegmatis.

Figure 1. Titration of DprE1 with DNBs.

Fluorescence spectra of DprE1 at increasing concentrations of DNB1 (A) and DNB3 (B); the fluorescence intensities are reported in arbitrary units. The dashed lines represent the fluorescence spectra of a blank sample containing 256 µM of ligand. All measurements were performed at 25°C, as reported in “Materials and Methods” section. (C) Titration curves of DprE1 with DNB1 (•), and with DNB3 (○). Lines are the fit of the data to equation 1.

Figure 2. LC/MS² analysis of DNB transformed by NfnB.

DNB1 was incubated with M. smegmatis NfnB in presence of NADH and the mixture was analyzed by LC/MS² A. DNB1; B. hydroxyl-amine form of DNB1 that is detected after a 30 min incubation at 37°C.