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/MS2 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.
Highlights
Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, is still a leading cause of death in developing countries and a resurgent disease in developed countries [1]
The selection and increasing spread of M. tuberculosis multidrug-resistant (MDRTB), extensively drug-resistant (XDR-TB) and, more recently, totally drug-resistant (TDR) or super-XDR strains is a serious threat to public health, in particular to immunocompromised patients [2,3,4,5]
The first series of M. smegmatis DNB resistant mutants were isolated onto 7H11 containing drug concentrations ranging from 150 to 300-fold the Minimal Inhibitory Concentration (MIC) for the wild-type strain (0.25 mg/ml) (Table 2) and the second series with concentrations ranging from 8 to 24-fold the MIC (Table 3)
Summary
Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, is still a leading cause of death in developing countries and a resurgent disease in developed countries [1]. 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. The M. smegmatis mutants GM22, AL49 and AL55, characterized by a low level of resistance to BTZ043 [14], were resistant to DNB (256 X MIC) (Table 3).
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