Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis

Abstract

PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.

Fig. 1.

Chemical structures of the 4-nitroimidazoles used in this study. Asterisk denotes the position of ¹⁴C in radiolabeled PA-824.

Fig. 2.

FGD and PA-824 activation assays of PA-824-resistant mutants. (A) Western blot analysis of cell lysates from FGD1^- mutant with polyclonal anti-FGD1 antibody (Upper) and polyclonal antibody to GyrA (Lower). (B) TLC analysis of ¹⁴C-labeled PA-824 metabolism. Whole bacterial cells were exposed to 1.8 μCi [¹⁴C]-PA-824 for 3 h and then lysed mechanically. Lysates were analyzed by silica gel TLC. Unconverted [¹⁴C]-PA-824 is denoted by the arrow. Strains are detailed in Table 1: Lanes 1-9 are H37Rv, H37Rv-T3, H37Rv-T3-fgd1, H37Rv-5A1, H37Rv-5A1-fbiC, H37Rv-T2, H37Rv-T2-Rv3547, H37Rv-14A1, and H37Rv-14A1-Rv3547, respectively.

Fig. 3.

F₄₂₀ and FO analysis. HPLC elution profiles of extracts from H37Rv (A), H37Rv-5A1 (B), H37Rv-7A2 (C), H37Rv-5A1-fbiC (D), and H37Rv-7A2-fbiAB (E). Strains are detailed in Table 1. The x axis is elution time in minutes, and the y axis is fluorescence intensity (FI).

Fig. 4.

Phenotype-based screening method for isolating oxazine-specific mutants. (A) Schematic representation of the protocol for identifying oxazine-specific resistant mutants. A pool of ΦMycoMarT7-infected H37Rv was selected for PA-824^R (2 μg/ml). Isolated PA-824^R mutants were counterscreened for CGI-17341^S sensitivity. (B) Identification of transposon-insertion sites in two PA-824^R/CGI-17341^S mutants. The point mutation subsequently discovered in Rv3547 in Tn824^R#1 is also indicated.

Fig. 5.

SignalMap (NimbleGen) representation of CGS analysis of H37Rv-5A1 (A) and H37Rv-T2 (B) mutants. The lower two traces are the signal intensity for the wild type and the mutant hybridizations, and third from the bottom is the ratio of these. The fourth trace shows genomic regions that were subjected to resequencing by using custom arrays, and, in B, the fifth line from the bottom illustrates SNPs confirmed by resequencing. In A, the fifth line shows the genomic deletion boundaries suggested by CGS in Rv1173 (fbiC). Only portions of the full genome hybridizations and resequenced regions are shown.

Fig. 6.

Summary of the role of FGD1, cofactor F₄₂₀, and Rv3547 in the bioreductive activation of nitroimidazo-oxazines, including PA-824. Mtb acquires resistance to all bicyclic 4-nitroimidazoles (4-NIs) upon loss of the ability to produce, or reduce, the deazaflavin cofactor F₄₂₀. Loss or mutation of Rv3547, in contrast, results in resistance only to 4-nitroimidazo-oxazines while retaining sensitivity to even closely related 4-nitroimidazo-oxazoles. This suggests that Rv3547, either alone or as part of a complex, is an F₄₂₀-dependent nitro-reductase with a high degree of ligand selectivity.