MICs of MTZ against Mtb under aerobic condition
We firstly assessed in vitro activity of MTZ against clinical MDR- and XDR-TB isolates. Of these 220 clinical isolates comprising 110 MDR-TB and 110 XDR-TB isolates, more than four-fifths isolates (81.8%, 180/220) had MIC values higher than 16 μg/mL, and the other 40 had MIC values of 16 μg/mL, demonstrating high-level resistance to MTZ under aerobic condition (Figure 1).
Intracellular activity of MTZ against Mtb under anaerobic condition
In one set of experiments, PZA and MTZ was added after xx h of intracellular growth of bacteria, respectively. Our results are illustrated in Figure 2. At a concentration of 10 μg/mL, PZA showed inhibition on the Mtb growth in macrophages, but the growth difference between PZA-treated group and control group (without any treatment) was not significant. However, MTZ significantly inhibited Mtb growth under anaerobic condition, and the number of viable bacteria in macrophages treated with MTZ was dramatically decreased by 71.3% after treated by MTZ for 5 days. Statistical analysis revealed that the percentage of viable bacteria in macrophages treated with MTZ was significantly lower than that of control group (P=0.0065).
Rv3131 is a nitroreductase conferring the activation of MTZ in Mtb
As a prodrug, the intracellular activation of MTZ requires an oxygen-insensitive NADPH nitroreductase. In order to find the possible nitroreductase responsible for MTZ activation, we searched Mtb proteins by BLAST using H. pylori RdxA as the query sequence The result showed that a putative nitroreductase Rv3131 with 344 amino acids, was the most similar nitroreductase to H. pylori RdxA. The query coverage, sequence identity and sequence similarity were 97.3%, 10.3% and 23.3%, respectively. Therefore, Rv3131 was selected as a potential candidate enzyme involving in the activation of MTZ in Mtb.
In vitro biochemical analysis demonstrated that Rv3131 exhibited the NADPH oxidase activity under anaerobic condition, and the absorbance of reaction mixture by the end of 30 second reaction was decreased by 34.7% compared to that in control group. In addition, the absence of glucose oxidase/catalase system resulted in no decrease in the concentration of substrate, indicating that the anaerobic condition was essential for catalytic activity (Figure 3).
Structural modeling and sequence alignment
To further predict the key residue for enzymatic activity, we modeled the structure of Rv3131 using SWISS-MODEL. As shown in Figure 4A, the structure of Rv3131 is formed of two domains. The N-terminal domain consists of a four-stranded antiparallel β sheet formed by β1, β2, β4 and β3, which are surrounded by α helices α1, α2, and α3. The C-terminal domain has a pattern of α/β/α sandwitch. It contains a central four-antiparallel β sheet (β5/β6/β8/β7), surrounded by helices α4 and α7 on one side and α5, α6 and α8 on the other side.
In previous study, Cys159 of H. pylori RdxA was proved to be required for the nitroreduction of metronidazole, suggesting that cysteines might be the key residues for enzymatic activity of Rv3131.18 The result of sequence alignment (Figure 4B) showed that Cys75 and Cys279 were conserved cysteines in Rv3131, which might be the potential key residues for the nitroreductase activity.
Comparison of wild-type and mutant Rv3131 for the nitroreductase activity
To investigate our hypothesis, the vectors with Cys75Ser and Cys279Ser point mutation in Rv3131 were constructed, respectively. As shown in Figure 5, the mutated Rv3131 showed a significant decrease in the NADPH nitroreductase activity. The Cys75Ser amino acid substitution maintained only 41.7% of its wild-type activity. The substitution of Cys279Ser could maintain 71.1% of enzyme activity compared to wild-type protein.