Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of Mycobacterium tuberculosis

Abstract

Nitazoxanide (Alinia(®)), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against Mycobacterium tuberculosis (Mtb): disruption of Mtb's membrane potential and pH homeostasis. Both actions were shared by a structurally related anti-mycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb's membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb's ultra-low frequency of resistance against nitazoxanide.

Figure 1

Targeted metabolomic analysis of NTZ and TIZ in Mtb: (a) Extracted ion chromatogram for NTZ-treated Mtb. The inset shows the blow up of the NTZ-peaks obtained from Mtb treated with 10-, 4-, 1-fold the MIC and untreated cells, at pH 5.5 + NaNO₂. Triplicates are shown. (b) Extracted ion chromatogram for NTZ-treated Mtb. The inset shows the blow up of the TIZ-peaks obtained from Mtb treated with 10-, 4-, 1-fold the MIC and untreated cells, at pH 5.5 + NaNO₂. Triplicates are shown. (c) Quantification of NTZ in Mtb as a function of the concentration of NTZ, cultured at pH 6.6 and pH 5.5 + 0.5 mM NaNO₂. Data shown are the average of three biological replicates, and the error is the standard deviation. (d) Quantification of TIZ in Mtb as a function of the concentration of NTZ, cultured at pH 6.6 and pH 5.5 + 0.5 mM NaNO₂. Two bars in each condition indicate average data from two independent experiments. Open and closed bars indicate data obtained at pH 6.6 and at pH 5.5 + NaNO₂, respectively.

Figure 2

NTZ and NCS disrupt Mtb’s membrane potential: (a and d) effect of NTZ, NCS, and control on the membrane potential at pH 7.4; (b and e) effect of NTZ, NCS, and control on the membrane potential at pH 5.5; (c and f) effect of NTZ, NCS, and control on the membrane potential at pH 5.5 + 0.5 mM NaNO₂. Data are the average of triplicates and are representative of two independent experiments. Error bars indicate standard deviation.

Figure 3

NTZ and NCS disrupt Mtb’s intrabacterial pH. (a) NTZ effect on pHIB in 7H9 media at different pH values; (b) NTZ effect on pHIB in 7H9 media containing 0.5 mM NaNO₂ at different pH values; (c) NCS effect on pHIB at different pH values; (d) NCS effect on pHIB in 7H9 media containing 0.5 mM NaNO₂ at different pH values. These results were obtained after 4 h of exposure to drug or vehicle alone. Black circles indicate pH 7.5, white circles indicate pH 6.5, and gray circles indicate pH 5.5.

Scheme 1. Structures of NTZ, TIZ, and NCS