N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

Localization of recombinant MSMEG_4947 and examination of its activity in membranes. (A) M. smegmatis mc²155/pJAM2-MSMEG_4947 was disrupted by sonication, and cell fractions were obtained by differential centrifugation. The presence of His-tagged MSMEG_4947 in cytosol, membrane fraction, and cell wall (P60) fractions was analyzed by SDS-PAGE (left) and Western blotting (right). (B) The activity of recombinant MSMEG_4947 was analyzed by enzymatic reaction mixtures containing membrane fractions from M. smegmatis mc²155/pJAM2 (control) and M. smegmatis mc²155 pJAM2-MSMEG_4947 (overproducer) and UDP-[¹⁴C]-GlcNAc. Reaction products [¹⁴C]-glycolipid 1 (GL1) and [¹⁴C]-glycolipid 2 (GL2) were extracted by organic solvents. Twenty percent of the lipid extract was loaded on silica-gel TLC plate, developed in CHCl₃-CH₃OH-NH₄OH-H₂O (65:25:0.5:3.6), and then exposed to autoradiography film for 3 days. (C) The amount of ¹⁴C-label incorporated into organic phase was quantified by scintillation counting. Data represent the means ± standard deviation (SD) of the results from two independent experiments (from two batches of cells) performed in triplicates.

ATc dose-dependent growth of wecA conditional mutant of M. tuberculosis. (A) Growth of wecA Tet-Off was monitored in standard Middlebrook 7H9 liquid broth supplemented with the indicated concentrations of ATc (in parentheses; ng/ml), using the H37Rv and wecA-SCO strains as controls. Data are means ± SD of the results from three independent experiments. (B) Growth of the wecA Tet-Off mutant is suppressed in the presence of ATc on agar. (C) Silencing of wecA transcript to ATc treatment. Logarithmic-phase cultures of the wecA Tet-Off or H37Rv strains were treated with ATc for 24 and 48 h, as indicated, and the concentrations of wecA transcript relative to sigA were determined by ddPCR, as described in Materials and Methods. All of the data generated by QuantaSoft software included the 95% confidence interval.

WecA is essential for the growth and survival of M. tuberculosis in both in vitro and intracellular environment. (A) The effect of wecA silencing on the viability of M. tuberculosis grown in vitro was assessed as described in Materials and Methods. The wecA Tet-Off and H37Rv strains were grown in the presence or absence of ATc (200 ng/ml), and the effect of silencing on viability was assessed by plating serial dilutions at the indicated times on 7H10 agar. (B) THP-1 cells were infected as described in Materials and Methods and grown in standard RPMI medium in the absence or presence of ATc (400 ng/ml), and the effect of wecA silencing on M. tuberculosis viability was assessed by plating serial dilutions as described in Materials and Methods. The data represent the mean ± SD from three biological replicates.

Optimization of WecA and translocase 1 assays. Organic and water phases were analyzed by TLC on silica-gel plates developed in 2-propanol–concentrated NH₄OH–H₂O (6:3:1), followed by autoradiography. To evaluate the activities of mycobacterial WecA and translocase 1, different buffer compositions were tested. (A) Buffer A (50 mM MOPS [pH 7.9], 10 mM MgCl₂, 5 mM 2-mercaptoethanol); (B) buffer B (50 mM Tris-HCl [pH 8.0], 40 mM MgCl₂, 0.5 mM EDTA, 50 mM sucrose, 5 mM 2-mercaptoethanol, 0.5% CHAPS, 50 μM undecaprenyl-P); (C) buffer C (100 mM Tris-HCl [pH 7.5], 30 mM MgCl₂, 0.15% Triton X-100, 50 μM undecaprenyl-P, 100 μg/ml phosphatidyl glycerol).

Effect of selected translocase I inhibitors on mycobacterial WecA and translocase I activities. Selectivity of tested compounds (200 μM) for WecA and translocase I activity was analyzed by radiometric assays using membrane fraction isolated from M. smegmatis mc²155 or M. tuberculosis H37Ra. Twenty percent of the lipid extract was loaded on a silica-gel TLC plate, developed in 2-propanol–concentrated NH₄OH–H₂O (6:3:1), and exposed to autoradiography film for 4 days. (A) Chemical structures of the compounds tested in this study. (B) Products of the reaction mixtures (GL1 or lipid I) after visualization by autoradiography.