Integration of biophysical and biological approaches to validate fragment-like compounds targeting l,d-transpeptidases from Mycobacterium tuberculosis

Phenotypic characterization of the ∆vioS and ∆vitR/vioS mutants. (A) Growth of the indicated strains in LB medium to verify the production of violacein and CAS tests showing the production of siderophores (orange halos) by the indicated strains inoculated on PSA-CAS plates. (B) Quantification of violacein production of the indicated strains. The strains were grown in LB medium under agitation at 37°C for 24 h. After incubation, 500 µL of culture was homogenized with 500 µL of acetone. After centrifugation, OD575 was measured for quantification of violacein. Data from three biological assays. ****P < 0.0001; when not indicated, not significant (n.s.). One-way ANOVA followed by Tukey’s multiple-comparison test. (C) Measurement of the CAS halo areas of the indicated strains. Data from three biological assays. Statistical analyses using one-way ANOVA followed by Dunnett’s multiple comparisons test. ****P < 0.0001, when not indicated, n.s. (D) Biofilm assay of the indicated strains. The strains were grown in LB medium for 24 h and the assay was performed with crystal violet to quantify biofilm. Data from six biological assays. Statistical analyses using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001; when not indicated, n.s. (E) Protease tests showing the production and secretion of proteases in M9 plates supplemented with 1.5% of powdered milk. Measurement of the protease halo areas of the indicated strains. Data from three biological assays. Statistical analyses using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05; when not indicated, n.s.