hada  (Bio-Techne corporation)

Journal: BMC Microbiology

Article Title: Impairment of two non-essential LytR-cpsA-psr (Lcp) cell wall ligases decelerate cell wall assembly in Mycolicibacterium smegmatis

doi: 10.1186/s12866-026-04722-4

Figure Lengend Snippet: Depletion of AG-PG cross-linkers in ΔlcpBΔlcpC . A Immuno dot blot analysis of the anti-glucan antibody binding to the whole bacterial cell and the ⍺-glucans in capsular polysaccharide extracts. This is a representative of two independent experiments. Full-length blots of the replicates are presented in Supplementary file 2 (Fig. 2A, Experiment-2). B (i) Schematic representation of the EB-A2 Mab binding to the exposed Galf-β-(1→5) units of the AG chain in ΔlcpBΔlcpC mutant. Unlike the presence of numerous branched AG chains linking the PG layer in native bacterium, only one branched chain of AG has been shown here for clarity. Abbreviations: MA – Mycolic acids, AG – arabinogalactan, PG – peptidoglycan, PM – plasma membrane. (ii) Data represents six independent experiments (each in triplicate) of direct ELISA showing the significant binding of EB-A2 Mab to the whole bacterial cell of ΔlcpBΔlcpC . Error bars indicate standard deviation, and statistical significance was determined by student’s T-test, in comparison with the WT strain. C Phase contrast and fluorescent images of HADA-stained WT, ΔlcpBΔlcpC and c-( lcpB + lcpC ) strains harvested at mid-log (OD 600 = 0.8) (i) and late-log phase (OD 600 = 1.5) (ii), showing major fluorescent binding of the dye at septum and poles of the mycobacterial strains. All images were minimally processed in ImageJ by simply subtracting the background at a rolling ball radius of 50 pixels with a dark background. The inbuilt CellSens dimension software of the Olympus BX53 was used to measure the length of the individual cells ( n = 300 for mid-log and n = 60 for late-log, across three independent experiments). Error bars represent standard deviation, and the statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level). D Determination of major sugar components in AG-PG complex of WT, ΔlcpBΔlcpC and c-( lcpB + lcpC ) M. smegmatis strains by TLC. PBS was used as blank. This is a representative of three independent experiments. E - F Quantification of AG-PG markers and major sugar components in the AG-PG complex of WT, ΔlcpBΔlcpC and c-( lcpB + lcpC ) strains by HILIC-MS. The intensity of the peaks for each component were measured in comparison to the peak of the standards (refer peak intensity histograms in Fig S2B). The data are presented as mean ± standard deviation and statistical significance determined by two-way ANOVA with Tukey post-hoc analysis (95% confidence level)

Article Snippet: In order to label the nascent PG in actively growing M. smegmatis strains, a final concentration of 250 μM fluorescent D-amino acid HADA (3-[7-hydroxycoumarin]-carboxamide-D-Alanine) (MedChemExpress, China) were incubated with the bacterial cells harvested at mid-log phase (OD 600 = 0.8) and at late-log phase (OD 600 = 1.5), on a shaking platform for 30 min at 37 °C [ ].

Techniques: Dot Blot, Binding Assay, Mutagenesis, Clinical Proteomics, Membrane, Direct ELISA, Standard Deviation, Comparison, Staining, Software, Hydrophilic Interaction Liquid Chromatography

Journal: BMC Microbiology

Article Title: Impairment of two non-essential LytR-cpsA-psr (Lcp) cell wall ligases decelerate cell wall assembly in Mycolicibacterium smegmatis

doi: 10.1186/s12866-026-04722-4

Figure Lengend Snippet: Effective bactericidal activity by combined drug strategy. A LcpA Mtb inhibition by kinase inhibitors. IC 50 determination of Lcp inhibitors, Entrectinib (i) and Sorafenib (ii) by in vitro ADP-Glo™ kinase assay using 2 µM purified LcpA Mtb protein, 135 µM GGPP and 10 µM ATP. The data is an average of three independent experiments each in duplicate and presented as mean ± standard deviation. B (i) MIC determination of Lcp inhibitors, cell-wall acting TB inhibitors and a combination of both on M. smegmatis plate culture. Abbreviation: VAN – vancomycin, INH – isoniazid, ENT – entrectinib and SORA – sorafenib. Final concentration of the inhibitor has been indicated, where combined inhibitors are in a ratio of 1:3 (VAN/INH: ENT/SORA); (ii) superimposed scatter plot showing M. smegmatis inhibition by combined drug strategy. Combined inhibitors are in a ratio of 1:3 (VAN/INH: ENT/SORA). Individual experimental values are plotted and represented as median with interquartile range, and statistical significance ($ − 50 µM, $$ − 100 µM and $$$ − 200 µM) determined by two-way ANOVA with Tukey post-hoc analysis (95% confidence level); (iii) phase contrast and fluorescent images of HADA-stained untreated and treated WT mc 2 155 strains harvested at mid-log phase (OD 600 = 0.8), showing major fluorescent binding of the dye at septum and poles. All images were minimally processed in ImageJ by simply subtracting the background at a rolling ball radius of 50 pixels with a dark background. The inbuilt CellSens dimension software of the Olympus BX53 was used to measure the length (iv) and width (v) of the individual cells ( n = 50 across three independent experiments). Error bars represent standard deviation, and the statistical significance was determined by two-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level). C HILIC-MS quantification of AG and PG markers (i-ii), and a representative intensity plot showing reduced PG components, GlcNAc and MurNAc in M. smegmatis sample treated with a combined dose of VAN and ENT in the ratio of 1:3 (iii); quantification of major sugar components in the AG-PG purified complex of untreated and treated WT mc 2 155 strains (iv-v). The intensity of the peaks for each component were measured in comparison to the peak of the standards (refer peak intensity histograms in Fig S5). The data is an average of three independent experiments and presented as mean ± standard deviation and statistical significance determined by two-way ANOVA with Tukey post-hoc analysis (95% confidence level). D Western blot images showing effective inhibition of M. smegmatis Lcp paralogs by combined drug strategy. 20 µg of the total protein lysate was used, and final concentration of the inhibitor has been indicated. For combined drug strategy, a constant concentration of 0.3 µM VAN/INH was used. Anti-flag Mab was used as the primary antibody. Full-length blots are presented in Supplementary file-2, Fig. 7D. E A representation of the M. smegmatis Lcp proteins interacting with two kinase inhibitors, Entrectinib (blue stick model) and Sorafenib (yellow stick model) (vii). The interacting amino acid residues are indicated for each interaction. Each protein chain is colored by individual colors as indicated. F Binding pockets of Lcp Msmeg in the catalytic Lcp domain showing LcpA-LcpD and LcpB-LcpC interacting residues that are common inhibitory targets of ENT/SORA. The Lcp domain of each Lcp Msmeg protein is represented as grey α-helix/β-sheet. The pocket surface is colored by hydrophobicity values in the Kyte-Doolittle scale (dodger blue for the most hydrophilic; white at 0.0; orange red for the most hydrophobic) in CASTpFold webserver . Sublets show magnified images of ENT/SORA binding sites as ball and stick model (ENT target site: Yellow; SORA target site: Green; Both ENT and SORA target site: Red)

Article Snippet: In order to label the nascent PG in actively growing M. smegmatis strains, a final concentration of 250 μM fluorescent D-amino acid HADA (3-[7-hydroxycoumarin]-carboxamide-D-Alanine) (MedChemExpress, China) were incubated with the bacterial cells harvested at mid-log phase (OD 600 = 0.8) and at late-log phase (OD 600 = 1.5), on a shaking platform for 30 min at 37 °C [ ].

Techniques: Activity Assay, Inhibition, In Vitro, Kinase Assay, Purification, Standard Deviation, Concentration Assay, Staining, Binding Assay, Software, Hydrophilic Interaction Liquid Chromatography, Comparison, Western Blot