Fig. 2: IleRS silencing compromises mycobacterial survival under stress and during macrophage infection.

A Growth dynamics of IleRS-silenced and empty vector strains in NR medium under simulated multi-stress conditions, mimicking host immune pressures. Cultures were grown to an OD₆₀₀ = 0.5 in 7H9, pelleted, and resuspended in NR medium with the indicated ATC concentrations for static incubation. Bacterial counts (CFU) were determined after 3–7 days by plating 10-fold serial dilutions on 7H10 agar (n = 3). B Survival of M. abscessus in RAW 264.7 and THP-1 macrophages infected at MOI = 1 with IleRS-silenced and empty vector strains. At 6, 24, 48, and 72 h post-infection, macrophages were washed, extracellular bacteria were removed with gentamicin (see Methods), cells were lysed with 0.01% SDS, and lysates were plated on 7H10 agar to quantify intracellular bacteria (n = 3). C Survival of M. marinum in RAW 264.7 and THP-1 macrophages under the same conditions as in (B). Both strains were pre-treated with ATC for 6 h and maintained under ATC throughout the infection period (n = 3). Error bars represent mean ± SD. Statistical significance was determined by two-way ANOVA. Differences were considered statistically significant at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****). “ns” denotes no significant difference.