Fig. 4: EccC3 is crucial for the uptake and intracellular replication of M. abscessus.

a Infection assays with THP-1 macrophages using fluorescent Mab S-derived strains (MOI of 2:1). CFU were determined at 4, 24, and 72 hpi. The data are presented as mean values from three independent experiments (n = 12). Statistical significance was determined using Tukey’s multiple comparisons test: ****, P < 0.0001. b THP-1 cells were infected with the different fluorescent Mab R (MOI of 2:1). CFU counts were similarly determined at 4, 24, and 72 hpi. The data are presented as mean values from three independent experiments (n = 12). Statistical significance was determined using Tukey’s multiple comparisons test: ****, P < 0.0001. c Percentage of infected macrophages was determined for each of the S- or R-derived mutants and their complemented strains. Data represent mean values from three independent experiments, each performed in triplicate (n = 9). Statistical significance was determined using Tukey’s multiple comparisons test: *, P < 0.05, ****, P < 0.0001. d Adhesion assays to evaluate the binding of eccC mutant strains to THP-1 macrophages. Macrophages were pre-cooled to prevent bacterial internalization and exposed to the bacterial strains at a MOI of 100 for 1 hr at 4 °C to enhance bacteria-cell contact. CFUs were quantified to assess adhesion. The data reflect mean values from three independent experiments, each in quadruplicate (n = 12). Statistical analysis was assessed using the one-tailed Mann-Whitney test: ***, P < 0.001, ****, P < 0.0001. e Immunofluorescence microscopy images were taken after 72 hpi, using a confocal microscope at 40x magnification, show macrophages (stained green) infected with various Mab strains (red). Yellow arrows point to mycobacteria-infected cells. Data are mean ± SD.