Figure 2.

Inactivation of oatA increases sensitivity to antimicrobial molecules that target bacterial cell wall and impairs L monocytogenes survival in macrophages. A, Lysozyme disk-diffusion assay. Growth inhibition caused by lysozyme loaded on a paper disk (1 mg/disk) was measured on BHI agar plates inoculated with EGDe (black bars), the ΔoatA mutant (white bars), or ΔoatA+oatA−complemented strains (hatched bars). B, Cefotaxime minimum inhibitory concentration was determined using E-test strips on BHI agar plates that were inoculated with EGDe (black bars), ΔoatA mutant (white bars), or ΔoatA+oatA−complemented strains (hatched bars). C, Gallidermin inhibitory activity was determined in 96-well plates. 10⁶ CFU/mL of EGDe (black bars) or ΔoatA (white bars) were incubated with increasing concentrations of gallidermin. The number of CFU in each well was assessed after overnight incubation at 37°C by plating serial dilutions on BHI agar plates. D, THP-1 cells (n = 4); E, peritoneal-elicited macrophages (n = 5); and F, bone marrow–derived macrophages (n = 4) were infected with EGDe (black circles) or ΔoatA (white squares). G, RAW264.7 cells (n = 5) were infected with EGDe (black circles), ΔoatA (white squares), or the ΔoatA+oatA complemented strain (gray triangles). The number of CFU was determined at different time points after cell lysis with 0.2% triton. Data are means ± SD. (H–I) Electron microscopy analysis of RAW264.7 cells infected with EGDe or ΔoatA. (H) RAW264.7 cells after 4 hours of infection with EGDe or ΔoatA. Left panel: EGDe in vacuoles and protrusions; right panel: ΔoatA free in the cytosol. Scale bars: 2 μm. I, The number of bacteria per cell was determined by counting intravacuolar and cytosolic bacteria 30 minutes and 4 hours postinfection. Data are means ± SD (n = 25). Student t test was performed to determine statistical significance (*** indicates P < .001). CFU, colony-forming unit.