Fig. 5.

Perforin-like protein of MEF increases susceptibility of intracellular bacteria to lysozyme. a-c Representative images of M. smegmatis were taken by phase light microscopy (magnification: ×50) of plated M. smegmatis on Middlebrook 7H11 agar plates. Insets for each image show a 3-fold electronic zoom. a M. smegmatis isolated from IFN-stimulated MEF 5 h after infection, incubated for 30 min with PBS on ice and then plated for 12 h. White arrows point to plump bodies of single mycobacterial cells. Black arrows show surviving slim mycobacteria. b M. smegmatis isolated from IFN-stimulated MEF 5 h after infection, incubated for 30 min with 35 µg/ml lysozyme in PBS on ice and then plated for 12 h; asterisk shows microcolonies forming from live cells. Plump bodies have disappeared due to lysis by lysozyme. c Freshly cultured M. smegmatis plated prior to incubation with MEF; black arrows point to M. smegmatis slim (healthy) morphology. d Quantification of plump and slim morphology of M. smegmatis isolated from MEF 5 h after infection without and with lysozyme treatment. 1,000 bacteria were counted in each experiment and characterized by morphology with the percentage of plump and slim M. smegmatis reported before and after lysoszyme treatment, respectively. e MEF were transfected 20 h prior to infection with scramble siRNA, P-2-specific siRNA, or P-2-RFP, stimulated with IFN-γ for 14 h, and then infected with MRSA. Bacteria were released by lysis of host cells at the times indicated and tested for lysozyme susceptibility as described in the Materials and Methods section. Graphs shown are representative of 3 independent experiments with 2 replicates (means and SEM), * p < 0.05 (Student's t test).