FIG 4.

Intracellular B. abortus extracted from macrophages and epithelial cells becomes highly infective for new cells. RAW 264.7 macrophages (A, B) or HeLa cells (C) were infected with B. abortus-RFP, and at 48 h postinfection, infected cells were washed three times with PBS and incubated for 4 h in DMEM without antibiotics. Bacteria from the supernatant (Sup) were collected by centrifugation. In parallel, intracellular bacteria were extracted and purified. Extracted intracellular bacteria (Int) and Sup bacteria were used as inocula for a new round of infection of HeLa cells (A) or RAW 264.7 macrophages (B, C) using a gentamicin protection assay with the indicated multiplicity of infection (MOI). As a control, HeLa cells or RAW 264.7 macrophages were infected at the indicated MOI with B. abortus-RFP grown in TSB in vitro (Ext). At 48 h postinfection, cells were processed by fluorescence microscopy, and the percentage of cells with >20 replicating bacteria was calculated. Each value is the average of at least three independent determinations. Statistical significance was calculated by analysis of variance and Tukey’s multiple-comparison test (*, P < 0.05; **, P < 0.005).