FIG 1.

Intracellular B. abortus becomes highly infective for new host cells. (A) HeLa cells were infected with B. abortus-RFP, and at 48 h postinfection, intracellular bacteria were extracted and purified (Int). Extracted intracellular bacteria were used as the inoculum for a new round of HeLa cell infection using a gentamicin protection assay with a multiplicity of infection (MOI) of 10. As a control, HeLa cells were infected with B. abortus-RFP grown in TSB in vitro (Ext) at a MOI of 10. At 48 h postinfection, cells were processed by fluorescence microscopy. (B) HeLa cells were infected with Int or Ext bacteria processed as for panel A at the indicated MOIs. At 48 h postinfection, the percentage of cells with replicating bacteria (>20 bacteria per cell) was determined. Each value is the average of three independent determinations. Statistical significance was calculated by analysis of variance and Tukey's multiple-comparison test (**, P < 0.005). (C) At 48 h postinfection, Int-GFP bacteria prepared as for panel A were used in a coinfection experiment of HeLa cells with GFP-negative extracellular bacteria (Ext). At the indicated times, cells were lysed, and the CFU count of intracellular bacteria was determined by plate counting using UV light to distinguish GFP-positive from GFP-negative bacteria. Each value is the average of three independent determinations. Statistical significance was calculated by Student’s t test. Asterisks indicate significant differences (**, P < 0.005) from counts of extracellular bacteria.