Figure 5

Entry and intracellular growth of wild-type strain U112 and its derivatives. (A–C) Intracellular growth of wild-type strain U112 and its derivatives. RAW264.7 (A), mouse bone-marrow-derived macrophages (B), and A549 (C) cell monolayers were infected with the wild-type U112 strain or its derivatives at an MOI of 100:1. At 3 and 24 h post infection, the cells were lysed and plated for enumeration. (D) Adhesion of the wild-type U112 strain and its derivatives to RAW264.7 cells. RAW264.7 cells were pretreated with 1 μg/ml cytochalasin D for 1 h before infection with the wild-type U112 strain or its derivatives at an MOI of 100:1 or 1000:1. After incubation for 2 h, the cells were lysed to enumerate the adherent bacteria CFU. (E) Invasion of RAW264.7 cells by the wild-type U112 strain or its derivatives. RAW264.7 cells were infected with the wild-type U112 strain or its derivatives at an MOI of 100:1 or 1000:1 for 2 h, and then treated with 100 μg/ml gentamycin for 1 h. The invasive bacteria were quantified by plating the cell lysates on TSA and counting the CFU. The values are mean log CFU/well ± SD of the results from triplicate samples as compared with those of the ΔgreA mutant. The experiments were repeated at least twice. Statistical significance was determined by one-way analysis of variance (ANOVA) test. *p < 0.05, **p < 0.01, and ***p < 0.001.