Figure 3.

BtpB is translocated into host cells during infection. (A) RAW macrophages were infected with wild type (wt) or ΔvirB9 B. abortus strains carrying N-terminal TEM-1 fused VceA, VceC, BtpA, and BtpB for 4 h and 24 h. Data represents the means ± standard errors of the percentage of cells with coumarin fluorescence from 5 independent experiments. (B) Representative confocal images of RAW cells infected with either wilt-type B. abortus (wt) or Δ virB9 mutant carrying TEM-fused BtpA, at 24 h after inoculation. Appearance of blue cells is indicative of translocated TEM lactamase. (C) and (D) Analysis of TEM-1 translocation assay for fixed samples of VceA, VceC, BtpA, and BtpB 24 h after infection. (E) Intracellular cAMP levels in J774.A1 cells infected for 4 h with isogenic strains with a functional (wt) or non-functional VirB system (virB10) expressing Btp proteins fused to CyaA. Non-infected cells and a wild type strain expressing the CyaA domain alone (pCyaA) were included as negative controls. A wild type strain expressing BPE123-CyaA was included as a positive control. Means and SD are shown for one representative out of three independent experiments.