(A and B) Quantification of the translocation of N-terminally- (A) or C-terminally- (B) TEM1-tagged Brucella proteins. The cytosolic translocation of β-lactamase by B. abortus strain 2308 expressing the different TEM1 fusion proteins was assessed by fluorescence microscopy in J774.A1 macrophages after 16 h of infection. Cells from ten independent random fields were counted and the percentage of blue cells calculated. Results shown are the mean ± SD of three independent experiments. TEM1-GST and GST-TEM1 were used as negative controls (green fluorescence) and TEM1-VceC as a positive control (blue fluorescence). (C and D) Representative fluorescence micrographs from individual assay wells showing control proteins (GST and VceC) and selected TEM1 fusion proteins tagged at either the N-terminus (C) or C-terminus (D). (E) Western blot analysis showing expression of TEM-1 fusions. Bacterial lysates from B. abortus 2308 strain expressing TEM-1 fusion proteins were resolved using SDS-PAGE and subjected to Western blot analysis with anti- β-lactamase TEM-1 antibody. Detection of Brucella Omp19 was used as a loading control.

https://doi.org/10.1371/journal.ppat.1013302.g001

A1 cells. (A) B. abortus strain 2308 (black bars) or the ΔvirB9 mutant strain (white bars) expressing the CyaA fusions to the indicated Brucella proteins were used to infect J774.A1 cells, and the cAMP level of infected cells was determined. A previously identified VirB effector, BPE123, was used as a positive control while vectors expressing the CyaA domain alone (CyaA-X or X-CyaA) were used as a negative control. Total cAMP levels resulting from translocation of protein fusions were quantified and are expressed as fmol.ml⁻¹/10⁶ CFUs. Results are means ± SD from a representative experiment performed in triplicate. (B) Schematic representation of Brucella secreted proteins (Bsp proteins), indicating the corresponding locus, protein lengths and predicted domains or motifs.

https://doi.org/10.1371/journal.ppat.1013302.g002