Figure 9.

Time course of VacA trafficking in HeLa cells expressing wild-type Rab7 or dominant-negative Rab7. HeLa cells were transiently transfected with either pEGFP-Rab7 (encoding wild-type Rab7) (A-L), or pEGFP-Rab7T22N (encoding dominant-negative mutant Rab7) (M-O) for 24 h. Then, cells were treated with 5 μg/ml acid-activated wild-type VacA (A-I and M-O) or VacAΔ6-27 (J-L). Intracellular localization of VacA was analyzed by indirect immunofluorescence methodology. The localization of VacA (left), EGFP-Rab7 (B, E, H, and K), and EGFP-Rab7T22N (N) at the indicated time points after VacA treatment is shown. Right, merged images of left and middle. In cells expressing wild-type EGFP-Rab7, wild-type VacA was internalized, clustered in the perinuclear region, and was colocalized with Rab7 (A-I). VacAΔ6-27 was targeted to Rab7-containing compartments in a manner similar to wild-type VacA, but it did not cause clustering of these compartments (J-L). In cells expressing EGFP-Rab7T22N, wild-type VacA did not cluster in the perinuclear region and did not colocalize with Rab7T22N (M-O). Bar, 10 μm.