FIG. 4.

Translocation of VgrG1 into HeLa cell cytoplasm. HeLa cells were infected with the A. hydrophila SSU Δact or Δact ΔvasH mutant strain expressing and producing full-length VgrG1::Bla (referred to as VgrG1::Bla) or VgrG1-NH₂::Bla. As a control, HeLa cells were infected with bacteria containing the empty vector. (A) Flow cytometric density plots showing disruption of CCF4 FRET (from green to blue) due to translocation of Bla into cytoplasm of HeLa cells infected with the A. hydrophila Δact parental strain (II and III) compared to its disruption in host cells infected with A. hydrophila SSU containing the empty vector (I and IV). HeLa cells infected with the A. hydrophila Δact ΔvasH mutant expressing and producing the fusion proteins were not able to translocate Bla and did not disrupt the CCF4 FRET (V and VI). For analysis, 2 × 10⁵ HeLa cells were acquired and gated in side forward/side scatter patterns to avoid aggregates. Ex, excitation wavelength; Em, emission wavelength. (B) Fold increases in percentages of blue HeLa cells with cleaved substrate versus green HeLa cells with uncleaved substrate (after infection with A. hydrophila SSU Δact and Δact ΔvasH mutant strains) compared to the results for HeLa cells infected with bacteria carrying the empty vector. The graph shows data from a representative experiment.