Translocation of C. trachomatis proteins into the cytoplasm of HeLa cells by Y. enterocolitica. HeLa cells were left uninfected (UI) or infected with Y. enterocolitica ΔHOPEMT strains expressing the indicated HA-tagged proteins. After 3 h of infection, extracellular bacteria were killed by the addition of gentamicin and the infected cells were incubated for additional 2 h. The infected cells were then fractionated into Triton-soluble and Triton-insoluble cell lysates that were subsequently analyzed by immunoblotting using anti-HA, anti-SycO and anti-tubulin antibodies, as indicated. Presence of HA-tagged proteins in the Triton-soluble cell lysates is indicative of translocation into the cytoplasm of HeLa cells. SycO is a strictly cytosolic Yersinia T3S chaperone [44,51] and its immunodetection ensured that the presence of HA-tagged proteins in the Triton-soluble cell lysates was not a result of bacterial lysis during the fractionation. Additionally, the incapacity to detect HA-tagged RplJ (a C. trachomatis ribosomal protein) in the Triton-soluble cell lysates further indicated that this fraction did not contain bacteria or non-translocated bacterial proteins. Tubulin served as a loading control of the Triton-soluble cell lysates. The images shown are representative of three independent experiments.
da Cunha et al. BMC Microbiology 2014 14:40 doi:10.1186/1471-2180-14-40