Figure 4. Y. pseudotuberculosis YopT Removes Multiple Rho Family Members from Nascent Phagosomes.

(A–D) COS1 cells transfected with plasmids encoding T7-Rac1, T7-Rac1C189S, myc-Cdc42, myc-RhoA, or HA-Arf6 were incubated at 37 °C for 30 min with Y. pseudotuberculosis YP17 (A and C) or YP17/pYopT (B and D) grown under described conditions (Materials and Methods). (A and B) To identify nascent phagosomes, bacteria were probed as described (Materials and Methods), and small GTPases were identified by immunoprobing against appropriate tags. Pink identifies the extracellular region of a bacterium and blue represents the internalized region. Green indicates the staining for small GTPases. (C and D) Quantifications of the percentage (mean ± SEM in triplicate) of partially internalized bacteria that stained positively for Rac1 (n = 33), Cdc42 (n = 33), RhoA (n = 33), Rac1C189S (n = 33), or Arf6-HA (n = 11).

(E) Representative localization of uninfected cells expressing mYFP-Rac1(WT), mYFP-Rac1C189S, or mYFP-Rac16Q, as well as a mYFP-Rac1(WT) transfectant challenged with YP17/pYopT for 30 min (WT; +YopT). White bar represents 1 μm.

(F) The presence of excess RhoGDI interferes with the activity of YopT. Y. pseudotuberculosis YP17/pYopT (+pYopT) was introduced for 30 min onto COS1 cells or transfectants overexpressing RhoGDI. Nuclear localization of Rac1 (defined as the ratio of the mean nuclear Rac1 intensity relative to the mean cytoplasmic Rac1 intensity) was determined in each case.