Figure 6. Cif virulence factor is delivered to the cytoplasm and localizes to the cytoplasmic face of early endosomes.

(A) Cif localizes to the early endosomal (Rab5 GTPase, early endosomal antigen (EEA)-1 labeled) compartment after entry into airway epithelial cells. Airway epithelial cells were treated with OMV for 10 min, cells lysed, and endosomes were purified. Cif was immunoprecipitated from the endosomal fraction, and Western blot analysis was performed for Rab5 GTPase and EEA-1, early endosomal markers. IgG IP is a non-immune control immunoprecipitation experiment. (B) The ability of proteinase K (PK) to degrade Cif from the early endosomal fraction reveals that the Cif virulence factor localizes to the cytoplasmic face of early endosomes, as measured by Western blot analysis. The transferrin receptor serves as a control for a luminal endosomal protein marker, which is only exposed to proteinase K after treatment with Triton X-100 (TX). (C) Cif entry into airway epithelial cells is not altered by disruption of endosomal acidification by NH₄Cl (5 mM). Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. (D) Entry of Cif virulence factor into airway cells is unaffected by inhibition of retrograde transport with Brefeldin A (1 µM), as measured by Western blot analysis. Rab5 GTPase and actin are endosomal and cytoplasmic markers, respectively. (E) Retrograde transport to the endoplasmic reticulum is not required for Cif to reduce plasma membrane CFTR in airway cells. Airway epithelial cells pretreated with Brefeldin A (1 µM), which inhibits retrograde transport, were treated with OMV. Brefeldin A had no effect on the ability of Cif to reduce plasma membrane CFTR, as measured by Western blot analysis. Data are presented as mean+/−SEM. n = 3, * p<0.05 versus Control.