Intracellular trafficking of diphtheria toxin and its mutated form, CRM197, in the endocytic pathway

OBJECTIVE: Diphtheria toxin (DTx) is a well-characterized bacterial toxin. However, the endocytic pathway of the mutant of DTx, CRM197, which is used as an immunological adjuvant, has not yet been fully explained. The aim of this study was to investigate the intracellular trafficking of CRM197-loaded endosomes. METHODS: Human umbilical vein endothelial cells (HUVECs) were used in a cell culture. The effective incubation time was determined by transmission electron microscopy in toxin-treated cells. Density gradient centrifugation and ADP-ribosylation assay were used to isolate and detect toxin-loaded endosomal fractions. Endosomal fractions from CRM197-treated cells were elicited after 15 minutes of incubation and the presence of fragment A was demonstrated using Western blot. Immunofluorescence microscopy was used to identify endosomes in CRM197-treated endothelial cells. RESULTS: DTx-loaded endosomes were detected as enlarged vesicles in the perinuclear area with 15 minutes of toxin treatment. DTx-loaded endosomal fractions were determined by ADP-ribosyltransferase activity test and Western blot analysis. Enzymatic activity of the toxin-loaded endosomal fraction increased by 20% in actin cytoskeletal-damaged cells treated with cytochalasin D. The steps for the toxin treatment of HUVECs with DTx and obtaining endosomal fractions were repeated for CRM197. In the CRM197-loaded endosomal fraction, actin and Hsp90 were identified in addition to fragment A. Fluorescent images revealed that CRM197-loaded endosomes were co-localized with actin filaments and that Rab11, which signals the return to the plasma membrane, was more prominent than Rab7, the lysosomal pathway indicator. CONCLUSION: These results suggest that CRM197-loaded endosomes participate in the recycling pathway.