Enhancement of internalization of diphtheria toxin recombinant fragments in sensitive cells mediated by toxin’s T-domain

Abstract

Subunit B of diphtheria toxin (DT) and its R-domain differ by the presence of T-domain. The aim of the present work was to analyze the interaction of these toxin fragments with mammalian cells in order to evaluate the T-domain’s influence on endocytosis in resistant cells. Internalization of recombinant fluorescent subunit B and R-domain was characterized in toxin-resistant L929 cells derived from mouse connective tissue and toxin-sensitive Vero cells from African green monkey kidney. It was found that during incubation of cells in the presence of both subunit B and R-domain in the culture medium, Vero cells internalize more molecules of subunit B than of R-domain. Under the same conditions, L929 cells internalize more molecules of R-domain than of subunit B. Colocalization of fluorescent subunit B and R-domain in L929 was rapid and proceeded almost completely at the early period of incubation compared to Vero cells in which it was slow and occurred gradually. The obtained data suggest that T-domain affects internalization and endosomal transport of DT in cells indirectly correlated with their toxin sensitivity. It was concluded that T-domain participates in intracellular endosomal transport and sorting of DT only in toxin-sensitive cells by enhancing the internalization of toxin molecules.

Keywords

• confocal microscopy
• diphtheria toxin
• endocytosis
• internalization
• proHB-EGF