Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on Int 407 and HeLa Cells

Abstract

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.