Aeromonas hydrophila Beta-Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells (HT-29/B6)

Abstract

ABSTRACT The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (I SC ) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (R t ) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant I SC (39 ± 3 μA/cm 2 ) and decreased the R t to ∼10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant I SC in totally stripped human colon. Tracer flux and ion replacement studies revealed the I SC to be mainly accounted for by electrogenic Cl − secretion. Supernatant applied serosally completely abolished basal I SC . The supernatant-induced I SC was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca 2+ chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical I SC and R t responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl − secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.