Role of Vibrio cholerae neuraminidase in the binding and penetration of cholera toxin

Abstract

Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the neuraminidase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wildtype and NANase V. cholerae 395 strains were then constructed using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT(CT-FITC) to C57BL/6 and C3H mouse fibroblasts exposed to NANase{dollar}\sp+{dollar} filtrates increased 5-fold and 8-fold respectively, relative to NANase{dollar}\sp-{dollar} filtrates. In addition, NANase{dollar}\sp+{dollar} filtrates increased the short-circuit current measured in Ussing chambers 65% relative to NANase{dollar}\sp-{dollar} filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation (FA) ratios was seen at doses from 10{dollar}\sp4{dollar} to 10{dollar}\sp8{dollar}, but NANase{dollar}\sp+{dollar} strains produced 18% higher FA ratios at 10{dollar}\sp9{dollar} than NANase{dollar}\sp-{dollar} strains when inoculated into non-fasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions.