Blood Group O-Dependent Cellular Responses to Cholera Toxin: Parallel Clinical and Epidemiological Links to Severe Cholera

Abstract

Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(-/-)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(-/-) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.

Figure 1.

(A) Confocal immunofluorescence images of enteroids grown on transwells demonstrating the presence of H antigen (group O) in ileal enteroids derived from a blood group O individual (top panel), or blood group A subject (bottom panel). (B) Cyclic adenosine monophosphate (cAMP) production after overnight stimulation of ileal enteroids with cholera toxin (CT, 0.1 μg/mL). Data for both blood groups represent composite data from two different subjects (**P value = 0.0096) (C) cAMP production in colonic enteroids from two blood group A and two blood group O subjects after CT stimulation as described in B (**P value = 0.016). Statistical calculations in B, C performed using two-tailed, Mann–Whitney U comparisons.

Figure 2.

(A) Confocal immunofluorescence images demonstrating the presence of group A antigen expressed on the surface of parental HT-29 cells (first row), but not the CRISPR engineered HT-29-A^−/− cells which lack the α1-3-N-acetylgalactosaminyltransferase required to form the A blood group (second row), but which retain expression of the core H (blood group O) antigen (third row). (B) Parental HT-29 (blue symbols), and the engineered HT-29-A^−/− cells (grey) exhibit similar response to the adenylate cyclase agonist forskolin. (C) HT-29 A/A⁻ cells exhibit enhanced cyclic adenosine monophosphate (cAMP) responses to cholera toxin (CT) compared with parent HT-29 cells. (**P = 0.002, two-tailed Mann–Whitney U comparisons).