Date Published: June 18, 2018
Publisher: Public Library of Science
Author(s): Frederico Alisson-Silva, Janet Z. Liu, Sandra L. Diaz, Lingquan Deng, Mélanie G. Gareau, Ronald Marchelletta, Xi Chen, Victor Nizet, Nissi Varki, Kim E. Barrett, Ajit Varki, Karla J.F. Satchell.
While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.
Cholera is a life-threatening, human-specific disease caused by the noninvasive enteric bacterium Vibrio cholerae that affects millions of people worldwide . Humans are infected after ingestion of food or water contaminated with the pathogen. Bacteria that survive passage through the acidic milieu of the stomach can colonize and multiply on the surface of the small intestinal epithelium  and induce severe watery diarrhea. This main symptom of the disease leads to loss of electrolytes and blood volume depletion, and can be fatal if the individual is not rehydrated rapidly . Cholera diarrhea is triggered after the B subunits of cholera toxin (Ctx) bind to the monosialoganglioside, GM1 [3–5], expressed on the outer leaflet of the apical membrane of intestinal epithelial cells, which facilitates toxin endocytosis and retrograde phagosomal transport to the endoplasmic reticulum (ER) [6, 7]. In the ER, the enzymatically active A subunit of Ctx (CtxA) is released from the B subunits and translocated into the cytoplasm, where it activates adenylate cyclase, leading to increased levels of cyclic adenosine monophosphate (cAMP). The rise in cAMP causes intense secretion of chloride ions through the cystic fibrosis transmembrane conductance regulator (CFTR) as well as inhibition of electroneutral sodium chloride absorption . These events are followed by passive water flow in response to osmotic gradients, resulting in profuse diarrhea . CtxA further induces epithelial cell barrier disruption by inhibiting exocyst-mediated trafficking of host proteins that make up the intercellular junctions of epithelial cells, a mechanism that may act in parallel with Cl− secretion to drive the pathophysiology of cholera .
There are multiple potential mechanisms by which the human evolutionary loss of epithelial Neu5Gc could contribute to the human-specific susceptibility to cholera. These include improved survival in gastric fluid (not studied here); colonization or preferential growth with Neu5Ac or Neu5Gc as carbon source (no effect seen here); decreased VcN degradation of inhibitory mucin sialic acids (not studied here); VcN remodeling of higher gangliosides into GM1 (a major impact seen here); improved delivery of the CtxA subunit into the cytosol (not studied here); and cAMP production and chloride channel activation (both shown here to be markedly enhanced). While some hypotheses were nullified, we discovered more than one likely contributory mechanism that could underlie the human-specific susceptibility to cholera.