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This chapter focuses on the disruption of transcellular chloride secretion by microbial pathogens, with emphasis on recent advances in this field. A brief review of normal chloride secretion is outlined in the chapter. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to mislocalization, altered function, or expression of this protein have serious pathophysiological consequences. Cholera toxin (CT) from Vibrio cholerae and the type I and type II heat-labile enterotoxins (LTI and LTII) from Escherichia coli are the primary agents that mediate the diarrhea caused by these organisms. These toxins belong to the AB5 enterotoxin family. Clostridium difficile, the leading cause of nosocomial enteric infections, is a noninvasive pathogen that causes colitis entirely by the action of two potent exotoxins, toxin A and toxin B. Unlike CT and E. coli enterotoxin, which elicit secretion without an acute inflammatory component, C. difficile toxin triggers marked intestinal inflammation. In the normal intestine, increases in cyclic guanosine monophosphate (cGMP) lead to the phosphorylation and activation of the CFTR by the membrane-bound cGMP-dependent protein kinase II (PKGII) or by cross-activation of the cyclic AMP (cAMP)-dependent protein kinase. Calcium-dependent chloride secretion is a transient response even in the continued presence of the agonist. Secretion via the transcellular pathway is an exquisitely regulated process. Various pathogens and their toxins can directly disrupt these pathways, frequently by invoking multiple mechanisms.
Intestinal epithelial cells display a polarized distribution of various ion transporters. Electroneutral transport of chloride across the basolateral surface is primarily driven by the sodium concentration gradient established by the Na+K+ ATPase. Potassium channels on the basolateral surface are involved in potassium recycling, thereby preventing cellular depolarization. Accumulation of chloride within the cell beyond its electrochemical equilibrium is the electrical driving force for chloride movement across apical chloride channels. While the bulk of this transport occurs via the CFTR, the CaCC also contribute, especially toward acute secretory responses. The intermediate messengers cAMP and cGMP potentiate chloride secretion by acting primarily on CFTR and NKCC1. Figure adapted from Barrett and Keely ( 4 ).
Cholera toxin and the heat-labile E. coli toxins bind to ganglioside receptors and enter epithelial cells as an AB5 complex by retrograde membrane trafficking through the Golgi and ER. Dissociation and cleavage of the A subunit result in the A1-peptide-mediated ADPribosylation of Gs α. This results in a sustained activation of adenylate cyclase and elevation of cAMP, which in turn increases electrogenic chloride secretion.
C. difficile toxins A and B bind to an as yet undefined receptor on human intestinal epithelial cells and enter cells via an endosomal compartment. These toxins inactivate low-molecular-weight GTPases of the Rho family by glucosylation. In addition, the toxins rapidly localize to the mitochondria, leading to cytotoxic effects. The effect of toxin A on secretion may involve the induction of COX-2-mediated elevation of PGE2 levels. PGE2, in turn, is known to induce cAMP-mediated chloride secretion in intestinal epithelial cells.
Guanylin, uroguanylin, and the homologous peptides from bacteria, EAST-1 and STa, bind to the guanylate cyclase C receptor, leading to the production of cGMP. cGMP mediates the phosphorylation and activation of CFTR by either the cGMP-or cAMP-dependent protein kinase.
TDH and TDH-related hemolysin of V. parahaemolyticus and NSP4 of rotavirus elevate intracellular calcium concentrations by a protein kinase C-dependent mechanism. This results in the activation of the CaCC.
Phosphorylated inositol derivatives are involved in regulating Ca2+-mediated chloride secretion and may have stimulatory or inhibitory effects. Derivatives such as Ins(3,4,5)P3 inhibit basolateral potassium channels, thereby elevating the positive charge within the cell, thus favoring the retention of chloride ions within the cell. Proteins injected into epithelial cells by Salmonella sp. promote the production of Ins(1,4,5,6)P4, which in turn blocks Ins(3,4,5)P3-mediated K channel inhibition. In addition, the Salmonella protein SopB also hydrolyzes Ins(3,4,5)P3. Removal of K+ channel inhibition results in the export of potassium ions and renders the cell with a net negative charge, thereby favoring the exit of chloride ions through the apical channels.
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