Microarray analyses of the small intestinal mucosa during experimental and clinical cholera

Abstract: Vibrio cholerae is a Gram-negative bacterium and the causative agent of cholera. The bacteria infect humans via contaminated water and food, and colonize the upper parts of the small intestine to cause disease. The disease is characterized by watery diarrhoea and most of its clinical features can be attributed to the secreted cholera toxin (CT). CT binds to the surface of epithelial cells by its B-subunit homopentamer and after internalization, the enzymatic active A-subunit catalyses the ADP-ribosylation of Gsá leading to the activation of adenylate cyclase. The following increase in cAMP modulates transporters in the epithelial cells resulting in the massive net secretion of electrolytes and water. Besides its toxic effect, CT is also known to be a strong mucosal adjuvant, causing augmented immune responses against co-administered antigens.We have here used the microarray technique to study how the gene expression is modulated in the small intestinal mucosa, both in rats after oral challenge with CT and in cholera patients during the acute stage of the disease. Observed differences in expression have to a large extent been confirmed with real-time PCR and/or immunohistochemistry.In rats it was shown that CT affected the expression of four different membrane transporters, of which at least three most likely counteract the secretory response. CT also induced the upregulation of two mucin genes, probably as a consequence of the CT-induced release of mucus from goblet cells. The microarray screening of the rat small intestinal mucosa also revealed the co-ordinated downregulation of a number of transcripts associated with CD8+ intraepithelial lymphocytes after CT challenge. Immunohistochemistry revealed that the decrease in the level of these transcripts in samples taken from the upper parts of the villi can be explained by a CT-induced migration of CD8+ cells from the epithelium to the lamina propria compartment, preferentially to the lower parts of the villi, and possibly also out from the villi.The whole genome screening of cholera patients showed a broad upregulation of innate defence factors, including genes encoding antibacterial proteins, as well as genes that contribute to the general protection of the mucosa, during the acute phase of the disease. In contrast, among the identified downregulated genes, a significant proportion is involved in metabolism and transport of lipids. Further, the screening revealed the differential expression of seven membrane transporters, two upregulated and five downregulated, suggesting changes that both counteract, as well as potentiate the secretory response. The two identified upregulated transporters encode two amino acid transporters, indicating a broad uptake of amino acids during cholera. Thus, the addition of amino acids to the oral rehydration solutions used for treatment of cholera should be reconsidered, since this may increase the absorption of cotransported electrolytes. Five of the genes encoding membrane transporters, which were shown to be differentially expressed during acute cholera, did show the same expression pattern in CT-stimulated CaCo-2 cells, a human intestinal epithelial cell line, pointing to direct effects of CT on the epithelial cells in these cases.In this work, we have, for the first time, applied the microarray technique to examine changes induced during cholera in vivo. The screening of thousands of genes revealed previously unknown modulations of transporters, the immune system, and the innate defence during experimental and clinical cholera. The results may add new clues to the secretory response during cholera, and how it is counteracted by the host, as well as suggesting new mechanisms that contribute to the immunomodulatory properties of CT.

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