Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins

摘要

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrheal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrheal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJ) and adherens junctions (AJ) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilizing in vitro, ex vivo, and in vivo models. CP infection (0.5 × 10⁶ oocysts/well in Transwell inserts, 24 h) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4 and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudin 1 and 2 and β-catenin were not altered. Substantial downregulation of occludin, claudin 4 and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers, and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP infected mouse ileum and jejunum, but were unaltered in Caco-2 cells. Further, bafilomycin, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhea.