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The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

ABSTRACT: The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial cells by using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca(2+)-dependent, saturable binding to HCT-8 and Caco-2 cells and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate. Binding was reduced after the removal of heparan sulfate and following the inhibition of glycosaminoglycan synthesis or sulfation in HCT-8 cells. Like CpClec-Fc binding, C. parvum attachment to and infection of HCT-8 cells were inhibited by glycosaminoglycans and were reduced after heparan sulfate removal or inhibition of glycosaminoglycan synthesis or sulfation. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca(2+)-dependent binding to sulfated proteoglycans on intestinal epithelial cells.

SUBMITTER: Ludington JG

PROVIDER: S-EPMC4862729 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

Ludington Jacob G JG Ward Honorine D HD

Infection and immunity 20160422 5

The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial ce ...[more]

PMID: 26975991

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Project description:The protozoan parasite Cryptosporidium sp. is a leading cause of diarrheal disease in those with compromised or underdeveloped immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium sp. invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection and then tested the isolated effects of these metabolites on C. parvum invasion and growth in intestinal epithelial cells. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, perhaps by negatively affecting the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Conversely, long-chain unsaturated fatty acids enhanced C. parvum invasion, possibly by modulating membrane fluidity. Hence, gut metabolites, either from diet or produced by the microbiota, influence C. parvum growth in vitro and may also contribute to the early susceptibility to cryptosporidiosis seen in young animals.IMPORTANCE Cryptosporidium sp. occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced the growth of Cryptosporidium parvum in vitro Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium sp. is unable to synthesize de novo The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids presented in this study may provide a framework for future studies into this enteric parasite's interactions with exogenous fatty acids during the initial stages of infection.

Project description:A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, beta-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with beta-defensin-related sequence [corrected] related to those of these alpha-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel beta-defensin, which we designate enteric beta-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized beta-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other beta-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other beta-defensin mRNAs were cloned. Most of these clones encoded previously characterized beta-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-beta-defensin. Northern blot evidence supported that all of these other beta-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these beta-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of beta-defensin expression in enteric tissue and that the mRNA encoding a major beta-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that beta-defensins may contribute to host defense of enteric mucosa.

Dataset Information

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

Publications

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

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