Interaction of Cryptosporidium hominis and Cryptosporidium parvum with primary human and bovine intestinal cells

Abstract

Cryptosporidiosis in humans is caused by the zoonotic pathogen Cryptosporidium parvum and the anthroponotic pathogen Cryptosporidium hominis. To what extent the recently recognized C. hominis species differs from C. parvum is unknown. In this study we compared the mechanisms of C. parvum and C. hominis invasion using a primary cell model of infection. Cultured primary bovine and human epithelial intestinal cells were infected with C. parvum or C. hominis. The effects of the carbohydrate lectin galactose-N-acetylgalactosamine (Gal/GalNAc) and inhibitors of cytoskeletal function and signal transduction mechanisms on entry of the parasites into host cells were tested. HCT-8 cells (human ileocecal adenocarcinoma cells) were used for the purpose of comparison. Pretreatment of parasites with Gal/GalNAc inhibited entry of C. parvum into HCT-8 cells and primary bovine cells but had no effect on entry of either C. parvum or C. hominis into primary human cells or on entry of C. hominis into HCT-8 cells. Both Cryptosporidium species entered primary cells by a protein kinase C (PKC)- and actin-dependent mechanism. Staurosporine, in particular, attenuated infection, likely through a combination of PKC inhibition and induction of apoptosis. Diversity in the mechanisms used by Cryptosporidium species to infect cells of different origins has important implications for understanding the relevance of in vitro studies of Cryptosporidium pathogenesis.

FIG. 1.

Effect of Gal/GalNAc and BSM on invasion of HCT-8 cells and primary human and bovine cells by C. hominis and C parvum. Gal/GalNAC and BSM significantly (*, P = 0.0002) reduced entry of C. parvum into HCT-8 cells and primary bovine cells but had no effect on entry of C. parvum into primary human cells or on entry of C. hominis into HCT-8 and primary human cells. The inhibitor used in each experiment is indicated at right.

FIG. 2.

Effect of cytochalasin D (CD) and cytochalasin B (CB) on invasion of human and bovine primary intestinal cells by C. parvum (A) and human cells by C. hominis (B). Pretreatment of cells prior to infection and infection of cells in the presence of inhibitors significantly reduced C. parvum entry into human and bovine cells and C. hominis entry into human cells. *, P < 0.005 compared to control (no inhibitor). The inhibitor used in each experiment is shown above the bars. Numbers in brackets indicate the time that cells were exposed to the inhibitors. H, human cells; Bo, bovine cells.

FIG. 3.

Phalloidin-FITC and lectin VVL staining of primary human cell monolayers infected with C. parvum (A) and C. hominis (B). Green represents phalloidin-FITC-stained actin, and red represents the intracellular stages of the parasite. Colocalization of the actin and parasite appears yellow (indicated by white lines). The slide was examined using confocal laser scanning microscopy and a 40× lens. Original magnification, ×400.

FIG. 4.

The effect of tyrosine phosphorylation inhibitors on invasion of HCT-8 cells by C. hominis (A) and C. parvum (B).

FIG. 5.

Effect of individual PKC and serine/threonine kinase inhibitors on invasion of HCT-8 cells by C. hominis (A) and C. parvum (B). *, P < 0.005 compared to control. Chel.Cl, chelerythrine chloride; Calph. C., calphostin C; Gö, Gö6976; Ro, Ro-32-0432; Bis, bisindolylmaleimide I; PKCI, PKC inhibitor.

FIG. 6.

Apoptotic effect of staurosporine on HCT-8 cells. Cellular apoptosis increased from 19.5% for nontreated cells (A) to 50.8% for 1.5 μM (B), 58.0% for 2.0 μM (C), and 67.0% for 2.5 μM (D) staurosporine-treated cells.

FIG. 7.

The effect of staurosporine and PKCα inhibitor Gö6976 on C. parvum entry into primary human and primary bovine intestinal cells (A) and on C. hominis entry into primary human cells (B).