Heparin interacts with elongation factor 1α of Cryptosporidium parvum and inhibits invasion

Abstract

Cryptosporidium parvum is an apicomplexan parasite that can cause serious watery diarrhea, cryptosporidiosis, in human and other mammals. C. parvum invades gastrointestinal epithelial cells, which have abundant glycosaminoglycans on their cell surface. However, little is known about the interaction between C. parvum and glycosaminoglycans. In this study, we assessed the inhibitory effect of sulfated polysaccharides on C. parvum invasion of host cells and identified the parasite ligands that interact with sulfated polysaccharides. Among five sulfated polysaccharides tested, heparin had the highest, dose-dependent inhibitory effect on parasite invasion. Heparan sulfate-deficient cells were less susceptible to C. parvum infection. We further identified 31 parasite proteins that potentially interact with heparin. Of these, we confirmed that C. parvum elongation factor 1α (CpEF1α), which plays a role in C. parvum invasion, binds to heparin and to the surface of HCT-8 cells. Our results further our understanding of the molecular basis of C. parvum infection and will facilitate the development of anti-cryptosporidial agents.

Figure 1. Inhibitory effects of sulfated polysaccharides on C. parvum sporozoite invasion of HCT-8 cells.

C. parvum sporozoites were inoculated to HCT-8 cells in RPMI-1640 medium containing each sulfated polysaccharide. The number of parasites left in the HCT-8 cells was counted per 100 fields of view by use of fluorescence microscopy. Each assay was performed in independent triplicates, and means ± standard deviations are shown. (a) Inhibitory efficacy of sulfated polysaccharides. Each sulfated polysaccharide was added at the concentration of 1, 10, or 100 μg /mL in RPMI-1640 medium. (b) Inhibitory efficacy of heparin tested over a wide range of concentrations. Heparin was added at the concentrations of ten-fold serial dilutions from 2,000,000 to 2 ng/mL in RPMI-1640 medium.

Figure 2. Inhibitory effects of heparin pre-incubated with sporozoites or HCT-8 cells.

To determine whether heparin affects C. parvum sporozoites or HCT-8 cells, a pre-incubation assay was conducted. The both panels show the number of parasites that invaded HCT-8 cells. HCT-8 cells pre-incubated with heparin prior to C. parvum infection showed no reduction in parasite invasion (left panel), whereas HCT-8 cells inoculated with C. parvum that had been pre-incubated with heparin showed a statistically significant decrease in parasite invasion (~18%) (right panel). Each assay was performed in independent triplicate, and means ± standard deviations are shown. Statistically significant differences in the number of parasites in the cells were determined by using the Welch’s T-test; P values less than 0.05 are shown by the asterisk.

Figure 3. Heparin-deficient CHO cell lines are less susceptible to C. parvum infection.

The invasion inhibition assay was also conducted using CHO K1 and CHO pgsD-677 strains. The number of parasites that invaded these cells is shown for each cell line. CHO pgs D-677 cells were less susceptible to C. parvum infection by 27% than were CHO K1 cells. Each assay was performed in independent triplicate, and means ± standard deviations are shown. Statistically significant differences in the number of parasites in the cells were determined by using the Welch’ s T-test; P values less than 0.05 are shown by the asterisk.

Figure 4. Identification of heparin-binding proteins of C. parvum sporozoites.

(a) Silver staining showing the whole lysates of C. parvum sporozoite (lane 1, Input), heparin-unbound proteins (lane 2, Flow through), and heparin-binding proteins (lane 3, Pull down). The proteins in the bands with molecular masses of 120 (#1), 90 (#2), and 45 (#3) kDa were specifically concentrated in the precipitated fractions, and proteins in these three bands were separately gel-extracted and subjected to mass spectrometry analysis. (b) Gene enrichment analysis of heparin-binding proteins. To functionally categorize the proteins that interacted with heparin, all of the proteins identified by mass spectrometry were assigned to a GO grouping. GO analysis was carried out by using Gene Ontology Enrichment embedded in the CryptoDB database (http://cryptodb.org/), where Fisher’s exact P values were used to determine the GO terms that were statistically significant (P < 0.05).

Figure 5. Heparin-binding property of rCpEF1α.

To examine whether recombinant CpEF1α binds to heparin, a pull down assay was conducted. Purified GST-CpEF1α and GST-CpActin were incubated with heparin agarose beads (Input; lanes 1 and 4), and beads were then pulled down. Heparin-unbound phase and heparin-binding proteins are shown for GST-CpEF1α (Lanes 2 and 3), and for GST-CpActin (Lanes 5 and 6), respectively. Arrows indicate GST-fused recombinant proteins.

Figure 6. HCT-8 binding property of rCpEF1α.

(a) The purified recombinant proteins were analyzed by SDS-PAGE and immunoblotting. The recombinant proteins were expressed in E. coli and purified by GST affinity chromatography. The purified proteins were concentrated by ultra-filtration. The black and white arrows indicate rCpEF1α and rGST, respectively. (b) Flow cytometry of HCT-8 cells incubated with recombinant protein. HCT-8 cells were incubated with each recombinant protein, reacted with rabbit α-GST antibody IgG, stained with Alexa 488-conjugated α-rabbit IgG goat antibody, and then subjected to flow cytometry analysis. The cells were gated on forward/side-light scatter to distinguish them from debris. The cells incubated with rCpEF1α (dark gray-filled histogram) showed higher fluorescence intensity than those incubated with rGST (bright gray-filled).