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

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.

relatively few studies on the molecular basis of the host-parasite interactions that are necessary for C. parvum invasion of host cells 12 . We do know that Gal/GalNac is recognized by C. parvum p30 14 , and that the 85-kDa protein on Caco-2 cells is a receptor for C. parvum circum sporozoite-like antigen 15 . But the remaining host factors that interact with C. parvum sporozoites at the invasion step remain largely unknown.
C. parvum infects mainly the gastrointestinal tract 16 . However, sporozoites cannot directly interact with intestinal epithelial cells because glycocalyx, a filamentous layer of branched carbohydrates 17 , is present on these cells and act as a defensive barrier. Glycocalyx contains high levels of transmembrane mucin glycoproteins 18 , a type of proteoglycan. Mucin is the major component of the intestinal barrier 19 and has been reported to reduce C. parvum attachment to intestinal epithelial cells in vitro 20 .
Proteoglycans (PGs) are composed of glycosaminoglycan (GAG) chains, which is a category of sulfated polysaccharides that are covalently bound to a protein core 21 . A variety of pathogens utilize them to invade their host cells [22][23][24] . Some sulfated polysaccharides have been shown to bind to several apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, and to inhibit their infection [25][26][27][28][29] . Therefore, we hypothesized that C. parvum interacts with GAGs on the host cells and that some sulfated polysaccharides may inhibit C. parvum infection. Despite many reports about the inhibitory effects of sulfated polysaccharides on various apicomplexan parasites, little is known about the interaction between C. parvum and sulfated polysaccharides 14,30 .
In this study, we evaluated the anti-cryptosporidial effects of five sulfated polysaccharides on parasite invasion, and found that heparin had the highest inhibitory effect on the C. parvum invasion of host cells. To gain further insight into the heparin-induced inhibitory mechanism of parasite invasion, we subsequently attempted to identify the C. parvum sporozoite proteins that physically bind to heparin.

Results
The effects of sulfated polysaccharides on C. parvum invasion of host cells. To examine the effects of sulfated polysaccharides on the invasion of host cells by C. parvum, we performed invasion inhibition assays with the following five sulfated polysaccharides: heparin, chondroitin sulfate A (CSA), dextran sulfate [DS high molecular weight (HMW) and DS low molecular weight (LMW)], and fucoidan (FDC) (Fig. 1A). All of these polysaccharides showed dose-dependent inhibitory effects on the C. parvum invasion of HCT-8 cells, inhibiting over 50% of invasion at a concentration of 100 μ g/mL; however, the inhibitory effect differed among the polysaccharides. Of the five polysaccharides tested, heparin inhibited the most C. parvum invasion at the lower concentration of 1 μ g/mL and its inhibitory effect was dose independent (Fig. 1B). Our data reveal that sulfated polysaccharides differ in their invasion inhibitory effects, with heparin having the highest and dose-dependent inhibitory effect on C. parvum invasion of 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 μ g /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.
Scientific RepoRts | 5:11599 | DOi: 10.1038/srep11599 HCT-8 cells among the sulfated polysaccharides tested. This result revealed that heparin competed with some factor(s) involved in the HCT-8 cell-parasite interaction in vitro.
C. parvum sporozoites are affected by heparin. To understand the mechanistic basis of the inhibitory effect of heparin on the invasion of culture cells by C. parvum, we conducted pre-incubation assays to elucidate whether heparin affects HCT-8 cells or parasites. No invasion inhibition was observed in the HCT-8 cells incubated with heparin prior to parasite infection (Fig. 2, left panel), whereas significant inhibition was observed in cells infected with parasites before incubation with heparin ( Fig. 2, left panel). These results suggest that heparin competed with some factor(s) on the parasites rather than the HCT-8 cells, and that these parasites' factors are involved in the invasion of HCT-8 cells in vitro.
Heparan sulfate is important for the efficient invasion by C. parvum. To determine whether heparin or heparin-like molecules are involved in the invasion of by C. parvum sporozoites, we compared the efficiency of parasite invasion of CHO pgs-D677 with that of wild-type CHO K1 cells. CHO pgsD-677 cells lacks both N-acetylglucosaminyl-and glucuronosyl-transferase, which are enzymes required for the polymerization of heparan sulfate or heparin 31 . Notably, heparin is mast cell polysaccharide 32 . Therefore, CHO pgsD-677 cells differ from CHO K1 cells in that they lack heparan sulfate, which is a kind of GAG and has a very similar structure to that of heparin. Cells were infected with C. parvum sporozoites, and the number of cells invaded by the parasites was compared. CHO pgsD-677 cells showed a statistically significant reduction in parasite invasion (27%) compared with that observed in wild-type CHO cells (Fig. 3). Thus, we demonstrated that heparan sulfate on the cell surface plays a role in invasion by C. parvum in vitro.

Identification of C. parvum-derived factors that interact with heparin.
To identify parasite factors that interact with heparin, we conducted pull down assays with cell lysates of C. parvum sporozoites by using heparin-agarose beads, and subsequently analyzed the precipitated parasite proteins by using liquid chromatography tandem mass spectrometry (LC-MS/MS). We found bands specifically concentrated with molecular masses of around 120, 90, and 45 kDa in the precipitated fraction (Fig. 4A). The 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. proteins in these three bands were gel-extracted and subjected to mass spectrometry analysis. For the heparin-binding proteins, we identified a total of 31 distinct proteins; 7 of these proteins were detected in the band with the molecular mass of 120 kDa, 11 proteins were detected for 90 kDa, and 13 for 45 kDa (Table 1).
To further categorize these proteins based on molecular function, we performed functional enrichment analysis by using Gene Ontology (GO) analysis. These 31 proteins were enriched for multiple biological processes, that is, translation, homeostasis, metabolism, and respiration ( Fig. 4B). Of note, the most enriched GO category for the heparin-binding proteins was translational elongation (P value = 4.1 × 10 −3 ), including elongation factor 1 alpha (EF1α , cgd6_3990) and translation elongation factor 2 (EF2, cgd8_2930). Thus, we identified 31 parasite proteins with the potential to interact with heparin that are involved in diverse biological processes.

C. parvum elongation factor 1α (CpEF1α) is a heparin ligand.
Among the parasite proteins that precipitated with heparin-agarose beads, C. parvum EF1α (CpEF1α ) and EF2 belong to the most enriched GO category. Matsubayashi, M. et al. have previously showed that CpEF1α plays a role in the invasion and could be a potential protective antigen of C. parvum 33 , yet its host receptor has not been identified. We focused on CpEF1α and tested whether it directly interact with heparin. The GST-fusion CpEF1α was expressed at the expected molecular weight of 74 kDa in E. coli, was purified by GST beads, incubated with heparin-agarose beads, and its binding was assessed by silver staining. Actin was selected as a negative control because it did not belong to any GO categories and is often detected by mass spectrometry due to its abundance as a house-keeping gene. As we expected, a 68-kDa protein representative of GST-fused CpActin was scarcely pulled down with heparin-agarose beads (Fig. 5, right panel). By contrast, GST-fused CpEF1α was precipitated with heparin-agarose beads and a clear band was observed at the 74 kDa position (Fig. 5, left panel). Thus, we demonstrated that CpEF1α directly binds to heparin.
Finally, to investigate the biological interaction between CpEF1α and the receptor on the surface of the HCT-8 cell, the number of HCT-8 cells that binds to rCpEF1α was counted by using flow cytometry. Prior to binding assay, recombinant rCpEF1α and rGST was purified by ultrafiltration and the expression of the recombinant proteins were confirmed by silver staining and immunoblot. rGST served as a negative control. The HCT-8 cells incubated with rCpEF1α showed more fluorescence intensity than that observed for cells incubated with rGST (Fig. 6B). We observed statistically significant differences 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.
in the fluorescence intensity between the cells incubated with rGST and those incubated with rCpEF1α (P < 0.05), demonstrating that rCpEF1α binds to HCT-8 cells.

Discussion
Here, we evaluated the inhibitory effect of sulfated polysaccharides on the invasion of HCT-8 cells by C. parvum, and found that heparin was the most effective inhibitor of C. parvum invasion among the five sulfated polysaccharides tested. To our knowledge, this is the first report of the effect of heparin on C. parvum invasion. We also showed that heparin competes with some factor(s) involved in C. parvum sporozoite invasion. In addition, we showed that heparin does not affect the HCT-8 cells but rather the C. parvum sporozoites. We further identified 31 parasite proteins that interact with heparin by using pull-down assays followed by mass spectrometry. We confirmed the binding of CpEF1α with a heparin-like molecule on the surface of HCT-8 cells. Taken together, our data suggest that a heparin-like molecule is important for the efficient invasion of HCT-8 cells by C. parvum sporozoites.
We observed a discrepancy in the inhibition efficacy of parasite invasion by heparin between the invasion inhibition assay and the pre-incubation assay. While 65% of parasite invasion was blocked by 1 μ g/mL heparin in the invasion inhibition assay, only 18% was blocked in the pre-incubation assay. This discrepancy could be attributed to the amount of heparin present in the medium. In the former experiment, heparin was abundant in the medium and bound to sporozoites when the sporozoites were inoculated. By contrast, in the latter experiment, only bound heparin was present because the sporozoites were washed three times after pre-incubation with heparin and then inoculated. In other words, the free heparin present in the medium might have contributed to the inhibition of parasite invasion of host cells. Heparin may, therefore, bind to two different types of C. parvum sporozoite factor: surface proteins of sporozoites and secreted proteins involved in the invasion of the host cells by the sporozoites, resulting in efficient invasion inhibition.
The CHO pgsD-677 cell line was used as a heparan sulfate-deficient cell in our study. This cell line was less susceptibility to C. parvum sporozoite invasion than were wild-type cells. This cell line, however, 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). expresses 3-to 4-fold higher levels of chondroitin sulfate than do wild-type cells 31 . Despite these higher levels of chondroitin sulfate, this cell line was much less susceptible to parasite invasion (Fig. 1A), indicating that heparan sulfate plays an important role in C. parvum invasion.
In our experiments, heparin did not completely inhibit C. parvum infection. Additionally heparan sulfate-deficient cells were less susceptibility to C. parvum. These results suggest heparan sulfate is not the sole receptor of C. parvum. Namely, other factors could cooperate to attach and invade into host cells. However, heparan sulfate is common molecule to mammals and this could help C. parvum is parasitic on many kinds of mammals. Therefore, heparan sulfate could be important molecule for infection and heparin is noticeable substance. Eukaryotic EF1α (eEF1α ) is involved in the first step of translation and elongation by binding and delivering aa-tRNAs to the A site of the ribosome 34 . In addition, eEF1-α has a multipleother functions 35 , including interaction with the actin cytoskeleton 36 and modulation of microtubules in a Ca 2+ /CaM-dependent manner in mammalian cells 37 . By contrast, CpEF1α is reported to localize to the apical region of sporozoites, and play an essential role in invasion of sporozoites 33 . However, the binding partner of CpEF1α has not yet known. In the present study, we identified heparin as a CpEF1α -binding partner, and confirmed that recombinant CpEF1α bound to heparin and HCT-8 cells in vitro. Our results thus further our understanding of host-parasite interactions that are essential for parasite invasion of host cells.
Studies of various pathogens, including parasites 27,38-40 , viruses [41][42][43] , and bacteria [44][45][46] , strongly indicate that heparin binds to a range of microorganism proteins. Furthermore, heparin is known to inhibit the infection of various kinds of pathogen 41,43,47,48 . Most of the existing anti-cryptosporidial medicines are antibiotics or ionophores [6][7][8][9][10] . But these medicines have side effects and there is little information about safe dosages. Our study showed that heparin has an inhibitory effect on C. parvum infection. Although heparin is a sulfated polysaccharide, it can be synthesized in Escherichia coli 49,50 . Therefore, heparin could serve as a new type of anti-cryptosporidial agent. For clinical use, experimental testing in both humans and livestock is necessary. In addition, there are potential concerns regarding drug delivery and side effects. Ingested heparin could be digested in the stomach and not reach the small intestine. Therefore, it may be necessary to protect the heparin molecule with some kind of capsule. Moreover, heparin has anticoagulant effects 51 , suggesting that it could affect the blood coagulation system if ingested as a drug. A possible plan to utilize heparin is employing chemically-modified heparins 52 which exhibit attenuated anticoagulant activity but, which keep an ability to inhibit C. parvum infection. If this substance is made practical, our research will contribute to progression of the treatment for cryptosporidiosis.
In summary, we revealed that heparin inhibits C. parvum invasion of host cells. We identified CpEF1α as a heparin-binding protein and characterized its heparin-binding property and affinity to HCT-8 cells. These results suggest that CpEF1α interacts with heparan sulfate on host cells and that this interaction is important for host cell invasion. Our findings help further our understanding of the molecular basis of C. parvum invasion and are of value for the development of novel anti-cryptosporidial agents.  cells (obtained from ATCC) were maintained in Ham's F-12 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and 50 μ g/mL streptomycin. All cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 .

Methods
C. parvum oocysts, strain HNJ-1, were kindly provided by Dr. K. Yagita (National Institute of Infectious Diseases). Oocysts were maintained by passage in experimentally infected nude mice (SLC, Shizuoka, Japan) and purified from feces by using discontinuous sucrose and cesium chloride gradients as described previously 53 .
Sporozoites excystation. C. parvum oocysts were treated with 10% (v/v) purelox (OyaloxCo.Ltd., Tokyo, Japan) for 10 min on ice, and then washed three times with phosphate buffered saline (PBS) by centrifugation at 7,000 rpm for 2 min at 4 °C. Purelox-treated and washed oocysts were treated with 0.1 M NaH 2 PO 4 -HCl (pH 2.0) for 30 min at 37 °C and then washed twice. The oocysts were further excysted in the PBS containing 0.75% sodium taurocholate and 0.25% trypsin for 1 h at 37 °C. Excysted sporozoites were separated from oocysts by filtration through a 5-μ m pore-size PVDF filter (Merck Millipore, Darmstadt, Germany) and then used in the study. Oocysts and sporozoites were counted with microscopy using hemocytometer.  Pre-incubation assay. C. parvum sporozoites or HCT-8 cells were incubated in RPMI-1640 medium containing heparin for 1 h prior to infection. Sporozoites or HCT-8 cells were washed with the medium without heparin three times, and subsequently mixed with HCT-8 cells or sporozoites, respectively, for 3 h. The number of parasites left in the cells was counted by following the protocol described above.

Pull-down assay.
To identify the parasite proteins that interact with heparin, excysted C. parvum sporozoites were lysed with 1% octylglucoside (OGS, Sigma-Aldrich) in PBS overnight at 4 °C 14 , and centrifugation at 10,000 rpm for 25 min. The supernatants were mixed with heparin-agarose beads (Sigma-Aldrich) at 4 °C for 1 h. The beads were then washed with 0.1% OGS in PBS three times.
To confirm the interaction of parasite proteins with heparin, the purified recombinant proteins were mixed with heparin-agarose beads (Sigma-Aldrich) at 4 °C for 1 h. The beads were washed with PBS three times. The beads were then boiled for 5 min in equal volumes of 2 × sample buffer that contained 0.125 M Tris (pH 6.8), 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol.
Silver staining, mass spectrometry and mascot search. The proteins eluted from the beads described above were separated by SDS-PAGE and then silver stained. The gel was stained by using the Ez stain silver kit (ATTO, Tokyo, Japan). Protein bands were excised from the gels, digested with trypsin and subjected to nano-LC/MS/MS analysis by following the standard protocol using QSTAR XL (Applied Biosystems, CA, USA) and Bio NanoLC (KYA Technologies, Tokyo, Japan). Protein digestion, nano-LC/ MS/MS analysis, and the mascot search were conducted by Japan Proteomics (Sendai, Japan).

Functional enrichment analyses.
To functionally categorize the proteins identified in our study, the proteins were assigned to a GO grouping. GO analysis was carried out by using the Gene Ontology Enrichment embedded in the CryptoDB database (http://cryptodb.org/); Fisher's exact P values were used to determine the GO terms that were significant (P < 0.05).
Flow cytometry. The purified rCpEF1α and rGST were concentrated by using Amicon Ultra filter units (50 K for rCpEF1α and 3 K for rGST) (Merck Millipore). The protein concentration was measured by using Protein Quantification Kit-Rapid (Dojindo laboratories, Kumamoto, Japan), and was diluted to 7.5 μ M with elution buffer. Semi-confluent HCT-8 cells were washed twice, and treated with 500 μ M EDTA in PBS for 5 min at 37 °C. Detached cells were washed with FACS buffer (PBS containing 2% fetal calf serum) by centrifugation at 1,500 rpm. Then 2 × 10 6 washed HCT-8 cells were incubated with 100 μ L of 7.5 μ M rCpEF1α or rGST for 2 h at 4 °C, and then washed twice with FACS buffer. The cells were then incubated with 100 μ L of rabbit α -GST antibody (Sigma-Aldrich) at 1:1000 dilution for 30 min at 4 °C, washed with FACS buffer containing 2% FCS twice, incubated with 100 μ L of Alexa 488-conjugated α -rabbit IgG goat antibody at 1:1000 dilution for 30 min at 4 °C, and washed with FACS buffer again. As a negative control, detached HCT-8 cells which were incubated with neither proteins nor antibodies were also prepared. The sample was then analyzed on BD FACSVerse (BD Bioscience) using BD FACSuite software (BD Biosciences). HCT-8 cells were gated on forward/side-light scatter to distinguish them from debris. Cells (10,000 events) were analysed by Alexa 488 channels.