Characterization of Dense Granule Metalloproteinase INS-16 in Cryptosporidium parvum

Abstract

The protozoan pathogen Cryptosporidium parvum infects intestinal epithelial cells and causes diarrhea in humans and young animals. Among the more than 20 genes encoding insulinase-like metalloproteinases (INS), two are paralogs with high sequence identity. In this study, one of them, INS-16 encoded by the cgd3_4270 gene, was expressed and characterized in a comparative study of its sibling, INS-15 encoded by the cgd3_4260 gene. A full-length INS-16 protein and its active domain I were expressed in Escherichia coli, and antibodies against the domain I and an INS-16-specific peptide were produced in rabbits. In the analysis of the crude extract of oocysts, a ~60 kDa fragment of INS-16 rather than the full protein was recognized by polyclonal antibodies against the specific peptide, indicating that INS-16 undergoes proteolytic cleavage before maturation. The expression of the ins-16 gene peaked at the invasion phase of in vitro C. parvum culture, with the documented expression of the protein in both sporozoites and merozoites. Localization studies with antibodies showed significant differences in the distribution of the native INS-15 and INS-16 proteins in sporozoites and merozoites. INS-16 was identified as a dense granule protein in sporozoites and macrogamonts but was mostly expressed at the apical end of merozoites. We screened 48 candidate INS-16 inhibitors from the molecular docking of INS-16. Among them, two inhibited the growth of C. parvum in vitro (EC₅₀ = 1.058 µM and 2.089 µM). The results of this study suggest that INS-16 may have important roles in the development of C. parvum and could be a valid target for the development of effective treatments.

Keywords: Cryptosporidium parvum; expression differences; invasion; metalloproteinase.

Figure 1

Domain structure and specific amino acid sequence of INS-16 of Cryptosporidium parvum. (A) Predicted tertiary structures of INS-16. INS-16 have four classic M16 domains; the ribbon model is colored based on the M16 active domain (MAD), inactive domain (ID), the middle domain (MTD), and M16 peptidase-like domain (MPP-like). Black arrows indicate the region of these domains in the model and the amino acid sequence. The red spheroid located between the active domain and the inactive domain indicates the zinc-binding site of INS-16. (B) Alignment of amino acid sequences of INS-15 and INS-16. The red box shows the amino acid sequence of INS-16-specific peptide (LRKTNNFVLKGKIG) used in the study.

Figure 2

Western blot analysis of cross-reactivity and native protein expression of INS-15 and INS-16. (A) Cross-reactivity of antibodies between INS-15 and INS-16. Lane M: protein marker; Lane 1: purified recombinant INS-15 domain I; Lane 2: purified recombinant INS-16 domain I. The image on the left shows the result by using anti-INS-15 domain I as the primary antibodies. The image on the right shows the result by using anti-INS-16 domain I as the primary antibodies. (B) Western blot analysis of the specificity of antibodies against INS-16 peptide. Lane M: protein marker; Lane 1: purified recombinant full-length INS-15. Lane 2: purified recombinant full-length INS-16. The picture on the left shows the full-length protein of INS-15 and INS-16 reacting with antibodies against the INS-15 peptide. The picture on the right shows the full-length protein of INS-15 and INS-16 reacting with antibodies against the INS-16 peptide. (C) Expression of native INS-16 protein in C. parvum sporozoites. Lane M: protein marker; Lane 1: purified recombinant full-length INS-16. Lane 2: C. parvum sporozoite lysate. The picture on the right shows the result of proteins reacting with pre-immune serum.

Figure 3

Patterns of INS-16 expression in oocysts, sporozoites, and intracellular stages of Cryptosporidium parvum in HCT-8 cells as indicated with immunofluorescence microscopy. (A) The localization of INS-16 with antibodies against INS-16 domain I. (B) The localization of INS-16 with antibodies against INS-16-specific peptide. The reactivity of the antibodies with oocysts, free sporozoites, and merozoites in infected HCT-8 cells is shown (red). Nuclei were counter-stained with DAPI (blue). Scale bars = 1 μm. (C) Co-localization of INS-15 and INS-16 in sporozoite, trophozoite, meront and free merozoite. The reaction of antibodies against INS-15-specific peptide is shown in green fluorescence, while the reaction of antibodies against INS-16-specific peptide is shown in red fluorescence. Nuclei were counter-stained blue with DAPI. Scale bars = 1 μm.

Figure 4

Differences in the distribution of INS-15 and INS-16 expression in organelles of developmental stages of Cryptosporidium parvum. The localization of INS-15 and INS-16 in subcellular structures was analyzed using immuno-transmission electron microscopy. The distribution of INS-16 (A) and INS-15 (B) in sporozoites and meronts. C. parvum oocysts and meronts in infected HCT-8 cells were fixed and stained with antibodies against the INS-16 or INS-15-specific peptide followed by 10 nm colloidal gold-conjugated goat anti-rabbit IgG. N, nucleus; DG, dense granule; C, crystalloid body; R, rhoptry. Scale bars, 500 nm.

Figure 5

Inhibitory efficacy of candidate INS-16 inhibitors on the development of Cryptosporidium parvum in HCT-8 cells. (A) Efficacy of all 48 compounds at 10 µM in primary evaluations. Ten compounds with high levels of efficacy (>50%) are marked as green squares. Thirty-eight compounds with low levels of efficacy (<50%) are marked as yellow dots. (B,C) Dose–response curves of compound 3805-1518 and F107-1944 on C. parvum growth. The two compounds can inhibit C. parvum growth by 50% at the concentration of 1.058 µM and 2.089 µM, respectively. The anti-cryptosporidial activities were determined by using qRT-PCR. (D,E) Dose–response curves of compound 3805-1518 and F107-1944 on HCT-8 cell growth. The maximum used on HCT-8 cells without any inhibitory effect above 100 µM for 3805-1518 and F107-1944. The data shown are means ± SD (n ≥ 3) from one representative of at least three independent experiments.