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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.

SUBMITTER: Cui H

PROVIDER: S-EPMC9315855 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.ResultsThe C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.ConclusionsIdentification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

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Characterization of Dense Granule Metalloproteinase INS-16 in Cryptosporidium parvum.

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