BackgroundCryptosporidium spp. are important diarrhea-causing pathogens in humans and animals. Comparative genomic analysis indicated that Cryptosporidium-specific MEDLE family proteins may contribute to host adaptation of Cryptosporidium spp., and a recent study of one member of this family, CpMEDLE-2 encoded by cgd5_4590, has provided evidence supporting this hypothesis. In this study, another member of the protein family, CpMEDLE-1 of Cryptosporidium parvum encoded by cgd5_4580, which is distinct from CpMEDLE-2 and has no signature motif MEDLE, was cloned, expressed and characterized to understand its function.MethodsCpMEDLE-1 was expressed in Escherichia coli and polyclonal antibodies against the recombinant CpMEDLE-1 protein were prepared in rabbits. Quantitative PCR was used to analyze the expression profile of cgd5_4580 in C. parvum culture. Immunofluorescence staining was used to locate CpMEDLE-1 expression in life-cycle stages, and in vitro neutralization assay with antibodies was adopted to assess the role of the protein in C. parvum invasion.ResultsThe results indicated that cgd5_4580 had a peak expression at 2 h of C. parvum culture. CpMEDLE-1 was located in the mid-anterior region of sporozoites, probably within the dense granules. The neutralization efficiency of anti-CpMEDLE-1 antibodies was approximately 40%.ConclusionsThe differences in protein and gene expression profiles between CpMEDLE-1 and CpMEDLE-2 suggest that MEDLE proteins have different subcellular locations, are developmentally regulated, could be potentially involved in the transcriptional regulation of the expression of parasite or host proteins and may exert their functions in different stages of the invasion and development process.
Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differences in host specificity. In this study, a Cryptosporidium parvum-specific member of the protein family MEDLE-2 encoded by cgd5_4590 was cloned and expressed in Escherichia coli. Immunofluorescent staining with antibodies generated from the recombinant protein showed the expression of the protein in sporozoites and development stages. In vitro neutralization assay with the antibodies partially blocked the invasion of sporozoites. These results support the potential involvement of MEDLE-2 in the invasion of host cells.
Cryptosporidium parvum and Cryptosporidium hominis share highly similar proteomes, with merely ~3% divergence in overall nucleotide sequences. Cryptosporidium -specific MEDLE family is one of the major differences in gene content between the two species. Comparative genomic analysis indicated that MEDLE family may contribute to differences in host range among Cryptosporidium spp. Previous studies have suggested that CpMEDLE-1 encoded by cgd5_4580 and CpMEDLE-2 encoded by cgd5_4590 are potentially involved in the invasion of C. parvum . In this study, we expressed in Escherichia coli, the C. hominis -specific member of the MEDLE protein family, ChMEDLE-1 encoded by chro.50507, and two C. parvum -specific members, CpMEDLE-3 encoded by cgd5_4600 and CpMEDLE-5 encoded by cgd6_5480 . Quantitative PCR, immunofluorescence staining and in vitro neutralization assay were conducted to assess their biologic characteristics. The expression of the cgd5_4600 gene was high during 12–48 h of the in vitro culture, while the expression of cgd6_5480 was the highest at 2 h. ChMEDLE-1 and CpMEDLE-3 proteins were mostly located in the anterior and mid-anterior region of sporozoites and merozoites, whereas CpMEDLE-5 was expressed over the entire surface of these invasive stages. Polyclonal antibodies against MEDLE proteins had different neutralization efficiency, reaching approximately 50% for ChMEDLE-1 and 60% for CpMEDLE-3, but only 20% for CpMEDLE-5. The differences in protein and gene expression and neutralizing capacity indicated the MEDLE proteins may have different roles during Cryptosporidium invasion and growth.
Calcium-dependent protein kinases (CDPKs) are considered promising targets for pharmaceutical intervention of cryptosporidiosis. Whole-genome sequencing has revealed the presence of several CDPKs (CpCDPKs) in Cryptosporidium parvum. In this study, we expressed recombinant CpCDPK3 encoded by the cgd5_820 gene in Escherichia coli. The biologic characteristics and functions of CpCDPK3 were examined using qRT-PCR, immunofluorescence microscopy, and in vitro neutralization assay. The expression of the cgd5_820 gene peaked in merozoites during in vitro culture while the CpCDPK3 protein was expressed in both sporozoites and merozoites. Polyclonal antibodies against CpCDPK3 showed no significant inhibitory effects on host invasion by the parasites. We assessed the inhibitory effects of 46 candidate compounds from molecular docking of CpCDPK3 on both C. parvum development and CpCDPK3 enzyme activities. One compound was identified to be effective. Results of these analyses suggest that CpCDPK3 might play an important role in the growth of C. parvum.
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