Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins.

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.

Characterization of MEDLE-1, a protein in early development of Cryptosporidium parvum.

BACKGROUND: Cryptosporidium 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. METHODS: CpMEDLE-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. RESULTS: The 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%. CONCLUSIONS: The 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.

Multilocus genotyping of Giardia duodenalis in Tibetan sheep and yaks in Qinghai, China.

Giardia duodenalis is a common gastrointestinal protozoon in mammals. Although many studies have been reported on the distribution of G. duodenalis genotypes in sheep and cattle raised under intensive farming, few studies are available on the distribution of G. duodenalis in Tibetan sheep and yaks, which are raised free ranging in a continental plateau climate. In this study, 495 fecal specimens from Tibetan sheep and 605 from yaks were collected from eight counties in Qinghai, China and analyzed for G. duodenalis by PCR targeting the ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. Based on PCR positivity at the bg locus, G. duodenalis occurrence rates were 13.1% (65/495) in Tibetan sheep and 10.4% (63/605) in yaks. DNA sequence analysis identified the presence of G. duodenalis Assemblages A (in 10 Tibetan sheep and 2 yaks) and E (in 51 Tibetan sheep and 60 yaks). In addition, mixed infections of the two were identified in four Tibetan sheep and one yak. Among the sequences obtained in this study, 1, 10, and 2 new subtypes of Assemblage E were detected at the bg, gdh and tpi loci, respectively. Based on sequences from the three loci, 28 multilocus genotypes (MLGs) were obtained, including 27 MLGs in Assemblage E and one MLG in Assemblage A. Each MLG was found in no more than seven animals, with most MLGs forming host-specific clusters in phylogenetic analysis except for one cluster including MLGs from both Tibetan sheep and yaks. Only two MLGs were found in both sheep and yaks. The above results demonstrate a high subtype diversity of G. duodenalis Assemblage E in Tibetan sheep and yaks raised in a traditional animal husbandry system and suggest that only limited cross-species transmission of G. duodenalis occurs between yaks and sheep sharing pastures.