Preliminary Characterization of MEDLE-2, a Protein Potentially Involved in the Invasion of Cryptosporidium parvum

Li, Baoling; Wu, Haizhen; Li, Na; Su, Jiayuan; Jia, Ruilian; Jiang, Jianlin; Feng, Yaoyu; Xiao, Lihua

doi:10.3389/fmicb.2017.01647

Cited by 16 publications

(16 citation statements)

References 22 publications

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“…In the native protein expression, CpMEDLE-3 is in the forms of monomer (~15 kDa) and dimer (~40 kDa), while CpMEDLE-5 forms only monomer (~15 kDa). In previous studies, CpMEDLE-2 did not form dimers either, while CpMEDLE-1 reacted with fragments of ~32, ~36, and ~50 kDa in C. parvum sporozoite extracts (Li et al., 2017; Fei et al., 2018). These differences in dimer formation among MEDLE proteins in sporozoites support the conclusion on their differential roles.…”

Section: Discussionmentioning

confidence: 79%

“…In previous studies, C. parvum -specific MEDLE-1 encoded by cgd5_4580 and MEDLE-2 encoded by cgd5_4590 were characterized by our group (Li et al., 2017; Fei et al., 2018). These studies supported the potential involvement of MEDLE proteins in the invasion of C. parvum .…”

Section: Introductionmentioning

confidence: 77%

“…The reactivity of anti-MEDLE antibodies to native proteins (in crude protein extract from 1 × 10 7 oocysts/lane) in C. parvum sporozoites was assessed by using western blot as described (Li et al., 2017). Polyclonal antibodies against MEDLE proteins were used as primary antibodies at the 1:4,000 dilution in PBS, while HRP-conjugated goat anti-rabbit IgG (Yeasen) as secondary antibodies at the 1:5,000 dilution in PBS, with pre-immune sera as controls.…”

Section: Methodsmentioning

confidence: 99%

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Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Jin

et al. 2019

Front. Microbiol.

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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.

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Section: Discussionmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

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Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Jin

et al. 2019

Front. Microbiol.

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“…In a previous study by our research group [ 13 ], we conducted preliminary characterization of CpMEDLE-2, a divergent member of the MEDLE family, and provided evidence to support the hypothesis that MEDLE proteins may play a potential role in the invasion of C. parvum . In order to gain more insight into the biological functions of MEDLE proteins, here we characterized CpMEDLE-1, which is the only one in the protein family of C. parvum that does not have the MEDLE motif at the C-terminus.…”

Section: Introductionmentioning

confidence: 80%

“…In comparison with CpMEDLE-2, CpMEDLE-1 has shown unique sequence characteristics and a very different expression profile [ 13 ]. In direct sequence comparison and phylogenetic analysis, CpMEDLE-2 is divergent from CpMEDLE-1 and other MEDLE proteins.…”

Section: Discussionmentioning

confidence: 99%

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

Fei

et al. 2018

Parasites Vectors

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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.

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Comparative genomics: how has it advanced our knowledge of cryptosporidiosis epidemiology?

2019

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Preliminary Characterization of MEDLE-2, a Protein Potentially Involved in the Invasion of Cryptosporidium parvum

Cited by 16 publications

References 22 publications

Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

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

Comparative genomics: how has it advanced our knowledge of cryptosporidiosis epidemiology?

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