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

Fei, Jilan; Wu, Haizhen; Su, Jiayuan; Jin, Chanchan; Li, Na; Guo, Yaqiong; Feng, Yaoyu; Xiao, Lihua

doi:10.1186/s13071-018-2889-2

Cited by 16 publications

(17 citation statements)

References 31 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: 80%

“…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%

“…CpMEDLE-3 is expressed in the anterior and mid-anterior regions of C. parvum sporozoites, as indicated by immunofluorescence analyses using both anti-CpMEDLE-3 and anti-ChMEDLE-1 antibodies. In contrast, CpMEDLE-1 and ChMEDLE-1 are similarly expressed, while CpMEDLE-2 and CpMEDLE-5 are expressed over almost the entire C. parvum sporozoites (Li et al., 2017; Fei et al., 2018). Previous studies reported that most invasion-associated proteins were located in anterior or mid-anterior regions of sporozoites (Singh et al., 2015).…”

Section: Discussionmentioning

confidence: 95%

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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: 80%

Section: Introductionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 95%

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Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Jin

et al. 2019

Front. Microbiol.

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“…At the same time, intracellular apicomplexans are known to use multiple strategies to invade cells (Sibley, 2004) and the invasion of zoites inevitably requires the synergy of various molecules including surface antigens and apical proteins (Carruthers and Blackman, 2010). As a result, the in vitro neutralization ability of antibodies against individual invasion-related proteins could be limited (Fei et al, 2018). Further studies are needed to verify the neutralization capacity of polyclonal antibodies against INS-5.…”

Section: Discussionmentioning

confidence: 99%

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum

Jia

Guo

et al. 2020

Front. Microbiol.

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The small Cryptosporidium genome (∼9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of ∼78 kDa, smaller fragments of ∼70, ∼55, and ∼30 kDa were simultaneously generated. After purification through a nickel-nitrilotriacetic acid affinity column, the full recombinant protein obtained was used to prepare polyclonal antibodies. Antibodies raised against INS-5 recognized the recombinant protein and native protein in sporozoite extracts. Further characterization of INS-5 included qRT-PCR assessment of gene expression; immunofluorescence localization of the protein expression in sporozoites, merozoites, and other developmental stages; and neutralization of invasion of C. parvum in vitro. The results obtained indicated that although INS-5 was expressed in sporozoites and merozoites, the high gene expression was from 36 to 48 h of the in vitro culture after invasion. Anti-INS-5 antibodies partially neutralized the invasion (inhibition rate = 38.5%). Results of this study suggest that INS-5 plays some role in the invasion and growth of C. parvum.

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“…Therefore, SPDs are suggested to be involved in differences in host range, tissue tropism and pathogenicity among Cryptosporidium species [ 10, 11 ]. Among them, MEDLE proteins were named after a conserved sequence motif at the C terminus and are expressed in the invasion stages of C. parvum [ 12, 13 ]. Insulinase-like proteases are widespread in apicomplexans, and are known to be involved in processing invasion-related proteins or modifying host cell activities [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Comparative genomic analysis of three intestinal species reveals reductions in secreted pathogenesis determinants in bovine-specific and non-pathogenic Cryptosporidium species

Guo

et al. 2020

Microbial Genomics

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The three common intestinal Cryptosporidium species in cattle differ significantly in host range, pathogenicity and public health significance. While Cryptosporidium parvum is pathogenic in pre-weaned calves and has a broad host range, C. bovis and C. ryanae are largely non-pathogenic and bovine-specific species in post-weaned calves. Thus far, only the genome of C. parvum has been sequenced. To improve our understanding of the genetic determinants of biological differences among Cryptosporidium spcies, we sequenced the genomes of C. bovis and C. ryanae and conducted a comparative genomics analysis. The genome of C. bovis has a gene content and organization more similar to C. ryanae than to other Cryptosporidium species sequenced to date; the level of similarity in amino acid and nucleotide sequences between the two species is 75.2 and 69.4 %, respectively. A total of 3723 and 3711 putative protein-encoding genes were identified in the genomes of C. bovis and C. ryanae, respectively, which are fewer than the 3981 in C. parvum. Metabolism is similar among the three species, although energy production pathways are further reduced in C. bovis and C. ryanae. Compared with C. parvum, C. bovis and C. ryanae have lost 14 genes encoding mucin-type glycoproteins and three for insulinase-like proteases. Other gene gains and losses in the two bovine-specific and non-pathogenic species also involve the secretory pathogenesis determinants (SPDs); they have lost all genes encoding MEDLE, FLGN and SKSR proteins, and two of the three genes for NFDQ proteins, but have more genes encoding secreted WYLE proteins, secreted leucine-rich proteins and GPI-anchored adhesin PGA18. The only major difference between C. bovis and C. ryanae is in nucleotide metabolism. In addition, half of the highly divergent genes between C. bovis and C. ryanae encode secreted or membrane-bound proteins. Therefore, C. bovis and C. ryanae have gene organization and metabolic pathways similar to C. parvum, but have lost some invasion-associated mucin glycoproteins, insulinase-like proteases, MEDLE secretory proteins and other SPDs. The multiple gene families under positive selection, such as helicase-associated domains, AMP-binding domains, protein kinases, mucins, insulinases and TRAPs could contribute to differences in host specificity and pathogenicity between C. parvum and C. bovis. Biological studies should be conducted to assess the contribution of these copy number variations to the narrow host range and reduced pathogenicity of C. bovis and C. ryanae.

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

Cited by 16 publications

References 31 publications

Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum

Comparative genomic analysis of three intestinal species reveals reductions in secreted pathogenesis determinants in bovine-specific and non-pathogenic Cryptosporidium species

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