Differential Expression of Three Cryptosporidium Species-Specific MEDLE Proteins

Abstract

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.

State of the Art and Future Directions ofCryptosporidiumspp.

“…Comparative genomic analysis revealed that one of the primary features differentiating Cryptosporidium species is the sequence diversity present in major secreted protein families, MEDLE, and insulinase-like proteases [184]. This is consistent with transcriptomic studies of C. parvum, which have demonstrated MEDLE proteins in different subcellular locations that may perform their functions in distinct stages of the invasion and development process [33]. Moreover, a reduction in the number of genes encoding secreted MEDLE and insulinase-like proteins was observed in C. ubiquitum and C. andersoni, whereas the mucin-type glycoproteins are highly divergent between the gastric C. andersoni and intestinal Cryptosporidium species [184].…”

“…No fully effective drug therapy or vaccine is available for Cryptosporidium, and the diagnosis of cryptosporidiosis has been based on the demonstration of oocysts in faeces, which present low sensibility [25]. However, the ability to culture relevant Cryptosporidium isolates in vitro, the development of novel gene-editing tools (knockout genes, CRISPR/ Cas9, and RNAi) [26][27][28][29][30], and 'omic' research (genomics, transcriptomics, and proteomics) represent essential paths towards significant advancements in the control of cryptosporidiosis [30][31][32][33][34][35][36][37][38]. In the future, those approaches will show a holistic view of the biology of Cryptosporidium.…”

Stem cell‐derived enteroid cultures as a tool for dissecting host‐parasite interactions in the small intestinal epithelium

“…They are known to be involved in host cell invasion but their presence and expression varies between Cryptosporidium species implying that they may have a role in defining host range. Although the precise function of this protein is still unclear, it gives an insight into how different Cryptosporidium species are able to infect different host species 111‐113 . Interestingly, with the establishment of species‐specific enteroid models and methods for creating transgenic parasites, it may be possible to determine how the MEDLE proteins determine host range.…”

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

“…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].…”