Abstract
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.
Highlights
Cryptosporidium spp. are apicomplexan parasites of the gastrointestinal epithelium, causing diarrhea in humans and various animals (Checkley et al, 2015)
No expression of the recombinant INS5 was detected in the supernatant of the E. coli lysate (Figure 1B)
The best insulinase-like proteases (INS)-5 expression as revealed by western blot analysis of lysates was achieved by culturing E. coli at 25◦C for 5 h after induction with 0.5 mM IPTG
Summary
Cryptosporidium spp. are apicomplexan parasites of the gastrointestinal epithelium, causing diarrhea in humans and various animals (Checkley et al, 2015). The Cryptosporidium molecules possibly involved in host cell attachment and invasion include GP40/15, GP900, P23, TRAP, and CSL (Singh et al, 2015). How these proteins are processed during sporozoite invasion, remains poorly elucidated. 12 INS genes are present in tandem in the 3 subtelomeric region of chromosome 3, suggesting the likelihood of regulated expression of INS proteins (Mauzy et al, 2012). Most of these INS are present in other intestinal Cryptosporidium species. As only 2% of Cryptosporidium genes have multiple copies, the expansion of this gene family suggests that the INS may have important biological functions during the invasion and development of Cryptosporidium spp. (Liu et al, 2016)
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