Preliminary Characterization of Two Small Insulinase-Like Proteases in Cryptosporidium parvum

Abstract

Cryptosporidium parvum is a major cause of moderate-to-severe diarrhea in humans and animals. Its compact genome contains 22 genes encoding divergent insulinase-like proteases (INS), which are poorly characterized. In this study, two small members of this family, INS-21 encoded by cgd7_2080 and INS-23 encoded by cgd5_3400, were cloned, expressed, and characterized to understand their functions. Recombinant INS-21 and INS-23 were expressed in Escherichia coli and polyclonal antibodies against these two proteins were prepared. The cgd7_2080 gene had a high transcription level during 0-2 h of in vitro C. parvum culture, while cgd5_3400 was highly transcribed at 0-6 h. INS-21 was mostly located in the apical region of sporozoites and merozoites whereas INS-23 was found as spots in sporozoites and merozoites. The immunoelectron microscopy confirmed the expression of INS-21 in the apical region of sporozoites while INS-23 appeared to be expressed in the dense granules of sporozoites. The neutralization efficiency was approximately 35%, when the cultures were treated with anti-INS23 antibodies. These results suggest that INS-21 and INS-23 are expressed in different organelles and might have different functions in the development of C. parvum.

Keywords: Cryptosporidium parvum; expression; insulinase-like protease; invasion; localization.

FIGURE 1

Sequence features of INS-21 and INS-23 of Cryptosporidium parvum. (A) Diagram of INS-21 (encoded by cgd7_2080) and INS-23 (encoded by cgd5_3400) of C. parvum. (B) Alignment of partial amino acid sequences of INS-21, INS-23 of C. parvum, and the α and β subunits of the mitochondrial processing peptidase (MPP) from Starmerella bombicola (yeast). Residues shown in the red box indicate the zinc-binding motif of the M16 family in MPPβ, while those in the green box represent the glycine-rich loop in MPPα. Thus, INS-23 contains the zinc-binding motifs while INS-21 contains the glycine-rich loop, indicating that both might act in concert in function. “*” indicates identical amino acid residue; “:” indicates high similarity; “.” indicates low similarity.

FIGURE 2

Expression of recombinant INS-21 and INS-23 in Escherichia coli. PCR amplification of the INS-21 gene (A) and INS-23 gene (B) of Cryptosporidium parvum. Lane M: 1000-bp molecular makers; Lane 1: PCR product. SDS-PAGE analysis of INS-21 protein (C) and INS-23 protein (D) expressed in E. coli. SDS-PAGE analysis of purified INS-21 protein (E) and INS-23 protein (F) from E. coli. Lane M, molecular weight markers; Lane 1, lysate from recombinant bacteria without the isopropyl β-D-thiogalactoside (IPTG) induction; Lane 2, lysate from recombinant bacteria after the IPTG induction, with the expected product being indicated by an arrow; Lane 3, purified recombinant proteins from the E. coli lysate using Ni-NTA affinity chromatography.

FIGURE 3

Expression of native INS-21 and INS-23 proteins in sporozoites of Cryptosporidium parvum. Western blot analysis of native INS-21 protein using antibodies against INS-21 (A) and pre-immune serum (B). Lane M, molecular weight markers; Lane 1, purified recombinant INS-21 protein; Lane 2, native proteins from sporozoites. Furthermore, the expression of native INS-23 protein was analyzed by Western blot using antibodies against INS-23 (C) and pre-immune serum (D). Lane M, molecular weight markers; Lane 1, native proteins from sporozoites; Lane 2, purified recombinant INS-23 protein. Arrows indicate native proteins from sporozoites reacting with polyclonal antibodies.

FIGURE 4

Transcription levels of the INS-21 (A) and INS-23 genes (B) in developmental stages of C. parvum. The relative expression of the INS gene at various C. parvum cultivation time was determined by reverse transcription-qPCR, with data being normalized with data from the expression of the Cp18S rRNA gene.

FIGURE 5

Expression of INS proteins in C. parvum life cycle stages indicated by immunofluorescence and transmission electron microscopy. Expression of INS-21 (A) and INS-23 (B) in C. parvum sporozoites and intracellular developmental stages in HCT-8 cell cultures, such as merozoites, type I meronts, and type II meronts, was examined using immunofluorescence microscopy. The images were taken under differential interference contrast (DIC), with the nuclei being counter-stained with 4’,6-diamidino-2-phenylindole (DAPI), parasites stained by immunofluorescence with anti-INS antibodies, and superimposition of the three images (Merged). Scale bars = 2 μm. In addition, the expression of the two INS in sporozoites was examined using transmission electron microscopy with anti-INS-21 antibodies (C) or anti-INS-23 antibodies (D) followed by the use of 18-nm colloidal gold-labeled goat anti-rabbit IgG. N, nucleus; AC, apical complex. Scale bars = 200 nm.

FIGURE 6

Inhibition efficiency of immune sera against INS-21 (A), INS-23 (B), and the combination of the two (C) on C. parvum invasion. Inhibition efficiency of C. parvum invasion by immune sera against INS was measured in HCT-8 cell culture. Data presented are mean ± SD from three independent experiments for both the expression and neutralization studies. Statistical analysis was performed using unpaired t-test for pairwise comparisons (*P < 0.05; **P < 0.01).