Search

Abstract Biological studies of the determinants of Cryptosporidium infectivity are lacking despite the fact that cryptosporidiosis is a major public health problem. Recently, the 60-kDa glycoprotein (GP60) has received attention because of its high sequence polymorphism and association with host infectivity of isolates and protection against reinfection. However, studies of GP60 function have been hampered by its heavy O-linked glycosylation. Here, we used advanced genetic tools to investigate the processing, fate, and function of GP60. Endogenous gene tagging showed that the GP60 cleavage products, GP40 and GP15, are both highly expressed on the surface of sporozoites, merozoites and male gametes. During invasion, GP40 translocates to the apical end of the zoites and remains detectable at the parasite-host interface. Deletion of the signal peptide, GPI anchor, and GP15 sequences affects the membrane localization of GP40. Deletion of the GP60 gene significantly reduces parasite growth and severity of infection, and replacement of the GP60 gene with sequence from an avirulent isolate reduces the pathogenicity of a highly infective isolate. These results have revealed dynamic changes in GP60 expression during parasite development. They further suggest that GP60 is a key protein mediating host infectivity and pathogenicity.

BackgroundThe determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences.Methodology/principal findingsIn this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-γ knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden.Conclusions/significanceThese results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice.

Summary: Cryptosporidium hominis and Cryptosporidium parvum are major causes of severe diarrhea. Comparative studies of them are hampered by the lack of effective cultivation and cryopreservation methods, especially for C. hominis. Here, we describe adapted murine enteroids for the cultivation and complete development of host-adapted C. parvum and C. hominis subtypes, producing oocysts infectious to mice. Using the system, we developed a cryopreservation method for Cryptosporidium isolates. In comparative RNA-seq analyses of C. hominis cultures, the enteroid system generated significantly more host and pathogen responses than the conventional HCT-8 cell system. In particular, the infection was shown to upregulate PI3K-Akt, Ras, TNF, NF-κB, IL-17, MAPK, and innate immunity signaling pathways and downregulate host cell metabolism, and had significantly higher expression of parasite genes involved in oocyst formation. Therefore, the enteroid system provides a valuable tool for comparative studies of the biology of divergent Cryptosporidium species and isolates.

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.

Cryptosporidiumparvum is a common protozoan pathogen responsible for moderate-to-severe diarrhea in humans and animals. The small genome of C. parvum has 22 genes encoding insulinlike proteases (INS) with diverse sequences, suggesting that members of the protein family may have different biological functions in the life cycle. In this study, two members of the INS family, CpINS-4 and CpINS-6 with the Zn2+-binding motif “HXXEH” but different numbers of function domains, were expressed in Escherichia coli and used in the generation of polyclonal antibodies. In both recombinant and native proteins, CpINS-4 and CpINS-6 were spliced into multiple fragments. The antibodies generated recognized their respective recombinant and native proteins and the spliced products, but had minimum cross-reactivity with each other. Anti-CpINS-4 antibodies reacted with the middle region of sporozoites and merozoites, while CpINS-6 had the highest reactivity to the apical region. Polyclonal anti-CpINS-4 antibodies produced 36% reduction in parasite load in HCT-8 cultures at 24 h, while those against CpINS-6, which has one of the function domains missing, failed in doing so. The genes encoding both CpINS-4 and CpINS-6 had the highest expression in the invasion phase of in vitro C. parvum culture. These data suggest that CpINS-4 and CpINS-6 might be expressed in different organelles and play different biological functions in the life cycle of C. parvum.

Cryptosporidiumfelis is an important cause of feline and human cryptosporidiosis. However, the transmission of this pathogen between humans and cats remains controversial, partially due to a lack of genetic characterization of isolates from cats. The present study was conducted to examine the genetic diversity of C. felis in cats in China and to assess their potential zoonotic transmission. A newly developed subtyping tool based on a sequence analysis of the 60-kDa glycoprotein (gp60) gene was employed to identify the subtypes of 30 cat-derived C. felis isolates from Guangdong and Shanghai. Altogether, 20 C. felis isolates were successfully subtyped. The results of the sequence alignment showed a high genetic diversity, with 13 novel subtypes and 2 known subtypes of the XIXa subtype family being identified. The known subtypes were previously detected in humans, while some of the subtypes formed well-supported subclusters with human-derived subtypes from other countries in a phylogenetic analysis of the gp60 sequences. The results of this study confirmed the high genetic diversity of the XIXa subtype family of C. felis. The common occurrence of this subtype family in both humans and cats suggests that there could be cross-species transmission of C. felis.