Cryptosporidium parasites are known to be highly divergent from other apicomplexan species at evolutionary and biological levels. Here we provide evidence showing that the zoonotic Cryptosporidium parvum also differs from other apicomplexans, such as Toxoplasma gondii, by possessing only two tubulin-based filamentous structures, rather than an array of subpellicular microtubules. Using an affinity-purified polyclonal antibody against C. parvum β-tubulin (CpTubB), we observed a long and a short microtubule that are rigid and stable in the sporozoites and restructured during the intracellular parasite development. In asexual development (merogony), the two restructuring microtubules are present in pairs (one pair per nucleus or merozoites). In sexual developmental stages, tubulin-based structures are detectable only in microgametes, but undetectable in macrogametes. These observations indicate that C. parvum parasites use unique microtubule structures that differ from other apicomplexans as part of their cytoskeletal elements.

Objectives To rapidly generate host cells with resistance to multiple compounds for differentiating drug action on parasite target or the host cell target (i.e. on-target or off-target effect) against the zoonotic enteric parasite Cryptosporidium parvum. Methods Transient overexpression of a multidrug resistance protein 1 (MDR1) gene in host cells (HCT-8 cell line) was explored to increase drug tolerance of the host cells to selected anti-cryptosporidial leads. In vitro cytotoxicity and anti-cryptosporidial efficacy of selected compounds were evaluated on the parasite grown in WT parental and transiently transfected HCT-8 cells. The approach was based on the theory that, for an epicellular parasite receiving consistent exposure to compounds in culture medium, overexpressing MDR1 in HCT-8 cells would increase drug tolerance of host cells to selected compounds but would not affect the anti-cryptosporidial efficacy if the compounds acted solely on the parasite target and the drug action on host cell target played no role on the antiparasitic efficacy. Results Six known anti-cryptosporidial leads were tested. Transient overexpression of MDR1 increased drug tolerance of HCT-8 cells on paclitaxel, doxorubicin HCl and vincristine sulphate (2.11- to 2.27-fold increase), but not on cyclosporin A, daunorubicin HCl and nitazoxanide. Increased drug tolerance in host cells had no effect on antiparasitic efficacy of paclitaxel, but affected that of doxorubicin HCl. Conclusions Data confirmed that, at efficacious concentrations, paclitaxel acted mainly on the parasite target, while doxorubicin might act on both parasite and host cell targets. This model can be employed for studying the action of additional anti-cryptosporidial leads, and adapted to studying drug action in other epicellular pathogens. The limitation of the model is that the anti-cryptosporidial leads/hits need to be MDR1 substrates.

Apicomplexan parasites possess several unique secretory organelles, including rhoptries, micronemes, and dense granules, which play critical roles in the invasion of host cells. The molecular content of these organelles and their biological roles have been well-studied in Toxoplasma and Plasmodium, but are underappreciated in Cryptosporidium, which contains many parasites of medical and veterinary importance. Only four proteins have previously been identified or proposed to be located in micronemes, one of which, GP900, was confirmed using immunogold electron microscopy (IEM) to be present in the micronemes of intracellular merozoites. Here, we report on the discovery of four new microneme proteins (MICs) in the sporozoites of the zoonotic species C. parvum, identified using immunofluorescence assay (IFA). These proteins are encoded by cgd3_980, cgd1_3550, cgd1_3680, and cgd2_1590. The presence of the protein encoded by cgd3_980 in sporozoite micronemes was further confirmed using IEM. Cgd3_980 encodes one of the three C. parvum rhomboid peptidases (ROMs) and is, thus, designated CpROM1. IEM also confirmed the presence of CpROM1 in the micronemes of intracellular merozoites, parasitophorous vacuole membranes (PVM), and feeder organelles (FO). CpROM1 was enriched in the pellicles and concentrated at the host cell–parasite interface during the invasion of sporozoites and its subsequent transformation into trophozoites. CpROM1 transcript levels were also higher in oocysts and excysted sporozoites than in the intracellular parasite stages. These observations indicate that CpROM1, an intramembrane peptidase with membrane proteolytic activity, is involved in host–parasite interactions, including invasion and proteostasis of PVM and FO.

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Background Cryptosporidium parvum is a zoonotic parasite and member of the phylum Apicomplexa with unique secretory organelles, including a rhoptry, micronemes and dense granules that discharge their contents during parasite invasion. The mucin-like glycoprotein GP900 with a single transmembrane domain is an immunodominant antigen and micronemal protein. It is relocated to the surface of excysted sporozoites and shed to form trails by sporozoites exhibiting gliding motility (gliding sporozoites). However, the biological process underlying its relocation and shedding remains unclear. The primary aim of this study was to determine whether GP900 is present as a transmembrane protein anchored to the plasma membrane on the surface of sporozoites and whether it is cleaved before being shed from the sporozoites. Methods Two anti-GP900 antibodies, a mouse monoclonal antibody (mAb) to the long N-terminal domain (GP900-N) and a rabbit polyclonal antibody (pAb) to the short C-terminal domain (GP900-C), were produced for the detection of intact and cleaved GP900 proteins in sporozoites and other parasite developmental stages by microscopic immunofluorescence assay and in discharged molecules by enzyme-linked immunosorbent assay. Results Both anti-GP900 antibodies recognized the apical region of unexcysted and excysted sporozoites. However, anti-GP900-N (but not anti-GP900-C) also stained both the pellicles/surface of excysted sporozoites and the trails of gliding sporozoites. Both antibodies stained the intracellular meronts, both developing and developed, but not the macro- and microgamonts. Additionally, the epitope was recognized by anti-GP900-N (but not anti-GP900-C) and detected in the secretions of excysted sporozoites and intracellular parasites. Conclusions GP900 is present in sporozoites and intracellular meronts, but absent in sexual stages. It is stored in the micronemes of sporozoites, but enters the secretory pathway during excystation and invasion. The short cytoplasmic domain of GP900 is cleaved in the secretory pathway before it reaches the extracellular space. The molecular features and behavior of GP900 imply that it plays mainly a lubrication role. Graphical Abstract