Discovery And Characterization Of A New Microneme Protein In Cryptosporidium Parvum

Apicomplexan parasites possesses several unique secretory organelles including rhoptries,micronemes and dense granules that play critical roles in the invasion of host cells.Molecular contents of these organelles and their biological roles are better studied in Toxoplasma and Plasmodium,but underappreciated in Cryptosporidium that contains many medically and veterinarily important parasites.For micronemes,only four proteins were previously identified or proposed to be located in this organelle,and one of them(GP900)was confirmed by immunogold electron microscopy(IEM)for its presence in the micronemes of intracellular merozoites.Here we report the discovery of four new microneme proteins(MICs)in the sporozoites of the zoonotic species C.parvum evidenced by immunofluorescence assay(IFA),including proteins encoded by cgd3＿980,cgd1＿3550,cgd1＿3680 and cgd2＿1590 genes.Among them,the presence of cgd3＿980 product in the micronemes of sporozoites was further confirmed by IEM.Cgd3＿980 encodes one of the three rhomboid peptidases(ROMs)encoded by the C.parvum genome,thus designated as Cp ROM1.Subsequent IEM also confirmed the presence of Cp ROM1 in the micronemes of intracellular merozoites,parasitophorous vacuole membranes(PVM)and feeder organelle(FO).Cp ROM1 was enriched to the pellicles and concentrated at the host cell-parasite interface during the invasion of sporozoites and its transformation to trophozoites.The transcript levels of Cp ROM1 gene were also much higher in oocysts and excysted sporozoites.These observations were indicative that Cp ROM1 as an intramembrane peptidase known for membrane proteolytic activity was involved in the parasite invasion and proteostasis of parasitophorous vacuole membrane and feeder organelle.