Dynamic surface exposure of the fungal-affiliated putative amino acid transporter CpAAT9 in Cryptosporidium parvum

Cryptosporidium parvum is an obligate intracellular parasite with highly reduced metabolic capacity and strong dependence on host-derived nutrients. Membrane transporters therefore play essential roles in parasite survival and development, yet amino acid transport systems in Cryptosporidium remain poorly characterized. In this study, we focused on CpAAT9 ( cgd8_3740 ), a putative amino acid transporter that lacks recognizable orthologs in most other apicomplexans and instead shows evolutionary affinity to a broader eukaryotic transporter family related to fungal homologs. Phylogenetic analysis suggests that AAT9-like transporters represent an ancient eukaryotic lineage that has been selectively retained in Cryptosporidium in the Phylum Apicomplexa. Using affinity-purified polyclonal antibodies, we examined the expression and subcellular distribution of CpAAT9 across multiple developmental stages by indirect immunofluorescence assays. CpAAT9 is expressed throughout the parasite life cycle. In sporozoites, CpAAT9 localizes primarily to the parasite plasma membrane and displays an irregular punctate distribution beneath the pellicle. Non-permeabilized staining reveals substantial surface exposure of CpAAT9 following excystation, and the protein is shed during sporozoite gliding motility, appearing along gliding trails. During intracellular development, CpAAT9 remains detectable and partially colocalizes with markers of the parasitophorous vacuole membrane (PVM) that faces the intestinal lumen. In contrast, extracellular exposure appears reduced during sexual development. These findings indicate that CpAAT9 is a membrane transporter candidate that undergoes dynamic redistribution and shedding or secretion during parasite development. The unusual localization pattern suggests that CpAAT9 may contribute to nutrient acquisition from the intestinal lumen or interaction with intestinal mucus. This study provides new insight into transporter biology in Cryptosporidium and identifies CpAAT9 as a potential target for future functional investigation. • CpAAT9 is a conserved fungal-affiliated putative amino acid transporter uniquely retained in Cryptosporidium . • The protein becomes surface-exposed during sporozoite activation and is shed by gliding sporozoites. • CpAAT9 partially localizes to the parasitophorous vacuole membrane (PVM) during intracellular development. • CpAAT9 may contribute to nutrient acquisition from the intestinal lumen or interaction with intestinal mucus.