Cryptosporidium parvum CpARO marks rhoptry envelope remnants associated with feeder organelle formation

Apicomplexans encode a single armadillo repeat-only (ARO) protein, exemplified by TgARO and PfARO, that anchors to the rhoptry envelope through N-terminal acylation and supports rhoptry positioning through interaction with an ARO-interacting protein (AIP). These AROs organize rhoptries but are not known to be secreted during invasion. Here, we show that Cryptosporidium parvum ARO (CpARO) localizes to the rhoptry envelope by immunofluorescence assay, ultrastructural expansion microscopy, and structured illumination microscopy. During sporozoite invasion, CpARO-positive rhoptry envelope structures shorten and condense into a discrete punctum after content discharge. Residual rhoptry membrane structures are subsequently detected between the host cell F-actin pad and parasite nucleus in developing trophozoites, consistent with a contribution to nascent feeder organelle formation. In contrast to TgARO and PfARO, CpARO is also detected in secreted fractions during excystation, gliding, invasion, and intracellular development, with no evidence of nuclear localization. Recombinant CpARO binds host cells with high affinity (Kd = 0.189 μM). Although C. parvum encodes an AIP homolog, this protein localizes to the sporozoite cytoplasm rather than to rhoptries; we therefore designate it AIP-like protein (CpAIP-L). Antibodies against both CpARO and CpAIP-L were detected in sera from C. parvum-infected mice. These findings reveal functional divergence of Cryptosporidium ARO-AIP-related proteins and identify CpARO as both a rhoptry envelope marker and a secreted host-interacting factor with potential roles in host interaction and virulence.