Abstract
The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.