A secreted Cryptosporidium parvum dipeptidyl peptidase (CpDPP1) is targetable for developing cryptosporidial...

Cryptosporidiosis is a major zoonotic diarrheal disease caused by Cryptosporidium spp. and is associated with severe diarrhea, growth faltering, malnutrition, and mortality in young children, immunocompromised individuals, and neonatal ruminants. Treatment options remain limited, and nitazoxanide has poor efficacy in immunocompromised hosts. Here, we identified and characterized a secreted dipeptidyl peptidase of Cryptosporidium parvum, CpDPP1. CpDPP1 contains an N-terminal signal peptide, an exclusion domain, and a cathepsin C-like catalytic region with conserved active-site-associated residues, consistent with a substrate-selective papain-like cysteine protease. CpDPP1 localized predominantly to the anterior end of sporozoites and was released during excystation, free-sporozoite incubation, and gliding. Parasite-derived supernatants and Pichia-expressed recombinant CpDPP1 (rCpDPP1) hydrolyzed Gly-Phe-AFC, confirming dipeptidyl peptidase activity. Active rCpDPP1 disrupted junctional E-cadherin and reduced E-cadherin abundance in Caco-2 cells, whereas heat-inactivated protein did not, linking CpDPP1 activity to epithelial barrier damage. An enzyme activity-based screen of 5000 bioactive compounds identified eight reproducible inhibitors. Hypocrellin A and fluazinam showed potent CpDPP1 inhibition (IC₅₀ = 4.67 and 6.82 μM, respectively), in vitro anti-C. parvum activity (EC₅₀ of 1.31 and 0.45 μM), in vitro selectivity indices ≥10, and protection against rCpDPP1-induced E-cadherin loss. In vivo, high-dose hypocrellin A and fluazinam reduced fecal oocyst shedding on day 3 of treatment by approximately 95.83% and 97.57%, respectively, in the chronic C. tyzzeri infection model, and on day 7 post-infection by approximately 55.39% and 52.94%, respectively, in the IFN-γ-KO C. parvum infection model, compared with vehicle controls. Treatment also reduced intestinal parasite burden, attenuated weight loss, improved survival, and ameliorated intestinal pathology. These findings identify CpDPP1 as a secreted host-interface protease linked to early infection-associated epithelial damage and support CpDPP1-directed screening as a strategy for anti-cryptosporidial inhibitor discovery.