BEZ235 inhibits Cryptosporidium parvum infection in mice by targeting the PI3K-NF-κB-c-MYB/BCL2A1 axis to restore host cell apoptosis.

Cryptosporidium parvum (C. parvum) is a zoonotic protozoan that causes cryptosporidiosis, primarily characterized by watery diarrhea. To clarify the molecular mechanisms of C. parvum-mediated host cell apoptosis regulation and identify potential therapeutics, we examined apoptosis rates, caspase activation, and apoptosis-related protein expression in infected NCM460 cells, along with the involvement of the phosphatidylinositol 3-kinase-nuclear factor kappa B (PI3K-NF-κB) signaling pathway. We further evaluated the efficacy of BEZ235, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, in both in vitro cell models and in vivo immunosuppressed mouse models. Results showed that C. parvum suppressed intrinsic apoptosis during 52-76 h post-infection (hpi) by inhibiting caspase-3 and caspase-9 activation and upregulating anti-apoptotic proteins c-MYB and BCL2A1. RNA interference-mediated knockdown of either c-MYB and BCL2A1 significant reversed this anti-apoptotic effect. Mechanistically, C. parvum infection robustly activated the PI3K-NF-κB axis between 40 and 76 hpi, as evidenced by increased AKT phosphorylation (p-AKT) and enhanced nuclear translocation of NF-κB subunit p65. BEZ235 treatment effectively suppressed PI3K-NF-κB activation, downregulated c-MYB and BCL2A1 expression, and restored apoptosis in infected cells. In a murine model of C. parvum infection, oral BEZ235 (15 mg/kg/day) exhibited superior therapeutic efficacy to paromomycin (50 mg/kg/day), reducing fecal oocyst shedding by 61.4% (vs. 42.9% with paromomycin) and ileal parasite burden by 60.3% (vs. 42.8% with paromomycin), and markedly alleviating intestinal epithelial damage and restoring villus‑crypt structure. These findings deepen our understanding of the intracellular survival strategy of C. parvum and highlight BEZ235 as a promising candidate for cryptosporidiosis treatment.