Induction of β-Defensin 1 (DEFB1) via synthetic mRNA reduces Cryptosporidium parvum infection in human intestinal cells.

Abstract

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, is a leading cause of severe diarrhea, morbidity, and mortality in immunocompromised patients and malnourished children. Because the only FDA-approved treatment, nitazoxanide, shows limited efficacy in malnourished children and is ineffective in the immunodeficient, novel therapeutics for vulnerable populations are urgently needed. Host-directed therapy (HDT) is a promising strategy that utilizes the induction of endogenous protective molecules. For example, intestinal epithelial cells defend the mucosa by upregulating antimicrobial peptides like β-defensins (DEFBs). Previous work has demonstrated that human DEFB1 possesses anti-cryptosporidial activity. In this work we used synthetic DEFB1-mRNA to induce DEFB1 protein in human intestinal cells and evaluate its protection against Cryptosporidium parvum infection in vitro. HCT-8 cells were transfected with synthetic, co-transcriptionally capped DEFB1-mRNA or control mRNAs complexed with Lipofectamine MessengerMAX. Transfection efficiency was validated via EGFP expression. Twenty-four hours post-transfection, cells were challenged with excysted C. parvum sporozoites. DEFB1 protein induction was quantified by ELISA, while parasite burden and cell viability were assessed via microscopy, RT-qPCR, and MTT assays. Our results show that transfecting HCT-8 cells with synthetic mRNA results in a twofold increase in DEFB1 protein expression. This induction protects cells from C. parvum infection, achieving an approximate 80% reduction in parasite burden with no detectable cytotoxicity. These findings highlight the potential of mRNA technology for developing HDTs against cryptosporidiosis and other enteric pathogens. Moreover, this study establishes a foundation for utilizing synthetic mRNA as a rapid platform to evaluate intestinal gene function during parasitic infections.