miR-375 Regulates Extracellular Vesicle Secretion From Giardia duodenalis via Targeting Rab1a.

Giardia duodenalis (G. duodenalis) represents a prevalent zoonotic protozoan pathogen responsible for diarrheal disorders across human and animal hosts. To date, no licensed effective vaccines have been developed against giardiasis, and the emergence of drug resistance progressively impairs the efficacy of conventional clinical chemotherapy. Extracellular vesicles (EVs) derived from G. duodenalis (GEVs) exert pivotal functions in mediating parasite immune evasion as well as the initiation of host inflammatory cascades; nevertheless, the molecular regulatory circuits underlying GEV biosynthesis and secretion remain poorly defined. Previous bioinformatic predictions implicate Rab1a in vesicular trafficking within G. duodenalis, whereas its concrete biological contribution to GEV secretion has not been experimentally validated. The present work was designed to screen microRNAs (miRNAs) targeting Rab1a and further elucidate their modulatory effects on GEVs biogenesis. GEVs were purified from G. duodenalis WB-strain trophozoites, and their identity was verified via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA, modal diameter: 164.7 nm), plus Western blot detection of classic extracellular vesicle biomarker proteins. High-throughput miRNA sequencing was subsequently conducted to filter candidate miRNAs capable of interacting with the Rab1a 3' untranslated region (3'UTR), from which miR-375, miR-133, and miR-999 were shortlisted as prospective regulatory molecules. Functional verification revealed that only miR-375 robustly repressed Rab1a expression at both mRNA and protein abundances. Dual-luciferase reporter assays further authenticated the direct physical interaction between miR-375 and the Rab1a 3'UTR. Consistently, ectopic overexpression of miR-375 resulted in a prominent decline in cellular GEV secretion. In summary, our experimental data corroborate that endogenous G. duodenalis miR-375 suppresses Rab1a expression at the post-transcriptional level to constrain downstream GEV release. The newly characterized miR-375-Rab1a-GEVs regulatory axis deepens our mechanistic understanding of post-transcriptional modulation governing vesicle formation in G. duodenalis.