True cyst formation underlies persistence and drug tolerance in Tritrichomonas foetus.

Tritrichomonas foetus, a protozoan parasite responsible for bovine and feline trichomonosis, has traditionally been considered to form only pseudocysts. Here, we demonstrate that T. foetus produces true cysts characterized by a chitin-like, fibrillar wall, cytoskeletal reorganization, and resistance to detergent treatment. Using scanning and transmission electron microscopy, sarkosyl resistance assays, lectin and chitin-binding staining, and monosaccharide composition analysis, we identify a polysaccharide-rich cyst wall enriched in N-acetylated hexosamines. Cystogenesis is induced by environmental stresses, including nutrient deprivation, alkaline pH mimicking the bovine preputial environment, and metronidazole exposure. Flow cytometry analyses reveal increased DNA content and multinucleation in cysts, consistent with endoreplication. Encysted parasites remain viable and revert to proliferative trophozoites upon restoration of favorable conditions. Pharmacological inhibition of cyst wall biosynthesis enhances metronidazole susceptibility, supporting a functional role for cyst formation in drug tolerance. Transcriptomic profiling identifies pathways associated with carbohydrate metabolism, Myb-like transcription factors, and calcium/calmodulin-dependent kinases during encystation. Together, our findings establish true cyst formation in T. foetus and define a previously unrecognized survival state linked to environmental persistence and reduced drug susceptibility.