A convergent proline biosynthetic step couples cell wall integrity to virulence in Cryptococcus neoformans

Metabolic adaptation is essential for the pathogenicity of the opportunistic fungus Cryptococcus neoformans . Although amino acid biosynthesis pathways are critical fitness determinants in fungal pathogens, the physiological contributions of proline biosynthesis remain incompletely understood, with most studies focusing on proline catabolism. Here, we characterized the proline biosynthetic enzymes Pro1, Pro2, and Pro3 in C. neoformans . Proline is produced by two routes that converge on Δ 1 -pyrroline-5-carboxylate (P5C): a de novo pathway from glutamate via Pro1 and Pro2, and an arginine-derived bypass via Car1 and Car2—both requiring Pro3 for the final reduction of P5C to proline. Although all biosynthetic mutants were strictly auxotrophic for proline, their requirements for virulence differed markedly: in a murine infection model, pro1 Δ and pro2 Δ mutants retained full virulence, whereas pro3 Δ exhibited significantly attenuated virulence accompanied by growth defects and impaired cell wall integrity. Genetic disruption of P5C-generating routes partially restored distinct pro3 Δ phenotypes, with PRO2 deletion alleviating delayed growth and combined PRO2 and CAR2 deletion partially restoring cell size, while cell wall defects remained unresolved. This dissociation indicates that growth requires proper balance of P5C-generating metabolic flux, whereas cell wall integrity depends on a distinct mechanism that is not resolved by blocking P5C production. Together, these findings identify Pro3 as a metabolic gatekeeper coupling proline biosynthesis to cell wall homeostasis, establishing the biosynthetic branch of proline metabolism as a previously unappreciated determinant of fungal pathobiology.