Phosphatidylcholine biosynthesis pathways in Cryptococcus neoformans: functional interplay and impact on virulence

As fungal diseases emerge, new studies aim to understand how different metabolic pathways, including the biosynthesis of phospholipids, influence the fungal pathogenicity. Therefore, to investigate the role of phosphatidylcholine (PC) in the biology of the human fungal pathogen Cryptococcus neoformans, a double mutant lacking OPI3 (phosphatidylethanolamine methyltransferase) from the de novo pathway, and PCT1 (choline phosphate cytidylyltransferase) from the salvage Kennedy pathway was generated using double-joint PCR coupled with biolistic technique for gene deletion. Phenotypic and virulence assays were performed, including growth viability in minimal nutrient, melanization, capsule expansion and titanization, lipid droplet analysis and in vivo infection in larval and murine models. The double mutant (opi3Δpct1Δ) exhibited normal growth in complex medium, but displayed severe growth defects and loss of viability under nutrient-limited conditions. Supplementation with L-α-glycerophosphorylcholine (GPC), PC or sorbitol fully restores growth, suggesting compensation of GPC-dependent reacylation pathway. Disruption of PC biosynthesis affected important virulence traits, including capsule formation, melanization, and titan cell development, and increased susceptibility to membrane stresses. In vivo, in both the Galleria mellonella and murine models, opi3Δpct1Δ was hypovirulent with reduced brain colonization. Other studies with C. neoformans and Candida albicans, another pathogenic yeast, showed no impact in deletion of either OPI3 or PCT1 alone for virulence and pathogenicity. Therefore, these findings highlight the critical role of PC biosynthesis for maintaining membrane integrity, morphological plasticity and host dissemination of C. neoformans.