Evolutionary uniqueN-glycan-dependent protein quality control system plays pivotal roles in cellular fitness and extracellular vesicle transport inCryptococcus neoformans

A conservedN-glycan-dependent endoplasmic reticulum protein quality control (ERQC) system has evolved in eukaryotes to ensure accuracy during glycoprotein folding. The human pathogenCryptococcus neoformanspossesses a uniqueN-glycosylation pathway that affects microbial physiology and interactions with the infected host. To investigate the molecular features and functions of the ERQC system inC. neoformans,we characterized a set of mutants with deletion of genes coding for the ERQC sensor UDP-glucose:glycoprotein glucosyltransferase (UGG1) and putative α1,2-mannose-trimming enzymes (MNS1,MNS101,MNL1, andMNL2). Theugg1Δ,mns1Δ,mns101Δ, andmns1Δ101Δ mutants showed alterations inN-glycan profiles, defective cell surface organization, decreased survival in host cells, and varying degrees of reducedin vivovirulence. Theugg1Δ strain exhibited severely impaired extracellular secretion of capsular polysaccharides and virulence-related enzymes. Comparative transcriptome analysis showed the upregulation of protein folding, proteolysis, and cell wall remodeling genes, indicative of induced ER stress. However, no apparent changes were observed in the expression of genes involved in protein secretion or capsule biosynthesis. Additionally, extracellular vesicle (EV) analysis combined with proteomic analysis showed significant alterations in the number, size distribution, and cargo composition of EVs inugg1Δ. These findings highlight the essential role of the functional ERQC system for cellular fitness under adverse conditions and proper EV-mediated transport of virulence factors, which are crucial for the full fungal pathogenicity ofC. neoformans.