Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.

Understanding how intracellular organelles are organized and dynamically remodeled is essential for elucidating fungal growth, differentiation, and pathogenicity. The human fungal pathogen <i>Cryptococcus neoformans</i> exhibits remarkable morphological diversity, yet the contribution of intracellular organelles to adaptation across environmental and host-relevant conditions has not been systematically examined. Here, we establish a comprehensive toolkit of fluorescent organelle marker plasmids and strains expressing mCherry or GFP fusions, enabling high-resolution live-cell imaging of ten major subcellular compartments: the nucleolus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisome, endosome, autophagosome, vacuole, the plasma membrane, and processing bodies (P-bodies). With this platform, we analyzed organelle organization under host-relevant conditions, including elevated temperature, 5% CO ₂ , and capsule- and melanin-inducing media, as well as throughout sexual development from zygote formation to basidiospore production. We further show that the toolkit supports colocalization analyses via diploid formation or dual-labeling strategies. Collectively, our results reveal condition- and stage-specific remodeling of organelle architecture during vegetative growth and mating. This imaging platform provides a robust framework for investigating how subcellular organization supports fungal adaptation, development, and virulence, and for inferring the functions of uncharacterized genes from their spatial localization.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved PP2A-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen <i>Cryptococcus neoformans</i> by combining genetic, genomic, virulence, and systems-level phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via <i>PPH22, FAR8, FAR9</i> , or <i>FAR11</i> mutations caused severe defects in growth, stress adaptation, cell-cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, <i>far11Δ</i> strains were avirulent, whereas <i>far9Δ</i> mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with all recovered isolates acquiring chromosome 11 tetraploidy despite no consistent in vitro fitness advantage. In striking contrast, deletion of <i>MOB3</i> produced a hypervirulent phenotype. <i>mob3Δ</i> cells exhibited enhanced transmigration across an <i>in vitro</i> blood-brain barrier model, increased survival in macrophages, and generated abundant small-cell morphotypes <i>in vitro</i> and <i>in vivo</i> , features associated with increased dissemination. Global phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal and cell-cycle control, chromatin and transcriptional regulation, RNA metabolism, and stress responses. By contrast, <i>mob3Δ</i> mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a central coordinator of genome stability, morphological plasticity, and virulence in <i>C. neoformans</i> , and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions.

A conserved N -glycan-dependent endoplasmic reticulum protein quality control (ERQC) system has evolved in eukaryotes to ensure accuracy during glycoprotein folding. The human pathogen Cryptococcus neoformans possesses a unique N -glycosylation pathway that affects microbial physiology and interactions with the infected host. To investigate the molecular features and functions of the ERQC system in C. 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 , and MNL2 ). The ugg1 Δ, mns1 Δ, mns101 Δ, and mns1 Δ 101 Δ mutants showed alterations in N -glycan profiles, defective cell surface organization, decreased survival in host cells, and varying degrees of reduced in vivo virulence. The ugg1 Δ 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 endoplasmic reticulum 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 in ugg1 Δ. 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 of C. neoformans .