Presence of UDP-glucose:glycoprotein glucosyltransferase (UGGT) and α1,2-mannosidases as endoplasmic reticulum protein quality control (ERQC) components in Cryptococcus neoformans.

(A) Schematic representation of the ERQC pathway in Homo sapiens, Saccharomyces cerevisiae, and Cryptococcus neoformans. In mammals, the oligosaccharyltransferase (OST) complex attaches Glc3Man9GlcNAc2 to nascent polypeptides, followed by glucose trimming by glucosidases I and II (GLS1/GLS2). This generates Glc1Man9GlcNAc2, which binds to calnexin (CNX) or calreticulin (CRT) for folding. UGGT reglucosylates misfolded proteins, allowing refolding, while properly folded proteins undergo mannose trimming by ERManI before Golgi transport. In contrast, fungal ERQC systems differ in key components. S. cerevisiae lacks UGGT, relying instead on Gls1/Gls2 and calnexin (Cne1). C. neoformans possesses UGGT but lacks ER glucosyltransferases (Alg6, Alg8 and Alg19) and CRT, resulting in a distinct ERQC system. Domain structures of proteins encoded by C. neoformans UGG1 (CNAG_03648), MNS1 (CNAG_02081), MNS101 (CNAG_03240), MNL1 (CNAG_01987), and MNL2 (CNAG_04498).

N-glycan profile analysis of C. neoformans ERQC mutant strains.

The HPLC and MALDI-TOF-based N-glycan structure analysis were carried out as described in Supplementary information. (A) HPLC-based analysis of N-glycan profiles of the ugg1Δ mutant. (B) Lectin blotting of sodium dodecyl sulphate (SDS)-polyacrylamide gels containing intracellular or secreted proteins into the culture supernatants of the wild type (WT) and ugg1Δ strains. Yeast cells were cultivated in YPD medium for 24 h, harvested, and subjected to sample preparation of soluble intracellular proteins and secreted proteins. The proteins (30 μg) were loaded on 15% SDS-polyacrylamide gel and analyzed using silver staining (left) or blotting (right) with Galanthus nivalis agglutinin conjugated to horseradish peroxidase (GNA-HRP, Roche). (C) HPLC analysis of total N-glycan profiles of mns1Δ, mns101Δ, and mns1Δ101Δ mutants. (D) MALDI-TOF profiles of neutral N-glycans of mns1Δ, mns101Δ, and mns1Δ101Δ mutants. The N-glycans of cell wall mannoproteins from C. neoformans cells were AA-labelled and analyzed using HPLC. For MALDI-TOF analysis, neutral N-glycan fractions were obtained from the HPLC fractionation of total N-glycans.

Growth phenotype of C. neoformans ERQC mutant strains.

(A) Expression analysis of ERQC genes in C. neoformans. Yeast cells were cultured in YPD medium to a mid-logarithmic phase and exposed to dithiothreitol (DTT; 20 mM), tunicamycin (TM; 5 µg/ml) or cultured at 37 °C for 1 h. The relative transcript levels of C. neoformans genes were analyzed using qRT-PCR and normalized with that of ACT1. Error bars represent standard deviation of duplicated assays. All statistical data were determined based on one-way ANOVA and Dunnett’s post-hoc test. *** P < 0.0005, ** P < 0.003, *** P < 0.005, * P < 0.05. (B) Spotting analysis of C. neoformans ugg1Δ mutant strains under various stress conditions such as heat stress (37 °C and 39 °C), ER stress (DTT and TM), cell-wall stress (CFW: calcofluor white, CR: Congo red, SDS: Sodium dodecyl sulfate, caffeine), osmotic stress (NaCl, KCl, sorbitol) and treatment with antifungal drugs (fluconazole, ketoconazole, fludioxonil). (C) Growth analysis in the presence of 5’,5’,5’-trifluoroleucine (TFL). Respective strains were spotted on SC-Leucine media with or without TFL supplementation. Plates were incubated for 3 days at 30 °C. (D) RT-PCR analysis of IRE1-dependent splicing of HXL1. Strains were cultured in YPD supplemented with 5 µg/ml TM.

In vitro and in vivo virulence-associated phenotypes of C. neoformans UGG1, MNS1, and MNS101 mutant strains.

(A) Melanin synthesis analysis on L-DOPA plates. WT, ugg1Δ, ugg1Δ::UGG1, mns1Δ, mns101Δ, mns1Δ101Δ, mns1Δ101Δ::MNS1, mns1Δ101Δ::MNS101, and cac1Δ (negative control) strains were serially diluted, plated on L-DOPA plates, and incubated at 30 °C and 37 °C. (B) Melanin synthesis activity per cell of the ugg1Δ mutant. WT, ugg1Δ, ugg1Δ::UGG1 were cultured in liquid L-DOPA medium. The amount of melanin in the culture supernatant was measured and normalized by cell density. Capsule formation. Cells were cultured for 2 days in 10% Sabouraud media at 30 °C and observed under the microscope. Statistical significance: ****, P < 0.0001, ns, not significant). In vivo virulence analysis. A/Jcr mice (n=8) were infected with 105 cells of WT, ugg1Δ, and ugg1Δ::UGG1, mns1Δ, mns101Δ, mns1Δ101Δ, mns1Δ101Δ::MNS1, and mns1Δ101Δ::MNS101 strains, and survival was monitored for 2 months, as described in the Supplementary Information. (E) Survival of C. neoformans in macrophages. Survival of C. neoformans cells within the J774A.1 macrophage-like cell line was determined by counting colony formation unit (CFU) obtained from lysed macrophages from two biologically independent experiment sets. **** P < 0.0001, *** P < 0.0005, * P < 0.05, ns, not significant. All statistical data were determined based on one-way ANOVA and Dunnett’s post-hoc test.

Capsule shedding and transfer analysis of C. neoformans UGG1, MNS1, and MNS101 mutant strains.

(A) Capsule shedding analysis. The presence of intracellular (left) and shed (right) glucuronoxylomannan (GXM) was assessed by blotting a cell culture filtrate using the monoclonal antibody 18B7. The arrow indicates the direction of electrophoresis. (B) Capsule transfer analysis using exogenous capsule material from WT. The capsule transfer assay was performed using the indicated strains as acceptors. Surface capsules were probed using the anti-GXM antibody 18B7 conjugated with AlexaFluor 488. Quantitative measurement of fluorescence intensity was calculated based on independent triplicate experiments with standard deviations presented as error bars. (C) Capsule transfer analysis using exogenous capsule material from WT or mns1Δ101Δ. Statistical significance: **** P < 0.0001. All statistical data were determined based on one-way ANOVA and Dunnett’s post-hoc test. (D) Transmission electron microscopy (TEM) of C. neoformans WT, ugg1Δ, and mns1Δ101Δ strains. Yeast cells were grown overnight at 30 °C in YPD medium and fixed in 2% glutaraldehyde and 2% paraformaldehyde. A Zeiss Axioscope (A1) equipped with an AxioCan MRm digital camera was used to visualize India ink-stained C. neoformans cells. Specimens were prepared using critical point drying prior to TEM microscopy. Capsule and yeast cell body diameters were measured using ImageJ (National Institute of Health).

Transcriptome analysis of C. neoformans WT and ugg1Δ cells.

(A) Volcano plot comparing a 2-fold differential gene expression between ugg1Δ and WT strains under normal growth conditions. (B) Number of genes upregulated and downregulated by ≥ 2-fold in ugg1Δ compared with that of the WT. (C) Gene ontology (GO) analysis of differentially expressed genes between WT and ugg1Δ strains. Significantly upregulated genes in ugg1Δ are shown in red, whereas significantly downregulated genes in ugg1Δ are shown in blue. Total RNA was extracted and subjected to RNAseq analysis as described in the Supplementary Information. (D) qRT-PCR analysis of mRNA expression levels of a set of genes responsible for capsule biosynthesis, cell wall remodeling, and both conventional and non-conventional secretion in ugg1Δ vs WT under normal growth conditions from three biologically independent experiment sets.

Analysis of protein secretion in C. neoformans UGG1, MNS1, and MNS101 mutant strains.

(A) Spot assay for urease analysis on Christensen’s urea agar. Absence of pink coloration indicates loss of urease activity. (B, C, D) Analysis of secretion for virulence-related enzymes such as urease, laccase, and acid phosphatase from three biologically independent experiment sets. Statistical significance: **** P < 0.0001, ** P < 0.003, *** P < 0.005, * P < 0.05. All statistical data were determined based on one-way ANOVA and Dunnett’s post-hoc test. (E, F) Analysis of secretion for non-virulence-related enzymes such as cellulase and α-amylase. Statistical significance: *** P<0.0005, ** P<0.003 * P < 0.05, ns, not significant. All statistical data were determined based on one-way ANOVA and Dunnett’s post-hoc test. (G) Analysis of the conventional secretion of Cda1 in C. neoformans. Presence of Cda1 was analyzed in total (T), soluble (S), and insoluble (I) fractions of intracellular extracts (left), along with the secreted fraction (right). Subcellular fractionations were performed as previously described (Thak et al., 2022), and the fractions were subjected to western blotting analysis using an anti-Cda1 antibody.

Analysis of extracellular vesicles purified from WT, ugg1Δ, and cap59Δ cells.

(A, B) Nanoparticle tracking analysis (NTA) of EVs extracted from WT, ugg1Δ, and cap59Δ strains and quantification of total extracellular vesicle (EV) concentration per cell density. Quantitative measurements were derived from three independent experiments with standard deviations presented as error bars. Statistical significance: *** P<0.0005, * P< 0.05. All statistical data were determined based on one-way ANOVA and Dunnett’s post-hoc test. (C, D) Cryo-TEM imaging of purified EVs and comparative analysis of EV size in WT, ugg1Δ, and cap59Δ strains. Scale bar, 100 nm. The outer EV diameter of a total number of 100 EVs per strain, captured using cryo-TEM, were measured. (E) Heatmap representation of fold change between WT and ugg1Δ EV-associated proteins, commonly detected in this study and in previously reported EV proteome datasets (ugg1Δ/WT). Upregulated proteins in ugg1Δ are shown in red, whereas downregulated proteins are shown in blue. The proteome data of whole-cell lysates (WCL), generated from the cell pellets obtained after EV separation, were included for comparison. (F) Blotting analysis of GXM in the C. neoformans cells and EVs of WT, ugg1Δ, and cap59Δ strains. The 8 M urea extracts were obtained from EVs and cell pellets, from which EVs are generated. The urea extracts (5 μg total proteins) were loaded on 8% SDS-polyacrylamide gel and subjected to silver staining or blotting analysis using the anti-GXM 18B7 (α-GXM) and anti-Cda1 (α-Cda1) antibodies, respectively. Left: total cell extract. Right: total EV extract.

Impact of ERQC disruption on glycoprotein folding and EV-mediated transport of virulence factors in C. neoformans.

(A) In WT strain, C. neoformans UGGT homolog, Ugg1, functions as a sensor for misfolded glycoproteins within the ER, playing a crucial role in protein quality control. Functional ERQC is essential not only for ensuring the proper folding of glycoproteins, which is critical for maintaining cellular fitness, but also for facilitating EV-mediated secretion of capsule polysaccharides and virulence-related enzymes necessary for pathogenicity. (B) In the UGGT-deficient strain (ugg1Δ), ER stress is increased because of misfolded protein accumulation within the ER lumen. This heightened stress leads to decreased cellular fitness, which negatively impacts EV biogenesis and cargo loading. Consequently, significant defects occur in EV-mediated transport, which ultimately leads to a complete loss of virulence. Nc: nucleus, Vc: vacuoles. This figure was partially created using BioRender (https://BioRender.com/3gzzput, https://BioRender.com/bgibgte).