Guanggan Hu

University of British Columbia - Vancouver | UBC

Fungal pathogens cause life-threatening diseases in humans, and the increasing prevalence of these diseases emphasizes the need for new targets for therapeutic intervention. Nutrient acquisition during infection is a promising target, and recent studies highlight the contributions of endomembrane trafficking, mitochondria, and vacuoles in the sensi...

DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones.

The pathogenic fungus Cryptococcus neoformans must overcome iron limitation to cause disease in mammalian hosts. Previously, we reported a screen for insertion mutants with poor growth on heme as the sole iron source. In this study, we characterized one such mutant and found that the defective gene encoded a Vam6/Vps39/TRAP1 domain-containing prote...

The capacity of opportunistic fungal pathogens such as Cryptococcus neoformans to cause disease is dependent on their ability to overcome an onslaught of stresses including elevated temperature under mammalian host conditions. Protein chaperones and co-chaperones play key roles in thermotolerance. In this study, we characterized the role of the end...

Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fun...

Cryptococcus neoformans is the causative agent of cryptococcal meningitis, a disease responsible for ∼15% of all HIV-related deaths. Unfortunately, development of antifungal drugs is challenging because potential targets are conserved between humans and C. neoformans . In this context, we characterized a unique J-domain protein, Mrj1, which lacks o...

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this st...

Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans , causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on c...

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source but the underlying mechanisms...

The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic traffick...

Restoration of vps45 mutant phenotypes by the fusion protein Vps45-GFP expressed from the safe haven location. The WT, mutants and complemented strains (VPS45 or VPS45-GFP) were grown on YPD in the presence of 10μg mL-1 fluconazole, 25μM aminodarone and 6mM chloroquine. Cells were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were...

Growth and survival of WT, vps45 mutants and complemented strains in defined low iron media (33). Cells were pre-grown in defined LIM for 48h at 30°C, washed and counted. 5X105 cells/mL were inoculated into LIM (A and C), LIM + 100 μM FeCl3 (B and D) and incubated at 30°C (A and B) or 37°C (C and D) for 48h. (TIF)

A) Localization of Cfo1-GFP in the absence of VPS45 at 37°C. Strains (WT, vps45Δ and complement) containing a CFO1-GFP construct were cultured overnight in YPD, washed 3 times and counted. 1X106 cells/mL were inoculated in YNB-BPS and incubated at 37°C for 1h. Then cells were stained with 5 μM FM4-64, transferred in a chamber slide and maintained a...

Influence of Vps45 on mitochondrial function, ROS sensitivity and mitochondrial membrane potential. A) Mean R values of positive correlation between VPS45-GFP and mitochondria of cells grown under different conditions. Colocalization analyses were performed on images of whole cells (15–45 cells) using the ImageJ coloc2 test. Positive correlations w...

Primers used in this study. (PDF)

Southern blot of genomic DNA for the WT strain H99, vps45 mutants and complemented strains. Extracted DNA was digested with BglI and genomic hybridization to detect the VPS45 locus yielded fragments of 4329 bp in the WT strain, 1582 bp in the deletion mutants, and 4478 bp and 1582 bp in complemented strains. (TIF)

Intracellular membrane staining with the lipophilic dye FM4-64. WT, vps45 mutant, complemented (A), and GFP-tagged (B) strains were grown for 24h in YNB-BPS at 37°C. 1X106 cells/mL were inoculated in YNB-BPS + 100μM FeCl3, stained with 5 μM FM4-64 and transferred in a chamber slide where the cells were maintained at 37°C. Confocal microscopy images...

The process of autophagy is conserved among all eukaryotes from yeast to humans, and is mainly responsible for bulk degradation of cellular contents and nutrient recycling during starvation. Autophagy has been suggested to play a role in the pathogenesis of the opportunistic human fungal pathogen Cryptococcus neoformans, potentially through a contr...

The acquisition of iron and the maintenance of iron homeostasis are important aspects of the virulence in the pathogenic fungus Cryptococcus neoformans. In this study, we identified the monothiol glutaredoxin Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans. Monothiol g...

The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans , employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic traffic...

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life-threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source but the underlying mechanisms...

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular z...

The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of...

Pathogenic fungi kill an estimated 1.3 million people each year. This number is predicted to rise as drug resistance spreads, thus antifungal drugs with novel modes of action are urgently required. Fungal endoglycoceramidase-related proteins 1 and 2 (EGCrP-1 and -2), which hydrolyse glucosylceramide and ergosteryl beta-glucoside, respectively, are...

The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisi...

Zinc is an essential element in living organisms and a cofactor for various metalloproteins. To disseminate and survive, a pathogenic microbe must obtain zinc from the host, which is an environment with extremely limited zinc availability. In this study, we investigated the roles of the ZIP family zinc transporters Zip1 and Zip2 in the human pathog...

The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and tricarboxylic acid (TCA) cycle components under lowiron conditions. The orthologous Hap2/3/4/5 com...

Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in...

Amino acid biosynthetic pathways that are absent in mammals are considered an attractive target for antifungal therapy. Leucine biosynthesis is one such target pathway, consisting of a five-step conversion process starting from the valine precursor 2-keto-isovalerate. Isopropylmalate dehydrogenase (Leu1) is an Fe-S cluster protein that is required...

Transition metals such as iron, zinc, and copper play key roles in numerous biological processes, including innate immunity in vertebrates. In fact, control of the availability of these essential metals is an important component of the immune response to limit the growth of pathogens during infection. In turn, pathogens must overcome the extreme li...

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesi...

Iron acquisition is critical for the ability of the pathogenic yeast Cryptococcus neoformans to cause disease in vertebrate hosts. In particular, iron overload exacerbates cryptococcal disease in an animal model, defects in iron acquisition attenuate virulence, and iron availability influences the expression of major virulence factors. C. neoforman...

Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulen...

Vertebrate hosts actively sequester iron, and fungal and other pathogens must therefore adapt to a severe limitation in iron availability to cause disease. Recent studies reveal that the pathogenic fungus Cryptococcus neoformans overcomes iron limitation by multiple mechanisms that target transferrin and heme. The regulation of iron uptake is media...

Iron acquisition is critical for virulence of the human pathogenic fungus Cryptococcus neoformans. The cryptococcal transcript for the extracellular mannoprotein Cig1 is highly regulated by iron and abundant in iron-starved cells, suggesting a role in iron acquisition. Indeed, loss of Cig1 resulted in delayed growth on heme at physiological pH. Exp...

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that...

The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and othe...

The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal sus...

The basidiomycete fungus Cryptococcus neoformans infects humans via inhalation of desiccated yeast cells or spores from the environment. In the absence of effective immune containment, the initial pulmonary infection often spreads to the central nervous system to result in meningoencephalitis. The fungus must therefore make the transition from the...

CGH analysis of 18 clinical and environmental strains (Figure S5). The strains were analyzed along with strains A5-35-17 and JP1086 (Figure 8) using the array for the reference strain H99.

Primer list for strain construction (Table S4). The primers employed to insert a selectable marker on chr 13 of strain H99 are listed in Table S4.

Analysis of three HC-6 isolates by fluorescence-activated flow cytometry (Figure S7). The ploidy of the three isolates from patient HC-6 were examined and the haploid strain H99 and the diploid strain KW5 were included as controls.

Genes up-regulated in the black strain B4 (Table S1). A microarray comparison of the transcriptomes of the black B4 isolate and the white W2 isolate identified genes with greater than two-fold elevated transcript levels in the B4 isolate.

Genes up-regulated in the white strain W2 (Table S2). A microarray comparison of the transcriptomes of the black B4 isolate and the white W2 isolate identified genes with greater than two-fold elevated transcript levels in the W2 isolate.

CGH analysis of additional strains obtained from mice (Figure S3). Passage of the CBS7779 variants W2, W3 and B6 in mice lead to variation at chr 4 and chr 13. As described in the text and presented in Figure 6, white and black strains collected from the lungs and brains of mice infected with CBS7779 variants were compared to the reference genome o...

Phenotypic characterization of variants of the laboratory strain H99 (Figure S4). (A) The screen for variants with reduced melanin production is shown for tagged strains on L-DOPA medium containing neomycin. This procedure yielded the strains analyzed in Figure 7. (B) The capsule size and cell morphology of the variants were examined with india ink...

CGH analysis of isolates from HIV/AIDS patients (Figure S6). Three isolated colonies were tested for patients HC-2, HC-3 and HC-5, and a representative colony is shown for the three colonies from patient HC-6. The analysis of another HC-6 colony is shown in Figure 8 (all three colonies had the same chromosome complement).

Comparison of gene expression for a second-generation black strain (Figure S1). Gene expression was compared for the CBS7779-B4 strain and the "second generation" black strain CBS7779-W2BA by microarray analysis. Strain CBS7779-W2BA was obtained from the white strain CBS7779-W2 (Figure 1). Two arrays were employed and two biological repeats were pe...

CGH analysis of additional strains with chromosome copy number variation (Figure S2). Passage of the white variant CBS7779-W2 (disomic for chr 13) in culture and analysis of black or white isolates by CGH revealed changes at additional chromosomes. The black variant W2-BB showed copy number increase for a segment of chr 12, and the white isolate W2...

Strain list (Table S3). The strains employed in the study are listed in Table S3.

Primer list for qPCR (Table S5). The primers employed for quantitative real time PCR to confirm the copy number of chromosomes 4 and 13 are listed in Table S5.

The adaptation of pathogenic fungi to the host environment via large-scale genomic changes is a poorly characterized phenomenon. Cryptococcus neoformans is the leading cause of fungal meningoencephalitis in HIV/AIDS patients, and we recently discovered clinical strains of the fungus that are disomic for chromosome 13. Here, we examined the genome p...

The basidiomycete fungus Cryptococcus neoformans is an important pathogen of immunocompromised people. The ability of the fungus to sense its environment is critical for proliferation and the generation of infectious propagules, as well as for adaptation to the mammalian host during infection. The conserved cAMP/protein kinase A pathway makes an im...

Growth rate measurement for the ubp5Δ mutant. WT, ubp5Δ and ubp5Δ+UBP5 strains were grown overnight at 30°C in liquid YPD medium. Cell numbers were counted by hemacytometer. 106 CFU from each culture were transferred to 30 ml fresh YPD medium in flasks and incubated at 30°C. OD600 was measured for each culture at four hour intervals. Mutant ubp5Δ s...

UBP5 deletion lead to increased expression of UBI4 in C. neoformans. Expression of polyubiquitin gene UBI4 at (A) 30°C and (B) 37°C. UBI4 was up-regulated in ubp5Δ mutant while the reconstituted strain showed similar expression level like WT strain. And high temperature exposure reinforced the up-regulation of UBI4 expression in ubp5Δ strain. (TIF)

Ubiquitin-proteasome genes identified by in vivo transcription profiling. Transcription profiles of in vivo C. neoformans cells from human or rabbit infections was compared to profiles from C. neoformans cells grown ex vivo in either pooled human cerebrospinal fluid or serum to identify genes that are differentially expressed in the host. (PDF)

Urease assay. Different DUB mutants grew on Christensen’s medium at 30°C for the detection of urease production. ure1Δ mutant strain was used as a negative control. (TIF)

Primers used in this study. Primers were designed using the Primer3 software package (http://frodo.wi.mit.edu/primer3/) for generating gene-deletion cassettes, gene-reconstitution cassettes, Southern hybridization probes, or real-time PCR products. (PDF)

Cryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS...

Cryptococcus neoformans is generally considered to be an opportunistic fungal pathogen because of its tendency to infect immunocompromised individuals, particularly those infected with HIV. However, this view has been challenged by the recent discovery of specialized interactions between the fungus and its mammalian hosts, and by the emergence of t...

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representin...

Construction of hap deletion mutations and confirmation of mutant genotypes by genomic hybridization. Diagrams are presented for the WT loci for HAP3 (A), HAP5 (B) and HAPX (C) as well as the deletion alleles in which the nourseothricin resistance gene was used to replace the open reading frame of each gene. D) Genomic hybridization results with th...

List of genes and their individual p values under each gene ontology term. (0.13 MB XLS)

Growth of the hap3Δ and hapXΔ mutants in liquid low-iron media supplemented with ferric chloride, hemin or transferrin. The densities of liquid cultures for the WT and mutant strains was monitored at OD600 during incubation in low-iron medium or low-iron medium supplemented with 100 µM FeCl3, 5 µM Transferrin or 10 µM Hemin. These growth conditions...

Differentially expressed genes (at least 2-fold) with statistical significance (q value less than 0.05) in the mutants versus wild-type in response to ferric chloride. (0.17 MB XLS)