Cryptococcus neoformans trehalose-6-phosphate synthase (tps1) promotes organ-specific virulence and fungal protection against multiple lines of host defenses.

Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.

Phenotypic characterization of HAM1, a novel mating regulator of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen responsible for >200,000 yearly cases with a mortality as high as 81%. This burden results, in part, from an incomplete understanding of its pathogenesis and ineffective antifungal treatments; hence, there is a pressing need to understand the biology and host interactions of this yeast to develop improved treatments. Protein palmitoylation is important for cryptococcal virulence, and we previously identified the substrates of its main palmitoyl transferase. One of them was encoded by the uncharacterized gene CNAG_02129. In the filamentous fungus Neurospora crassa, a homolog of this gene named hyphal anastomosis protein 13 plays a role in proper cellular communication and filament fusion. In Cryptococcus, cellular communication is essential during mating; therefore, we hypothesized that CNAG_02129, which we named hyphal anastomosis protein 1 (HAM1), may play a role in mating. We found that ham1Δ mutants produce more fusion products during mating, filament more robustly, and exhibit competitive fitness defects under mating and non-mating conditions. Additionally, we found several differences with the major virulence factor, the polysaccharide capsule, that may affect virulence, consistent with prior studies linking virulence to mating. We observed that ham1Δ mutants have decreased capsule attachment and transfer but exhibit higher amounts of exopolysaccharide shedding and biofilm production. Finally, HAM1 expression is significantly lower in mating media relative to non-mating conditions, consistent with it acting as a negative regulator of mating. Understanding the connection between mating and virulence in C. neoformans may open new avenues of investigation into ways to improve the treatment of this disease. IMPORTANCE: Fungal mating is a vital part of the lifecycle of the pathogenic yeast Cryptococcus neoformans. More than just ensuring the propagation of the species, mating allows for sexual reproduction to occur and generates genetic diversity as well as infectious propagules that can invade mammalian hosts. Despite its importance in the biology of this pathogen, we still do not know all of the major players regulating the mating process and if they are involved or impact its pathogenesis. Here, we identified a novel negative regulator of mating that also affects certain cellular characteristics known to be important for virulence. This gene, which we call HAM1, is widely conserved across the cryptococcal family as well as in many pathogenic fungal species. This study will open new avenues of exploration regarding the function of uncharacterized but conserved genes in a variety of pathogenic fungal species and specifically in serotype A of C. neoformans.

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP3-4 kinase activity.

Inositol tris/tetrakis phosphate kinases (IP3-4K) in the human fungal priority pathogens, Cryptococcus neoformans (CnArg1) and Candida albicans (CaIpk2), convey numerous virulence functions, yet it is not known whether the IP3-4K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) CnArg1 strain (dkArg1). We verified that, although dkARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP3-4K activity. Using recombinant CnArg1 and CaIpk2, we confirmed that, unlike the IP3-4K homologs in humans and Saccharomyces cerevisiae, CnArg1 and CaIpk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP3 conversion is blocked in the dkArg1 and ARG1 deletion (Cnarg1Δ) strains and that 1-IP7 and a recently discovered isomer (4/6-IP7) are made by wild-type C. neoformans. Importantly, the dkArg1 and Cnarg1Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP3-4K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since CnArg1 is dispensable for growth on different nitrogen sources. IP3-4K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP3-4K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans, and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP3-4K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP3-4K catalytic activity of CnArg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion, reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP3-4K catalytic site, as a new antifungal drug class.

Aspartyl peptidase May1 induces host inflammatory response by altering cell wall composition in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcal meningoencephalitis, a disease that kills more than 180,000 people annually. Contributing to its success as a fungal pathogen is its cell wall surrounded by a capsule. When the cryptococcal cell wall is compromised, exposed pathogen-associated molecular pattern molecules (PAMPs) could trigger host recognition and initiate attack against this fungus. Thus, cell wall composition and structure are tightly regulated. The cryptococcal cell wall is unusual in that chitosan, the acetylated form of chitin, is predominant over chitin and is essential for virulence. Recently, it was shown that acidic pH weakens the cell wall and increases exposure of PAMPs partly due to decreased chitosan levels. However, the molecular mechanism responsible for the cell wall remodeling in acidic pH is unknown. In this study, by screening for genes involved in cryptococcal tolerance to high levels of CO2, we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO2 due to acidification of unbuffered media. Overexpression of MAY1 increases the cryptococcal cell size and elevates PAMP exposure, causing a hyper-inflammatory response in the host while MAY1 deletion does the opposite. We discovered that May1 weakens the cell wall and reduces the chitosan level, partly due to its involvement in the degradation of Chs3, the sole chitin synthase that supplies chitin to be converted to chitosan. Consistently, overexpression of CHS3 largely rescues the phenotype of MAY1oe in acidic media. Collectively, we demonstrate that May1 remodels the cryptococcal cell wall in acidic pH by reducing chitosan levels through its influence on Chs3. IMPORTANCE: The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

A Galleria Model of Cryptococcus neoformans Infection.

Galleria mellonella larvae are a popular and simple model organism for infectious disease research. Last instar larvae can be purchased inexpensively from commercial suppliers and infected with Cryptococcus. Injection into the proleg of larvae results in systemic infections. Larvae may then be monitored for survival or homogenized to determine fungal burden. Fixation of infected larvae produces samples suitable for histological staining and analysis.

Proteomic Profiling of Samples Derived from a Murine Model Following Cryptococcus neoformans Infection.

Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.

Models for Inducing Experimental Cryptococcosis in Mice.

Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.

Measuring Urease and Phospholipase Secretion in Cryptococcus neoformans.

Urease and phospholipase are enzymes that are important virulence factors for Cryptococcus neoformans. These are two of the most studied enzymes involved in how C. neoformans breaches the blood-brain barrier. Additionally, phospholipase secretion also supports dissemination from the lungs. This chapter describes the methods used to measure the secretion of these enzymes, which may be used to characterize strain invasiveness and virulence.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans.IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Regulatory basis for reproductive flexibility in a meningitis-causing fungal pathogen.

Pathogenic fungi of the genus Cryptococcus can undergo two sexual cycles, involving either bisexual diploidization (after fusion of haploid cells of different mating type) or unisexual diploidization (by autodiploidization of a single cell). Here, we construct a gene-deletion library for 111 transcription factor genes in Cryptococcus deneoformans, and explore the roles of these regulatory networks in the two reproductive modes. We show that transcription factors crucial for bisexual syngamy induce the expression of known mating determinants as well as other conserved genes of unknown function. Deletion of one of these genes, which we term FMP1, leads to defects in bisexual reproduction in C. deneoformans, its sister species Cryptococcus neoformans, and the ascomycete Neurospora crassa. Furthermore, we show that a recently evolved regulatory cascade mediates pre-meiotic unisexual autodiploidization, supporting that this reproductive process is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern hallmark events distinguishing unisexual and bisexual reproduction in Cryptococcus.

Unraveling the Pathobiological Role of the Fungal KEOPS Complex in Cryptococcus neoformans.

The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target. IMPORTANCE The cellular function and structural configuration of the KEOPS complex have been elucidated in some eukaryotes and archaea but have never been fully characterized in fungal pathogens. Here, we comprehensively analyzed the pathobiological roles of the KEOPS complex in the globally prevalent fungal meningitis-causing pathogen C. neoformans. The CnKEOPS complex, composed of a linear arrangement of Pcc1-Kae1-Bud32-Cgi121, not only played evolutionarily conserved roles in growth, sexual development, stress responses, and tRNA modification but also had unique roles in controlling virulence factor production and pathogenicity. Notably, a unique extended loop structure in CnBud32 is critical for the KEOPS complex in C. neoformans. Supporting its pleiotropic roles, transcriptome analysis revealed that the CnKEOPS complex governs several hundreds of genes involved in carbon and amino acid metabolism, pheromone response, and ergosterol biosynthesis. Therefore, this study provides novel insights into the fungal KEOPS complex that could be exploited as a potential antifungal drug target.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Caenorhabditis elegans DAF-16 regulates lifespan and immune responses to Cryptococcus neoformans and Cryptococcus gattii infections.

BACKGROUND: Cryptococcosis is a life-threatening infection is primarily caused by two sibling species Cryptococcus neoformans and Cryptococcus gattii. Several virulence-related factors of these cryptococci have been widely investigated in Caenorhabditis elegans, representing a facile in vivo model of host-pathogen interaction. While recent studies elucidated cryptococcal virulence factors, intrinsic host factors that affect susceptibility to infections by cryptococci remain unclear and poorly investigated. RESULTS: Here, we showed that defects in C. elegans insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) pathway influenced animal lifespan and mechanisms of host resistance in cryptococcal infections, which required the activation of aging regulator DAF-16/Forkhead box O transcription factor. Moreover, accumulation of lipofuscin, DAF-16 nuclear localization, and expression of superoxide dismutase (SOD-3) were elevated in C. elegans due to host defenses during cryptococcal infections. CONCLUSION: The present study demonstrated the relationship between longevity and immunity, which may provide a possibility for novel therapeutic intervention to improve host resistance against cryptococcal infections.

Role of the anillin-like protein in growth of Cryptococcus neoformans at human host temperature.

Cryptococcus neoformans, a basidiomycete yeast, causes lethal meningitis in immunocompromised individuals. The ability of C. neoformans to proliferate at 37°C is essential for virulence. We identified anillin-like protein, CnBud4, as essential for proliferation of C. neoformans at 37°C and for virulence in a heterologous host Galleria mellonella at 25°C. C. neoformans cells lacking CnBud4 were inviable at 25°C in the absence of active calcineurin and were hypersensitive to membrane stress and an anti-fungal agent fluconazole, phenotypes previously described for C. neoformans mutants lacking septins. CnBud4 localized to the mother-bud neck during cytokinesis in a septin-dependent manner. In the absence of CnBud4, septin complex failed to transition from a collar-like single ring to the double ring during cytokinesis. In an ascomycete yeast, Saccharomyces cerevisiae, the anillin-like homologue ScBud4 participates in the organization of the septin ring at the mother-bud neck and plays an important role in specifying location for new bud emergence, known as axial budding pattern. In contrast to their role in S. cerevisiae, neither septins nor CnBud4 were needed to direct the position of the new bud in C. neoformans, suggesting that this function is not conserved in basidiomycetous yeasts. Our data suggest that the requirement of CnBud4 for growth at 37°C and pathogenicity in C. neoformans is based on its conserved role in septin complex organization.

Proteome and secretome profiling of zinc availability in Cryptococcus neoformans identifies Wos2 as a subtle influencer of fungal virulence determinants.

BACKGROUND: Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. RESULTS: Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. CONCLUSIONS: Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

Inositol Metabolism Regulates Capsule Structure and Virulence in the Human Pathogen Cryptococcus neoformans.

The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen. IMPORTANCE The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. Our recent studies revealed that inositol utilization by the fungus promotes C. neoformans development in nature and invasion of the central nervous system during infection. The mechanisms of the inositol regulation in fungal virulence remain incompletely understood. In this study, we found that C. neoformans has three genes encoding myo-inositol oxygenase, a key enzyme in the inositol catabolic pathway. Expression of these genes is highly induced by inositol, and they are highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. Our mutagenesis analysis indeed demonstrates that inositol catabolism is required for C. neoformans virulence by altering the growth and structure of polysaccharide capsule, a major virulence factor. Considering the abundance of free inositol and inositol-related metabolites in the brain, our study reveals an important mechanism of host inositol-mediated fungal pathogenesis for this neurotropic fungal pathogen.