Loss of the putative Rab GTPase, Ypt7, impairs the virulence of Cryptococcus neoformans.

Small GTPases of the Rab family coordinate multiple membrane fusion and trafficking events in eukaryotes. In fungi, the Rab GTPase, Ypt7, plays a critical role in late endosomal trafficking, and is required for homotypic fusion events in vacuole biogenesis and inheritance. In this study, we identified a putative YPT7 homologue in Cryptococcus neoformans, a fungal pathogen causing life threatening meningoencephalitis in immunocompromised individuals. As part of an ongoing effort to understand mechanisms of iron acquisition in C. neoformans, we established a role for Ypt7 in growth on heme as the sole iron source. Deletion of YPT7 also caused abnormal vacuolar morphology, defective endocytic trafficking and autophagy, and mislocalization of Aph1, a secreted vacuolar acid phosphatase. Ypt7 localized to the vacuolar membrane and membrane contact sites between the vacuole and mitochondria (vCLAMPs), and loss of the protein impaired growth on inhibitors of the electron transport chain. Additionally, Ypt7 was required for robust growth at 39°C, a phenotype likely involving the calcineurin signaling pathway because ypt7 mutants displayed increased susceptibility to the calcineurin-specific inhibitors, FK506 and cyclosporin A; the mutants also had impaired growth in either limiting or high levels of calcium. Finally, Ypt7 was required for survival during interactions with macrophages, and ypt7 mutants were attenuated for virulence in a mouse inhalation model thus demonstrating the importance of membrane trafficking functions in cryptococcosis.

Loss of Opi3 causes a lipid imbalance that influences the virulence traits of Cryptococcus neoformans but not cryptococcosis.

The basidiomycete fungus Cryptococcus neoformans is a useful model for investigating mechanisms of fungal pathogenesis in mammalian hosts. This pathogen is the causative agent of cryptococcal meningitis in immunocompromised patients and is in the critical priority group of the World Health Organization fungal priority pathogens list. In this study, we employed a mutant lacking the OPI3 gene encoding a methylene-fatty-acyl-phospholipid synthase to characterize the role of phosphatidylcholine (PC) and lipid homeostasis in the virulence of C. neoformans. We first confirmed that OPI3 was required for growth in nutrient limiting conditions, a phenotype that could be rescued with exogenous choline and PC. Additionally, we established that loss of Opi3 and the lack of PC lead to an accumulation of neutral lipids in lipid droplets and alterations in major lipid classes. The growth defect of the opi3Δ mutant was also rescued by sorbitol and polyethylene glycol (PEG), a result consistent with protection of ER function from the stress caused by lipid imbalance. We then examined the impact of Opi3 on virulence and found that the dependence of PC synthesis on Opi3 caused reduced capsule size and this was accompanied by an increase in shed capsule polysaccharide and changes in cell wall composition. Further tests of virulence demonstrated that survival in alveolar macrophages and the ability to cause disease in mice were not impacted by loss of Opi3 despite the choline auxotrophy of the mutant in vitro. Overall, this work establishes the contribution of lipid balance to virulence factor elaboration by C. neoformans and suggests that host choline is sufficient to support proliferation during disease.

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.

The Monothiol Glutaredoxin Grx4 Influences Iron Homeostasis and Virulence in Ustilago maydis.

The corn smut fungus, Ustilago maydis, is an excellent model for studying biotrophic plant-pathogen interactions, including nutritional adaptation to the host environment. Iron acquisition during host colonization is a key aspect of microbial pathogenesis yet less is known about this process for fungal pathogens of plants. Monothiol glutaredoxins are central regulators of key cellular functions in fungi, including iron homeostasis, cell wall integrity, and redox status via interactions with transcription factors, iron-sulfur clusters, and glutathione. In this study, the roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis were investigated by constructing strains expressing a conditional allele of grx4 under the control of the arabinose-inducible, glucose-repressible promoter Pcrg1. The use of conditional expression was necessary because Grx4 appeared to be essential for U. maydis. Transcriptome and genetic analyses with strains depleted in Grx4 revealed that the protein participates in the regulation of iron acquisition functions and is necessary for the ability of U. maydis to cause disease on maize seedlings. Taken together, this study supports the growing appreciation of monothiol glutaredoxins as key regulators of virulence-related phenotypes in pathogenic fungi.

Organic acids and glucose prime late-stage fungal biotrophy in maize.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis. These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

A life-history trade-off gene with antagonistic pleiotropic effects on reproduction and survival in limiting environments.

Although life-history trade-offs are central to life-history evolution, their mechanistic basis is often unclear. Traditionally, trade-offs are understood in terms of competition for limited resources among traits within an organism, which could be mediated by signal transduction pathways at the level of cellular metabolism. Nevertheless, trade-offs are also thought to be produced as a consequence of the performance of one activity generating negative consequences for other traits, or the result of genes or pathways that simultaneously regulate two life-history traits in opposite directions (antagonistic pleiotropy), independent of resource allocation. Yet examples of genes with antagonistic effects on life-history traits are limited. This study provides direct evidence for a gene-RLS1, that is involved in increasing survival in nutrient-limiting environments at a cost to immediate reproduction in the single-celled photosynthetic alga, Chlamydomonas reinhardtii. Specifically, we show that RLS1 mutants are unable to properly suppress their reproduction in phosphate-deprived conditions. Although these mutants have an immediate reproductive advantage relative to the parental strain, their long-term survival is negatively affected. Our data suggest that RLS1 is a bona fide life-history trade-off gene that suppresses immediate reproduction and ensures survival by downregulating photosynthesis in limiting environments, as part of the general acclimation response to nutrient deprivation in photosynthetic organisms.

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen.

Histone chaperoning ensures genomic integrity during routine processes such as DNA replication and transcription as well as DNA repair upon damage. Here, we identify a nuclear J domain protein, Dnj4, in the fungal pathogen Cryptococcus neoformans and demonstrate that it interacts with histones 3 and 4, suggesting a role as a histone chaperone. In support of this idea, a dnj4Δ deletion mutant had elevated levels of DNA damage and was hypersensitive to DNA-damaging agents. The transcriptional response to DNA damage was also impaired in the dnj4Δ mutant. Genes related to DNA damage and iron homeostasis were upregulated in the wild-type strain in response to hydroxyurea treatment; however, their upregulation was either absent from or reduced in the dnj4Δ mutant. Accordingly, excess iron rescued the mutant's growth in response to DNA-damaging agents. Iron homeostasis is crucial for virulence in C. neoformans; however, Dnj4 was found to be dispensable for disease in a mouse model of cryptococcosis. Finally, we confirmed a conserved role for Dnj4 as a histone chaperone by expressing it in Saccharomyces cerevisiae and showing that it disrupted endogenous histone chaperoning. Altogether, this study highlights the importance of a JDP cochaperone in maintaining genome integrity in C. neoformans. IMPORTANCE 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. In this study, we show that a histone chaperone, Dnj4, is required for genome integrity and for the response to DNA damage. The gene encoding this protein in Cryptococcus neoformans lacks an ortholog in Saccharomyces cerevisiae; however, it is conserved in humans in which its ortholog is essential. Since it is not essential in C. neoformans, we were able to generate deletion mutants to characterize the roles of Dnj4. We also expressed Dnj4 in S. cerevisiae, in which it was able to bind S. cerevisiae histones and interfere with existing histone chaperoning machinery. Therefore, we show a conserved role for Dnj4 in histone chaperoning that suggests that C. neoformans is useful to better understand aspects of this important biological process.

Dnj1 Promotes Virulence in Cryptococcus neoformans by Maintaining Robust Endoplasmic Reticulum Homeostasis Under Temperature Stress.

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 endoplasmic reticulum (ER) J-domain containing co-chaperone, Dnj1, in the virulence of C. neoformans. A strain expressing a Dnj1-GFP fusion protein was used to confirm localization to the ER, and a dnj1∆ deletion mutant was shown to be hypersensitive to the ER stress caused by tunicamycin (TM) or 4µ8C. Dnj1 and another ER chaperone, calnexin were found to coordinately maintain ER homeostasis and contribute to maintenance of cell wall architecture. Dnj1 also contributed to thermotolerance and increased in abundance at elevated temperatures representative of febrile patients (e.g., 39°C) thus highlighting its role as a temperature-responsive J domain protein. The elaboration of virulence factors such as the polysaccharide capsule and extracellular urease activity were also markedly impaired in the dnj1∆ mutant when induced at human body temperature (i.e., 37°C). These virulence factors are immunomodulatory and, indeed, infection with the dnj1∆ mutant revealed impaired induction of the cytokines IL-6, IL-10, and MCP-1 in the lungs of mice compared to infection with wild type or complemented strains. The dnj1∆ mutant also had attenuated virulence in an intranasal murine model of cryptococcosis. Altogether, our data indicate that Dnj1 is crucial for survival and virulence factor production at elevated temperatures. The characterization of this co-chaperone also highlights the importance of maintaining homeostasis in the ER for the pathogenesis of C. neoformans.