Amoeba predation of Cryptococcus: A quantitative and population genomic evaluation of the accidental pathogen hypothesis.

The "Amoeboid Predator-Fungal Animal Virulence Hypothesis" posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor Bzp4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions.

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22-modified FK520 analog against C. neoformans.

IMPORTANCE: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited, and the development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally active FDA-approved therapy. This research advances the development of much-needed newer antifungal treatment options with novel mechanisms of action.

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22 modified FK520 analog against C. neoformans.

Fungal infections are of mounting global concern, and the current limited treatment arsenal poses challenges when treating such infections. In particular, infections by Cryptococcus neoformans are associated with high mortality, emphasizing the need for novel therapeutic options. Calcineurin is a protein phosphatase that mediates fungal stress responses, and calcineurin inhibition by the natural product FK506 blocks C. neoformans growth at 37°C. Calcineurin is also required for pathogenesis. However, because calcineurin is conserved in humans, and inhibition with FK506 results in immunosuppression, the use of FK506 as an anti-infective agent is precluded. We previously elucidated the structures of multiple fungal calcineurin-FK506-FKBP12 complexes and implicated the C-22 position on FK506 as a key point for differential modification of ligand inhibition of the mammalian versus fungal target proteins. Through in vitro antifungal and immunosuppressive testing of FK520 (a natural analog of FK506) derivatives, we identified JH-FK-08 as a lead candidate for further antifungal development. JH-FK-08 exhibited significantly reduced immunosuppressive activity and both reduced fungal burden and prolonged survival of infected animals. JH-FK-08 exhibited additive activity in combination with fluconazole in vivo . These findings further advance calcineurin inhibition as an antifungal therapeutic approach. Importance: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited and development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally-active FDA approved therapy. This research advances the development of much needed newer antifungal treatment options with novel mechanisms of action.

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.

Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex.

.Fungi are enigmatic organisms that flourish in soil, on decaying plants, or during infection of animals or plants. Growing in myriad forms, from single-celled yeast to multicellular molds and mushrooms, fungi have also evolved a variety of strategies to reproduce. Normally, fungi reproduce in one of two ways: either they reproduce asexually, with one individual producing a new individual identical to itself, or they reproduce sexually, with two individuals of different 'mating types' contributing to produce a new individual. However, individuals of some species exhibit 'homothallism' or self-fertility: these individuals can produce reproductive cells that are universally compatible, and therefore can reproduce sexually with themselves or with any other cell in the population.Homothallism has evolved multiple times throughout the fungal kingdom, suggesting it confers advantage when population numbers are low or mates are hard to find. Yet some homothallic fungi been overlooked compared to heterothallic species, whose mating types have been well characterised. Understanding the genetic basis of homothallism and how it evolved in different species can provide insights into pathogenic species that cause fungal disease.With that in mind, Passer, Clancey et al. explored the genetic basis of homothallism in Cryptococcus depauperatus, a close relative of C. neoformans, a species that causes fungal infections in humans. A combination of genetic sequencing techniques and experiments were applied to analyse, compare, and manipulate C. depauperatus' genome to see how this species evolved self-fertility.Passer, Clancey et al. showed that C. depauperatus evolved the ability to reproduce sexually by itself via a unique evolutionary pathway. The result is a form of homothallism never reported in fungi before. C. depauperatus lost some of the genes that control mating in other species of fungi, and acquired genes from the opposing mating types of a heterothallic ancestor to become self-fertile.Passer, Clancey et al. also found that, unlike other Cryptococcus species that switch between asexual and sexual reproduction, C. depauperatus grows only as long, branching filaments called hyphae, a sexual form. The species reproduces sexually with itself throughout its life cycle and is unable to produce a yeast (asexual) form, in contrast to other closely related species.This work offers new insights into how different modes of sexual reproduction have evolved in fungi. It also provides another interesting case of how genome plasticity and evolutionary pressures can produce similar outcomes, homothallism, via different evolutionary paths. Lastly, assembling the complete genome of C. depauperatus will foster comparative studies between pathogenic and non-pathogenic Cryptococcus species.

Fungi are enigmatic organisms that flourish in soil, on decaying plants, or during infection of animals or plants. Growing in myriad forms, from single-celled yeast to multicellular molds and mushrooms, fungi have also evolved a variety of strategies to reproduce. Normally, fungi reproduce in one of two ways: either they reproduce asexually, with one individual producing a new individual identical to itself, or they reproduce sexually, with two individuals of different 'mating types' contributing to produce a new individual. However, individuals of some species exhibit 'homothallism' or self-fertility: these individuals can produce reproductive cells that are universally compatible, and therefore can reproduce sexually with themselves or with any other cell in the population. Homothallism has evolved multiple times throughout the fungal kingdom, suggesting it confers advantage when population numbers are low or mates are hard to find. Yet some homothallic fungi been overlooked compared to heterothallic species, whose mating types have been well characterised. Understanding the genetic basis of homothallism and how it evolved in different species can provide insights into pathogenic species that cause fungal disease. With that in mind, Passer, Clancey et al. explored the genetic basis of homothallism in Cryptococcus depauperatus, a close relative of C. neoformans, a species that causes fungal infections in humans. A combination of genetic sequencing techniques and experiments were applied to analyse, compare, and manipulate C. depauperatus' genome to see how this species evolved self-fertility. Passer, Clancey et al. showed that C. depauperatus evolved the ability to reproduce sexually by itself via a unique evolutionary pathway. The result is a form of homothallism never reported in fungi before. C. depauperatus lost some of the genes that control mating in other species of fungi, and acquired genes from the opposing mating types of a heterothallic ancestor to become self-fertile. Passer, Clancey et al. also found that, unlike other Cryptococcus species that switch between asexual and sexual reproduction, C. depauperatus grows only as long, branching filaments called hyphae, a sexual form. The species reproduces sexually with itself throughout its life cycle and is unable to produce a yeast (asexual) form, in contrast to other closely related species. This work offers new insights into how different modes of sexual reproduction have evolved in fungi. It also provides another interesting case of how genome plasticity and evolutionary pressures can produce similar outcomes, homothallism, via different evolutionary paths. Lastly, assembling the complete genome of C. depauperatus will foster comparative studies between pathogenic and non-pathogenic Cryptococcus species.

Epistatic genetic interactions govern morphogenesis during sexual reproduction and infection in a global human fungal pathogen.

Cellular development is orchestrated by evolutionarily conserved signaling pathways, which are often pleiotropic and involve intra- and interpathway epistatic interactions that form intricate, complex regulatory networks. Cryptococcus species are a group of closely related human fungal pathogens that grow as yeasts yet transition to hyphae during sexual reproduction. Additionally, during infection they can form large, polyploid titan cells that evade immunity and develop drug resistance. Multiple known signaling pathways regulate cellular development, yet how these are coordinated and interact with genetic variation is less well understood. Here, we conducted quantitative trait locus (QTL) analyses of a mapping population generated by sexual reproduction of two parents, only one of which is unisexually fertile. We observed transgressive segregation of the unisexual phenotype among progeny, as well as a large-cell phenotype under mating-inducing conditions. These large-cell progeny were found to produce titan cells both in vitro and in infected animals. Two major QTLs and corresponding quantitative trait genes (QTGs) were identified: RIC8 (encoding a guanine-exchange factor) and CNC06490 (encoding a putative Rho-GTPase activator), both involved in G protein signaling. The two QTGs interact epistatically with each other and with the mating-type locus in phenotypic determination. These findings provide insights into the complex genetics of morphogenesis during unisexual reproduction and pathogenic titan cell formation and illustrate how QTL analysis can be applied to identify epistasis between genes. This study shows that phenotypic outcomes are influenced by the genetic background upon which mutations arise, implicating dynamic, complex genotype-to-phenotype landscapes in fungal pathogens and beyond.

Pleiotropy and epistasis within and between signaling pathways defines the genetic architecture of fungal virulence.

Cryptococcal disease is estimated to affect nearly a quarter of a million people annually. Environmental isolates of Cryptococcus deneoformans, which make up 15 to 30% of clinical infections in temperate climates such as Europe, vary in their pathogenicity, ranging from benign to hyper-virulent. Key traits that contribute to virulence, such as the production of the pigment melanin, an extracellular polysaccharide capsule, and the ability to grow at human body temperature have been identified, yet little is known about the genetic basis of variation in such traits. Here we investigate the genetic basis of melanization, capsule size, thermal tolerance, oxidative stress resistance, and antifungal drug sensitivity using quantitative trait locus (QTL) mapping in progeny derived from a cross between two divergent C. deneoformans strains. Using a "function-valued" QTL analysis framework that exploits both time-series information and growth differences across multiple environments, we identified QTL for each of these virulence traits and drug susceptibility. For three QTL we identified the underlying genes and nucleotide differences that govern variation in virulence traits. One of these genes, RIC8, which encodes a regulator of cAMP-PKA signaling, contributes to variation in four virulence traits: melanization, capsule size, thermal tolerance, and resistance to oxidative stress. Two major effect QTL for amphotericin B resistance map to the genes SSK1 and SSK2, which encode key components of the HOG pathway, a fungal-specific signal transduction network that orchestrates cellular responses to osmotic and other stresses. We also discovered complex epistatic interactions within and between genes in the HOG and cAMP-PKA pathways that regulate antifungal drug resistance and resistance to oxidative stress. Our findings advance the understanding of virulence traits among diverse lineages of Cryptococcus, and highlight the role of genetic variation in key stress-responsive signaling pathways as a major contributor to phenotypic variation.

Genome-wide functional analysis of phosphatases in the pathogenic fungus Cryptococcus neoformans.

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.

Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro.

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.

Cryptococcus neoformans Recovered From Olive Trees (Olea europaea) in Turkey Reveal Allopatry With African and South American Lineages.

Cryptococcus species are life-threatening human fungal pathogens that cause cryptococcal meningoencephalitis in both immunocompromised and healthy hosts. The natural environmental niches of Cryptococcus include pigeon (Columba livia) guano, soil, and a variety of tree species such as Eucalyptus camaldulensis, Ceratonia siliqua, Platanus orientalis, and Pinus spp. Genetic and genomic studies of extensive sample collections have provided insights into the population distribution and composition of different Cryptococcus species in geographic regions around the world. However, few such studies examined Cryptococcus in Turkey. We sampled 388 Olea europaea (olive) and 132 E. camaldulensis trees from seven locations in coastal and inland areas of the Aegean region of Anatolian Turkey in September 2016 to investigate the distribution and genetic diversity present in the natural Cryptococcus population. We isolated 84 Cryptococcus neoformans strains (83 MATα and 1 MATa) and 3 Cryptococcus deneoformans strains (all MATα) from 87 (22.4% of surveyed) O. europaea trees; a total of 32 C. neoformans strains were isolated from 32 (24.2%) of the E. camaldulensis trees, all of which were MATα. A statistically significant difference was observed in the frequency of C. neoformans isolation between coastal and inland areas (P < 0.05). Interestingly, the MATaC. neoformans isolate was fertile in laboratory crosses with VNI and VNB MATα tester strains and produced robust hyphae, basidia, and basidiospores, thus suggesting potential sexual reproduction in the natural population. Sequencing analyses of the URA5 gene identified at least five different genotypes among the isolates. Population genetics and genomic analyses revealed that most of the isolates in Turkey belong to the VNBII lineage of C. neoformans, which is predominantly found in southern Africa; these isolates are part of a distinct minor clade within VNBII that includes several isolates from Zambia and Brazil. Our study provides insights into the geographic distribution of different C. neoformans lineages in the Mediterranean region and highlights the need for wider geographic sampling to gain a better understanding of the natural habitats, migration, epidemiology, and evolution of this important human fungal pathogen.

Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

Genetic and Genomic Analyses Reveal Boundaries between Species Closely Related to Cryptococcus Pathogens.

Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes >200,000 new human infections annually. Elucidation of how these species evolved into important human-pathogenic species remains challenging and can be advanced by studying the most closely related nonpathogenic species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii However, these species have only four known isolates, and available data were insufficient to determine species boundaries within this group. By analyzing full-length chromosome assemblies, we reappraised the phylogenetic relationships of the four available strains, confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. The genomes of the three species are ∼6% divergent and exhibit significant chromosomal rearrangements, including inversions and a reciprocal translocation that involved intercentromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can still undergo sexual reproduction with C. amylolentus However, most of the resulting spores were inviable or sterile or showed reduced recombination during meiosis, indicating that intrinsic postzygotic barriers had been established. Our study and genomic data will foster additional studies addressing fungal speciation and transitions between nonpathogenic and pathogenic Cryptococcus lineages.IMPORTANCE The evolutionary drivers of speciation are critical to our understanding of how new pathogens arise from nonpathogenic lineages and adapt to new environments. Here we focus on the Cryptococcus amylolentus species complex, a nonpathogenic fungal lineage closely related to the human-pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analyses, we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. Their genomes are ∼6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) were still able to interbreed, the resulting hybrid progeny were usually inviable or sterile, indicating that barriers to reproduction had already been established. These results advance our understanding of speciation in fungi and highlight the power of genomics in assisting our ability to correctly identify and discriminate fungal species.

In Vitro and In Vivo Assessment of FK506 Analogs as Novel Antifungal Drug Candidates.

FK506 (tacrolimus) is an FDA-approved immunosuppressant indicated for the prevention of allograft rejections in patients undergoing organ transplants. In mammals, FK506 inhibits the calcineurin-nuclear factor of activated T cells (NFAT) pathway to prevent T-cell proliferation by forming a ternary complex with its binding protein, FKBP12, and calcineurin. FK506 also exerts antifungal activity by inhibiting calcineurin, which is essential for the virulence of human-pathogenic fungi. Nevertheless, FK506 cannot be used directly as an antifungal drug due to its immunosuppressive action. In this study, we analyzed the cytotoxicity, immunosuppressive activity, and antifungal activity of four FK506 analogs, 31-O-demethyl-FK506, 9-deoxo-FK506, 9-deoxo-31-O-demethyl-FK506, and 9-deoxo-prolyl-FK506, in comparison with that of FK506. The four FK506 analogs generally possessed lower cytotoxicity and immunosuppressive activity than FK506. The FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against Cryptococcus neoformans and Candida albicans, which are two major invasive pathogenic yeasts, due to the inhibition of the calcineurin pathway. Furthermore, the FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against the invasive filamentous fungus Aspergillus fumigatus Notably, 9-deoxo-31-O-demethyl-FK506 and 31-O-demethyl-FK506 exhibited robust synergistic antifungal activity with fluconazole, similar to FK506. Considering the antifungal efficacy, cytotoxicity, immunosuppressive activity, and synergistic effect with commercial antifungal drugs, we selected 9-deoxo-31-O-demethyl-FK506 for further evaluation of its in vivo antifungal efficacy in a murine model of systemic cryptococcosis. Although 9-deoxo-31-O-demethyl-FK506 alone was not sufficient to treat the cryptococcal infection, when it was used in combination with fluconazole, it significantly extended the survival of C. neoformans-infected mice, confirming the synergistic in vivo antifungal efficacy between these two agents.

Evolutionarily Conserved and Divergent Roles of Unfolded Protein Response (UPR) in the Pathogenic Cryptococcus Species Complex.

The unfolded protein response (UPR) pathway, consisting of the evolutionarily conserved Ire1 kinase/endonuclease and the bZIP transcription factor Hxl1, is critical for the pathogenicity of Cryptococcus neoformans; however, its role remains unknown in other pathogenic Cryptococcus species. Here, we investigated the role of the UPR pathway in C. deuterogattii, which causes pneumonia and systemic cryptococcosis, even in immunocompetent individuals. In response to ER stress, C. deuterogattii Ire1 triggers unconventional splicing of HXL1 to induce the expression of UPR target genes such as KAR2, DER1, ALG7, and ERG29. Furthermore, C. deuterogattii Ire1 is required for growth at mammalian body temperature, similar to C. neoformans Ire1. However, deletion of HXL1 does not significantly affect the growth of C. deuterogattii at 37 °C, which is in contrast to the indispensable role of HXL1 in the growth of C. neoformans at 37 °C. Nevertheless, both C. deuterogattii ire1Δ and hxl1Δ mutants are avirulent in a murine model of systemic cryptococcosis, suggesting that a non-thermotolerance phenotypic trait also contributes to the role of the UPR pathway in the virulence of pathogenic Cryptococcus species. In conclusion, the UPR pathway plays redundant and distinct roles in the virulence of members of the pathogenic Cryptococcus species complex.

Whole-Genome Analysis Illustrates Global Clonal Population Structure of the Ubiquitous Dermatophyte Pathogen Trichophyton rubrum.

Dermatophytes include fungal species that infect humans, as well as those that also infect other animals or only grow in the environment. The dermatophyte species Trichophyton rubrum is a frequent cause of skin infection in immunocompetent individuals. While members of the T. rubrum species complex have been further categorized based on various morphologies, their population structure and ability to undergo sexual reproduction are not well understood. In this study, we analyze a large set of T. rubrum and T. interdigitale isolates to examine mating types, evidence of mating, and genetic variation. We find that nearly all isolates of T. rubrum are of a single mating type, and that incubation with T. rubrum "morphotype" megninii isolates of the other mating type failed to induce sexual development. While the region around the mating type locus is characterized by a higher frequency of SNPs compared to other genomic regions, we find that the population is remarkably clonal, with highly conserved gene content, low levels of variation, and little evidence of recombination. These results support a model of recent transition to asexual growth when this species specialized to growth on human hosts.

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans.

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans.

Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect- or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes.

Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus.

The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species. IMPORTANCE: Species in the genus Malassezia are a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culture in vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species of Malassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

Metal Chelation as a Powerful Strategy to Probe Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis.

Fungal pathogens have evolved diverse strategies to sense host-relevant cues and coordinate cellular responses, which enable virulence and drug resistance. Defining circuitry controlling these traits opens new opportunities for chemical diversity in therapeutics, as the cognate inhibitors are rarely explored by conventional screening approaches. This has great potential to address the pressing need for new therapeutic strategies for invasive fungal infections, which have a staggering impact on human health. To explore this approach, we focused on a leading human fungal pathogen, Candida albicans, and screened 1,280 pharmacologically active compounds to identify those that potentiate the activity of echinocandins, which are front-line therapeutics that target fungal cell wall synthesis. We identified 19 compounds that enhance activity of the echinocandin caspofungin against an echinocandin-resistant clinical isolate, with the broad-spectrum chelator DTPA demonstrating the greatest synergistic activity. We found that DTPA increases susceptibility to echinocandins via chelation of magnesium. Whole genome sequencing of mutants resistant to the combination of DTPA and caspofungin identified mutations in the histidine kinase gene NIK1 that confer resistance to the combination. Functional analyses demonstrated that DTPA activates the mitogen-activated protein kinase Hog1, and that NIK1 mutations block Hog1 activation in response to both caspofungin and DTPA. The combination has therapeutic relevance as DTPA enhanced the efficacy of caspofungin in a mouse model of echinocandin-resistant candidiasis. We found that DTPA not only reduces drug resistance but also modulates morphogenesis, a key virulence trait that is normally regulated by environmental cues. DTPA induced filamentation via depletion of zinc, in a manner that is contingent upon Ras1-PKA signaling, as well as the transcription factors Brg1 and Rob1. Thus, we establish a new mechanism by which metal chelation modulates morphogenetic circuitry and echinocandin resistance, and illuminate a novel facet to metal homeostasis at the host-pathogen interface, with broad therapeutic potential.

Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets.