Black yeasts in the omics era: Achievements and challenges

Black yeasts in the omics era: Achievements and challenges Abstract Black yeasts (BY) comprise a group of polyextremotolerant fungi, mainly belonging to the order Chaetothyriales, which are capable of colonizing a wide range of extreme environments. The tolerance to hostile habitats can be explained by their intrinsic ability to survive under acidic, alkaline, and toxic conditions, high temperature, low nutrient availability, and osmotic and mechanical stress. Occasionally, some species can cause human chromoblastomycosis, a chronic subcutaneous infection, as well as disseminated or cerebral phaeohyphomycosis. Three years after the release of the first black yeast genome, the number of projects for sequencing these organisms has significantly increased. Over 37 genomes of important opportunistic and saprobic black yeasts and relatives are now available in different databases. The whole-genome sequencing, as well as the analysis of differentially expressed mRNAs and the determination of protein expression profiles generated an unprecedented amount of data, requiring the development of a curated repository to provide easy accesses to this information. In the present article, we review various aspects of the impact of genomics, transcriptomics, and proteomics on black yeast studies. We discuss recent key findings achieved by the use of these technologies and further directions for medical mycology in this area. An important vehicle is the Working Groups on Black Yeasts and Chromoblastomycosis, under the umbrella of ISHAM, which unite the clinicians and a highly diverse population of fundamental scientists to exchange data for joint publications. black yeast, genome, transcriptome, proteome, pathogenesis Introduction Species of the order Chaetothyriales, better known under their popular descriptor ‘black yeasts and filamentous relatives’ (BY), display a wide diversity of lifestyles. In contrast to frequent statements in the literature, they are not commonly found in soil and plants, but their habitats always share a certain degree of hostility: hot, dry,1 toxic aromatic hydrocarbons,2 poor in nutrients,3 or exposed to irradiation.4,5 For this ecology the term ‘polyextremotolerance’ was introduced.6 An ecological explanation for this behavior was an assumption of low competitive ability with the multitude of microbes in more permissive environments.7 Melanization and tolerance of reactive oxygen would allow these fungi to survive phagocytosis in case they would accidentally be introduced into animal tissue, but the human body is not the prime niche of most of these fungi. In addition, the melanized cell wall confers protection against a broad range of environmental stressors, including resistance against UV irradiation, enzymatic lysis and, in some instances, extremes of temperatures.8,9 Once they have entered the host, they may cause recalcitrant infections, in immunocompromised as well as in immunocompetent individuals. But they seem to lack mechanisms to enter the host, as is exemplified by the ubiquitous occurrence, for example, of Exophiala dermatitidis in wet cells of the indoor environment combined with its rarity of infection in otherwise healthy patients.10 This character determines the black yeasts as classical opportunists: able to cause infection, but infection does not contribute to their fitness. Etiology The order Chaetothyriales includes both environmental isolates, not causing disease, and clinically relevant species recognized as agents of severe chromoblastomycosis and phaeohyphomycosis. Recently, the chromoblastomycosis has been added to the World Health Organization (WHO) portfolio of neglected tropical diseases (NTDs). This disease was grouped together with mycetoma and other deep mycoses into a category that receives sources from the NTD Department, as well as notorious exposure on their website and literature, opening new funding opportunities, which are important for actions on prevention, early diagnosis and better therapies (www.who.int). Chromoblastomycosis (CBM), however, a chronic endemic mycosis involving skin and subcutaneous tissues, is puzzling. Without exception, agents of CBM are phylogenetically accommodated in the Chaetothyriales, classified in divergent genera: Cladophialophora, Exophiala, Fonsecaea, Phialophora, and Rhinocladiella, with Cladophialophora carrionii, Fonsecaea pedrosoi, and F. monophora being the prevalent species. Although the precise route of infection remains unclear, the current hypothesis proposes that rural workers acquired the infection by cutaneous inoculation of fungal material from an environmental source. In tissue, the fungi change to large, muriform, thick-walled dematiaceous cells, considered to be the pathogenic form of the fungus.11 Badali et al.12 demonstrated that under appropriate conditions muriform cells are also produced by strictly environmental species of the above genera. Therefore, agents of CBM, as well as their environmental siblings,7 seem to have evolved similar factors that have as yet unknown functions in the environment but enhance accidental, traumatic infection in vertebrate hosts. Some species are remarkably successful as infectious agents: they are prevalent on humans but are difficult to isolate from sites where their strictly saprobic siblings are found. The infective potential thus differs between closely related species, and the concept of opportunism perhaps has to be revised for CBM. Phaeohyphomycosis is characterized by the presence of melanized, septate hyphal elements or sometimes yeast-like cells in tissue. Innate immune response to these elements leads to necrosis, which may become destructive.13 Black yeasts have also been involved infections of cold-blooded animal. Frog, toad, and fish infections have been frequently reported,12 and Vicente et al.14 descried an extensive epizootic caused by the black yeast Fonsecaea brasilienses and which led to massive death of the mangrove-land crab Ucides cordatus (Brachyura: Ocypodidae) along the Brazilian coast. Remarkably, infections in warm-blooded vertebrates other than humans are rare.15,16 Ecology of black fungi Black yeasts (Exophiala species, particularly E. dermatitidis) thrive in man-made habitats, such as dishwashers,17 saunas,18 sinks,19 and bathing facilities.20 Their close vicinity to human hosts would pose a public health risk if the fungi were genuinely pathogenic. But household-acquired infections are either mild21 or are restricted to susceptible patient populations with genetic disorders such as those with cystic fibrosis.22 Nevertheless, the occurrence of severe infections, for example, in patients with primary immunodeficiencies, remains an area of concern. Among the primary immunodeficiency diseases, inborn errors in the gene that codes for caspase recruitment domain-containing protein 9 (CARD9) have been reported in patients with phaeohyphomycosis.23 In addition, acquired conditions, such as human immunodeficiency virus (HIV)-infected patients, as well as patients receiving solid-organ transplants or under immunosuppressive therapies, have also been responsible for disseminated infections. Genome and transcriptome analyses may contribute to understanding of the genes enabling black fungal opportunistic infection in general, and solve questions concerning the possible adaptation of CBM agents toward pathogenicity. Next-generation sequencing (NGS) The application of high-throughput sequencing technologies to elucidate the genetic bases of pathogenicity and niche adaptation in BY started in 2011 when the first whole-genome sequence belonging to a chaetothyrialean fungus, Exophiala dermatitidis, was released as a part of the Broad Institute Fungal Genome Initiative (http://www.broadinstitute.org/annotation/genome/Black_Yeasts/MultiHome.html) and published by Chen et al.20 Since that publication, efforts have led to sequencing and analysis of an additional 37 genomes and transcriptomes, producing an avalanche of data for comparative genomics (Fig. 1, Table 1). Overall, these genomes have been determined using short-read approaches, based on the Illumina, Ion Torrent and/or 454 instruments (Table 1). These NGS sequencing technologies rely on cyclic reversible termination, single nucleotide addition and pyrosequencing, respectively. For a general survey on NGS technologies, please check the review paper by Goodwin et al.24 Although the majority of projects only used a single NGS platform to determine the genome, the combination of different technologies could avoid systemic bias generated by using a single sequencing approach.24 In addition, the use of distinct library preparation methods might prevent bias caused by the PCR amplification, an important source of bias from DNA-seq library preparation protocols. At the transcription level, the dUTP second strand marking method25 has been adopted in at least eight transcriptome studies26 and turned out to be the leading protocol for RNA sequencing of black yeast species. Figure 1. View largeDownload slide Overview of black yeast genome studies between 2014 and 2017. The time line shows important landmarks of black yeast genome sequencing projects, mainly through the Fungal Genome Initiative of the BROAD institute of MIT and Harvard. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Overview of black yeast genome studies between 2014 and 2017. The time line shows important landmarks of black yeast genome sequencing projects, mainly through the Fungal Genome Initiative of the BROAD institute of MIT and Harvard. This Figure is reproduced in color in the online version of Medical Mycology. Table 1. Whole-genome sequencing of black yeast and relative deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/genome). Species  Collection number  Instrument  Sequencing center  Reference  Capronia coronata  CBS 617.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia epimyces  CBS 606.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia semiimmersa  CBS 27337  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  CBS 173.52  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  UM 956  Illumina - HiSeq 2000  University Malaya  Kuan et al.54  Cladophialophora carrionii  dH 23894  454  LNCC  Teixeira et al.26  Cladophialophora carrionii  CBS 160.54  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 834.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 110551  ION Proton Technology  University of Natural Resources and Life Sciences  Sterflinger et al.74  Cladophialophora psammophila  CBS 110553  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora yegresii  CBS 114405  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Coniosporium apollinis  CBS 100218  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala alcalophila  JCM 1751  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala aquamarina  CBS 119918  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala calicioides  JCM 6030  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala dermatitidis  NIH/UT8656  Illumina - HiSeq 2000  Broad Institute  Chen et al.43  Exophiala mesophila  CBS 120910  ION Proton Technology  University of Natural Resources and Life Sciences  Tafer et al.75  Exophiala mesophila  CBS 402.95  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala oligosperma  CBS 725.88  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala sideris  CBS121828  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  CBS 899.68  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  JCM 15939  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala xenobiotica  CBS 118157  Illumina - HiSeq 2000/ION Proton Technology  Broad Institute  Teixeira et al.26  Exophiala xenobiotica  CBS 102455  ION Proton Technology  University of Natural Resources and Life Sciences  N/A  Fonsecaea erecta  CBS 125763  Illumina - HiSeq 2000  Federal University of Paraná  Vicente et al.55  Fonsecaea monophora  CBS 269.37  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Bombassaro et al.76  Fonsecaea multimorphosa  CBS 102226  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Fonsecaea multimorphosa  CBS 980.96  Illumina - MiSeq  Federal University of Paraná  Leao et al.77  Fonsecaea nubica  CBS 269.64  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Costa et al.78  Fonsecaea pedrosoi  CBS 271.37  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Ochroconis mirabilis  UM 578  Illumina - HiSeq 2000  University Malaya  Yew et al.79  Phialophora attae  CBS 131958  ION Proton Technology  Westerdijk Institute  Moreno et al.80  Phialophora europaea  CBS 101466  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Rhinocladiella mackenziei  IHM 22877  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  dH 24460  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  CBS 650.93  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Verruconis gallopava  CBS 437.64  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Species  Collection number  Instrument  Sequencing center  Reference  Capronia coronata  CBS 617.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia epimyces  CBS 606.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia semiimmersa  CBS 27337  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  CBS 173.52  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  UM 956  Illumina - HiSeq 2000  University Malaya  Kuan et al.54  Cladophialophora carrionii  dH 23894  454  LNCC  Teixeira et al.26  Cladophialophora carrionii  CBS 160.54  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 834.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 110551  ION Proton Technology  University of Natural Resources and Life Sciences  Sterflinger et al.74  Cladophialophora psammophila  CBS 110553  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora yegresii  CBS 114405  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Coniosporium apollinis  CBS 100218  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala alcalophila  JCM 1751  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala aquamarina  CBS 119918  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala calicioides  JCM 6030  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala dermatitidis  NIH/UT8656  Illumina - HiSeq 2000  Broad Institute  Chen et al.43  Exophiala mesophila  CBS 120910  ION Proton Technology  University of Natural Resources and Life Sciences  Tafer et al.75  Exophiala mesophila  CBS 402.95  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala oligosperma  CBS 725.88  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala sideris  CBS121828  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  CBS 899.68  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  JCM 15939  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala xenobiotica  CBS 118157  Illumina - HiSeq 2000/ION Proton Technology  Broad Institute  Teixeira et al.26  Exophiala xenobiotica  CBS 102455  ION Proton Technology  University of Natural Resources and Life Sciences  N/A  Fonsecaea erecta  CBS 125763  Illumina - HiSeq 2000  Federal University of Paraná  Vicente et al.55  Fonsecaea monophora  CBS 269.37  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Bombassaro et al.76  Fonsecaea multimorphosa  CBS 102226  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Fonsecaea multimorphosa  CBS 980.96  Illumina - MiSeq  Federal University of Paraná  Leao et al.77  Fonsecaea nubica  CBS 269.64  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Costa et al.78  Fonsecaea pedrosoi  CBS 271.37  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Ochroconis mirabilis  UM 578  Illumina - HiSeq 2000  University Malaya  Yew et al.79  Phialophora attae  CBS 131958  ION Proton Technology  Westerdijk Institute  Moreno et al.80  Phialophora europaea  CBS 101466  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Rhinocladiella mackenziei  IHM 22877  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  dH 24460  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  CBS 650.93  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Verruconis gallopava  CBS 437.64  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  View Large Genomic patterns in BY With the whole-genome sequence of 27 distinctive species already published, the Chaetothyriales is currently the seventh most sequenced order of the division Pezizomycotina. The ensemble of available genomes comprises species that display a wide diversity of lifestyles, including the human opportunists involved in disseminated and neurotropic infections Cladophialophora bantiana, Fonsecaea monophora, Exophiala dermatitidis, and Rhinocladiella mackenziei; the etiologic agents of chromoblastomycosis Cadophialophora carrionii and Fonsecaea pedrosoi and their environmental counterparts Cl. yegresii and F. erecta, respectively; the species associated with the degradation of toxic hydrocarbons Exophiala oligosperma, E. xenobiotica, E. spinifera, Cl. immunda and Cl. psammophila; the ant-associated Phialophora attae; and several saprobic species occasionally reported causing mild infections. All BY genomes are similar in size, ranging from 25.8 Mb in Capronia coronata to 43 Mb in Cladophialophora immunda CBS 834.96 and a typical genomic feature seems to be the exceptionally high GC content (49%–54.3%), with comparable values only having been reported in Sporothrix. Similar genome sizes have been described in the genus Aspergillus, while reduced genomes are present in the order Onygenales.27 With few exceptions, the largest black yeast genomes belong to the members of the bantiana-clade, and smaller genomes are found in species of the dermatitidis-clade. Interestingly, an association seems apparent between genome size and ecological preferences. A potential explanation for the larger genomes in many species of the bantiana-clade is the striking abundance of the protein families short chain dehydrogenase, CYP P450, aldehyde dehydrogenase, sugar transporters, and membrane transporter proteins.28 It is not a surprise for black yeasts to contain expanded protein families associated to acquisition of nutrients, transporters and environmental stresses, since these families tend to be overrepresented in extremely tolerant fungi such as the halophile Hortaea werneckii.29 Protein family expansion as the result of gene duplication might have generated raw genetic material and consequently enhanced the metabolic plasticity and increased the fitness of black yeasts during the colonization of hostile ecological niches. Another interesting feature is the presence of physically linked genes, forming metabolic clusters, as patterns of selections for reduced accumulation of toxic intermediate compounds (ICs). The order and orientation of genes participating in metabolic pathways has been proposed as an important genomic adaptation against the accumulation of ICs in fungi.30 For example, in Rhinocladiella mackenziei the pathway involved in the conversion of choline to the osmoprotectant glycine betaine produce the toxic IC betaine aldehyde.31 In this fungus, the enzymes choline dehydrogenase and betaine-aldehyde dehydrogenase are adjacent pairs. Such gene organization may synchronize their activity by means of co-expression or prevent independent gene loss that could lead to disturbance in the metabolic cluster.32 The physical linkage of choline metabolism genes has also been described in other fungi.30 Numerous other metabolic gene clusters have been described in BY, many of which are involved in the degradation of xenobiotics, such as toluene and in secondary metabolite biosynthesis.26,33 In contrast to unrelated fungi such as Alternaria brassicicola and Cladosporium sphaerospermum,34 BY genes are not organized in clusters in any of the three pathways for the production of melanin.26 Melanin is the pigment responsible for the dark coloration of BY cells; it is produced by the oxidation of tyrosine in a multistep reaction involving enzymes that are often organized into supergenes or clusters in most fungi. Evolutionary genomics Orthologs and paralogs represent subcategories of homologous genes, which evolved, respectively, by vertical descent (speciation) from a single gene in the last common ancestor, or by gene duplication.35 Although the biological function of these groups of genes, and their products, is not necessarily maintained after such evolutionary processes, ortholog and paralog classification has widely been used to make inferences about the relationship between genes.36 For example, analysis of inparalogs, a subtype of paralogs originated by gene duplication after a speciation event, is important for detecting lineage-specific adaptations and emergence of functional novelties.37 Teixeira et al.26 reported extensive gene duplication processes in several protein families in 23 BY genomes. Among the expanded families, cytochromes p450 (CYP), drug efflux pumps, alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDHs) seem to be broadly distributed across black yeasts, and their substrate promiscuity suggests that they have played critical roles in niche adaptation. Gene duplication of these families might have provided raw material for new open reading frames (ORFs), often with different selective pressures, which could be used to enhance flexibility in acquisition and protection against stressful environments. The duplicated genes could increase fitness in an environment where gene dosage is important. Interestingly, in many opportunistic fungi, such as in Aspergillus fumigatus38 and Candida albicans,39 paralogous genes implicated in infection appear to be located in telomere-proximal regions or even in mobile pathogenicity chromosomes, as verified in the plant pathogen Fusarium oxysporum.40 The location of duplicated genes in BY has not yet been assessed. Similarly, the identification of fast-evolving genes, which are most likely to play a role in host-pathogen interaction, has not yet been addressed in black yeast. Moreno et al.31 reported that in the neurotrope Rhinocladiella mackenziei the paralogous genes accumulate more important mutations, that is, with probably severe impact on their biological function, than orthologous genes. Ancestral state reconstruction analysis determined that the expansions of these enzymes took place during the Cretaceous period < 80 million years ago (Mya),26 likely before the radiation of the families Herpotrichiellaceae and Cyphellophoraceae, which is believed to have occurred in the presence of a new substratum.41 The ecology of black yeast is too complex for a single hypothesis, but some striking aspects may have an intriguing explanation. The preference of many BY for ecological habitats polluted with harmful aromatic hydrocarbons may have been enhanced by the massive gene duplication in ADH, ALDH, drug efflux pumps, and CYP p450 that might have formed the basis for the metabolic versatility observed in modern black yeasts. The ancestor of these BY may have colonized extreme habitats such as hydrocarbon-rich ant- and termite-nests, and in part may have diversified along with these insects.26 Similarly, other important events of genome evolution, such as horizontal gene transfer (HGT), gene rearrangements, and gene loss have been subject to researches to explain central evolutionary processes in BY.33,42 Considered a key source of metabolic gene innovation in fungi, significant cases of HGT took place in black yeasts, particularly involving xenobiotic catabolism,33 inteins,26 and precursors for glycosyltransferase enzymes.43 Events of gene loss are rare in black yeasts, only having been reported in a few protein families in Rhinocladiella mackenziei.31 Ant-association lifestyle and the origin of the polyextremotolerance Phylogenomic studies, in combination with classical phylogenies based on taxon-rich data matrices, have shed light onto the history of life and evolutionary patterns in fungi.44 Phylogenies have been extensively used to explain biological interactions, such as the mutualistic association between fungi and ants.45 These insects are able to produce natural substances with antimicrobial activities that are used to protect their colonies from disease. In many ant species, phenylacetic acid seems to be a major active component against pathogens.46 The list of secreted toxins produced by ants also includes indole-3-acetic acid, 3-hydroxydecanoic acid, and 3-hydroxy acids, in addition of plenty of phenol derivates, such as 3-propylphenol, 3-pentylphenol, 5-propylresorcinol, 5-pentylresorcinol.47,48 As a consequence, only a small number of fungi adapted to tolerate such toxins are able to live in association with these insects. An updated phylogeny, recently published by Vasse et al.,49 revealed that ant-associated black yeasts are extremely frequent and scattered through the phylogeny of the order Chaetothyriales. This interaction was previously reported by Voglmayr et al.50 and Nepel et al.,51 who uncovered at least three distinct clades that accommodated BY from ant-cartons or -domatia. A speculative scenario to explain the ant-BY symbiosis might be found in the harsh and toxic environmental conditions of lichen-colonization, which produce toxic chemicals, and a subsequent environmental jump after manipulation by ants. This hypothesis was firstly proposed by Voglmayr et al.50 using phylogenies of Gueidan et al.52 and gained support with comparative genomic analyses that have recently come to light. Teixeira et al.26 identified a putative route for the catabolism of phenolic compounds via phenylacetic acid and homogentisate and reported a striking abundance of transporters and cytochromes p450 potentially involved in detoxification processes. The authors also predicted that these genetic adaptations were present in the common ancestor of the studied species and were maintained throughout the evolution of the BY. Since the calibrated phylogenetic tree estimated that the radiation of the Chaetothyriales species took place circa 75–50 MYA, during/after the Cretaceous-Paleogene (K-Pg) extinction event, and later than the origin of the ants (estimated in Early Cretaceous to Middle Jurassic, 140–168 MYA), the ant-association as an ancestral trait seems to be a plausible scenario to explain the origin of metabolic plasticity in the black yeasts. Because none of the comparative analysis studies thus far identified striking differences between clinical species and saprobes, we hypothesize here that the entire order has maintained similar ecological preferences, which were acquired during ancestral ant-interactions; species virulence is based on secondary characters, such as ability to survive phagocytosis or to be carried by blood. Free-living lifestyles versus infection While the identification of virulence factors is relatively common in bacterial comparative genomic studies, the prediction of fungal genes involved in infection is challenging.53 None of the recent publications reporting whole genome sequencing of the BY and subsequent comparative genome analysis have discovered unambiguous pathogen-specific genes. The candidates predicted as potential virulence factors, such as enzymes involved in adaptation to new nutrient sources and environmental stresses, are more associated with saprobic lifestyles than with pathogenicity. In part this may be explained by the fact that all the genomes sequenced so far belong to two derived families of Chaetothyriales: Cyphellophoraceae and Herpotrichiellaceae. In order to provide insights into the origin of infectivity it is essential to sequence species that are taxonomically not too remote but have consistently different ecological preferences. Studies thus far unintentionally addressed species with similar genetic make-up, with only gradational differences in, for example, predilection for aromatic hydrocarbons. Species causing disease in warm- versus cold-blooded animals may share traits that permit their growth in the environment, and if this is physiologically stressful this might also allow their survival inside the human body. In an attempt to identify genes that are associated with cerebral phaeohyphomycosis caused in Rhinocladiella mackenziei, Moreno et al.31 proposed a putative route for the degradation of aromatic compounds, which may be environmental contaminants such as styrene and ethylbenzene, but also neurotransmitters that are abundant in the brain. Similarly, Kuan et al.54 performed a genomic comparison of Cladophialophora bantiana, the prevalent cause of brain infection in healthy patients, with other neurotropic fungi and concluded that this fungus has probably evolved as an environmental saprobe. Although these two studies have identified several genes that potentially engaged in pathogenicity, it remains unclear why these species show predilection for the central nervous system. Additionally, other comparisons of opportunistic BY and their environmental counterparts, such as Cl. bantiana versus Cl. psammophila and Cl. carrionii versus Cl. yegresii, did not reveal striking genomic differences or host adaptations that could explain their infective potential.26 The genomes of Cl. carrionii and Cl. yegresii share more than 90% of their proteins, and functional annotation revealed high similarities in carbohydrate-active enzymes and peptidases.26 In line with this, a recent study of survival tests using Galleria mellonella larvae infected with clinical F. pedrosoi, and F. monophora, with saprobic F. erecta showed that the environmental species is equally able to infect and survive in animal host tissue.55 We may state that the terms pathogenic and nonpathogenic have been inaccurately used at the BY species level, causing misleading interpretations. The term ‘opportunism’ is more adequate for most BY, which implies that the fungus is basically saprobic but can survive in animal tissue. Comparative genomic analyses of true human pathogens, such as Coccidioides immitis and C. posadasii against closely related, nonpathogenic relatives, have revealed pathogen-specific adaptations,56 the fungi having retained or acquired enzymes involved in the scavenging of iron and in energy metabolism, while genes associated with plant degradation, such as cellulose, cutinase, pectins as well as genes required for carbohydrate metabolism, were lost. The study concluded that these are true pathogens that are associated with animal hosts.56 In contrast, black yeasts are found in niches that are hostile to microbial competitors, and therefore they exhibit a significant degree of extremotolerance. This general stress tolerance enables them to survive inside living host tissue and cause infection, but as most of them are unable to be transmitted from the host this is not a strategy to enhance their evolutionary fitness. Given the remarkable prevalence of some agents of CBM on human hosts, it may be speculated that some BY have adapted to pathogenicity by being able to escape from the host and transmit acquired mutations to next generations. Black yeasts in the post-genomic era Describing the sequence of a wide variety of black yeast genomes is only a first step to shed light on adaptive processes, including shifts to pathogenicity. The next stage of research is the so-called post-genomic era, which already started for BY, translating knowledge from DNA to transcriptome and proteome analyses. Attempts have been made to elucidate the transcriptional response of BY to stressors such as temperature, pH, radiation, and toxicity.33,43,57,58 The first BY with an assessed transcriptome was Exophiala dermatitidis, often considered as a model for melanized agents of disease.58,59 Robertson et al.58 analyzed gene expression in responses to low-dose ionizing radiation and discussed the importance of the cell wall pigmentation for tolerance; melanin was associated with overexpression of ribosomal biogenesis and transporter genes.58 Several other studies using BY have underlined the overproduction of melanin as a key response to stress. Chen et al.43 compared messenger RNA expression in E. dermatitidis upon alteration of the pH of the growth medium. Three pathways for melanin production were detected: via 1,8-dihydroxynaphthalen (DHN), via 3,4-dihydroxyphenylalanine (DOPA), and via l-tyrosine degradation, and all were activated during pH-stress. Similar results were obtained when Cladophialophora immunda was grown in the presence of toluene as sole carbon and energy source.33 Overall, these results demonstrate the importance of cell wall pigmentation for extremotolerance, providing a barrier against oxidative damage. A role of melanin in skin infection was assessed by Poyntner et al.,60 identifying genes that were transcriptionally altered during the artificial infection of an ex vivo skin model by E. dermatitidis. The transcriptome of an albino strain of Fonsecaea monophora was compared with its melanized parent by Li et al.,61 who identified 2283 differentially expressed genes between the genotypes. Extensive down-regulation of key genes in the DHN pathway was shown in the albino mutant, which was also more susceptible to low pH, high UV radiation, and oxidative stress.62 Down-regulation of melanin-associated genes was also verified in E. dermatitidis under conditions of low temperature (1°C for 1 week), indicating that melanin production changes in response to temperature variation.57 Not only transcriptional studies have been used to better understand the extremophilic character of BY. At proteomic level, Teset et al.63 analyzed the repertoire and abundance of proteins of E. dermatitidis under temperature stress and reported striking changes in its proteome. Long-term exposure to the cold (1°C) reduced metabolic activities of carbon and pyruvate metabolism, but high temperature (45°C) seemed not to induce any stress response, likely reflecting the high level of adaptation of E. dermatitidis to thrive at elevated temperature.63 Down-regulation of genes involved in metabolic processes, including metabolism of amino acids, carbohydrates and glycolysis, are consistent with and complementary to earlier reports in E. dermatitidis transcriptomics.58 In addition, the assessment of the proteins is an important tool to construct phylogenies using a large number of proteins shared by all sequenced black yeasts. As a consequence, geological time-calibrated trees, in addition to specific protein family expansions, provided important insights into the genomic pre-adaptations that might have driven the ecological preferences observed nowadays. Interestingly, species phylogenies based on amino acid sequences, corresponding to multiple single copy orthologous genes, produced similar topologies compared to the previously published phylogenetic trees.64,65 Future directions The employment of ‘omics’ technology, such as genomics, transcriptomics, and proteomics, to assess the genetic information of BY, is now a reality, and an enormous amount of data has already been generated. In addition, the application of other omics technologies, including metagenomics and metabolomics, offers a unique opportunity to interrogate the diversity of this group of fungi and will certainly be soon incorporated into high-throughput analysis using black yeast like-fungi (Fig. 2). A crucial step to keep these projects running is the development of a repository to provide access to this information for comprehensive curated analyses. Similar initiatives have been set up for other fungi, such as Candida66 and Aspergillus.67 In BY a collaborative effort has been made with the launch of a beta-version of a database: the Black Yeast HUB (www.blackyeasthub.com) was developed using GMOD68 and Tripal,69 a combination of database and interactive web pages for manipulating and displaying genome annotations. The current version of the Black Yeast HUB provides the complete genomes of 37 strains and 35 protein sets of chaetothyrialean fungi from the families Herpotrichiellaceae and Cyphellophoraceae. Additional information about gene structure, gene products, phylogenetic analyses, metabolic pathways, gene families and groups of orthologous genes is available for the 35 strains where gene annotations were previously performed. Figure 2. View largeDownload slide Potential applications of “omics” technology in black yeast research. ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; MS, mass spectrometry; qPCR, quantitative polymerase chain reaction; SNP, single nucleotide polymorphism; TLC, thin-layer chromatography. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Potential applications of “omics” technology in black yeast research. ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; MS, mass spectrometry; qPCR, quantitative polymerase chain reaction; SNP, single nucleotide polymorphism; TLC, thin-layer chromatography. This Figure is reproduced in color in the online version of Medical Mycology. With declining costs of next-generation sequencing and the availability of high standard reference genomes, one can expect that in the coming years members belonging to the Trichomeriaceae, Chaetothyriaceae, and Epibryaceae, considered as ‘ancestral’ families of Chaetothyriales, will be sequenced. Inclusion of ancestral species will considerably improve molecular dating of the ordinal tree and possibly confirm raised hypotheses on ant association and aromatic hydrocarbon degradation. The application of post-genomic transcriptomics and proteomics has raised our understanding of stress response in BY to a new level. Although the molecular machinery of pathogenicity is far from being elucidated, important indications have been obtained.60 Other initiatives, such as the use of in vivo models for mimicking the state of infection must be encouraged. The combination of omics techniques and advanced computational analyses cannot only be used to elucidate the origin of pathogenicity, it might also be applied as a diagnostic tool. Smeekens et al. highlighted the importance of omic approaches, such as transcriptomics, to classify and distinguish candidemia from Staphylococcus aureus infection.70 In addition, infection progression could be monitored assessing the transcription profile of specific genes, in which the expression is time-dependent.71 Similarly, proteomics has been proved to be effective to identify clinical pathogens, including Aspergillus species72 as well as candida isolates with reduced caspofungin susceptibility.73 In disseminated infections caused by black yeasts, where the rapid and accurate diagnosis is needed for therapeutic decisions, the discovery of specific biomarkers from omics data is of high importance, from which efficient detection methods, such as in situ hybridization, can be developed. For instance, cerebral phaeohyphomycosis has only been diagnosed based on conventional histopathology and culturing from brain biopsy, which delays the treatment and results in high mortality. In addition, the assessment of the substances or fungal exoantigens released by these organisms into the host, for example via metabolomic studies, can speed up pathogen detection and consequently perform adequate treatment. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Selbmann L, de Hoog GS, Mazzaglia A, Friedmann EI, Onofri S. Fungi at the edge of life: cryptoendolithic black fungi from Antarctic desert. Stud Mycol . 2005; 51: 1– 323. 2. Zhao J, Zeng J, de Hoog GS, Attili-Angelis D, Prenafeta-Boldú FX. Isolation and identification of black yeasts by enrichment on atmospheres of monoaromatic hydrocarbons. Microb Ecol . 2010; 60: 149– 56. 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Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Black yeasts in the omics era: Achievements and challenges

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Abstract

Abstract Black yeasts (BY) comprise a group of polyextremotolerant fungi, mainly belonging to the order Chaetothyriales, which are capable of colonizing a wide range of extreme environments. The tolerance to hostile habitats can be explained by their intrinsic ability to survive under acidic, alkaline, and toxic conditions, high temperature, low nutrient availability, and osmotic and mechanical stress. Occasionally, some species can cause human chromoblastomycosis, a chronic subcutaneous infection, as well as disseminated or cerebral phaeohyphomycosis. Three years after the release of the first black yeast genome, the number of projects for sequencing these organisms has significantly increased. Over 37 genomes of important opportunistic and saprobic black yeasts and relatives are now available in different databases. The whole-genome sequencing, as well as the analysis of differentially expressed mRNAs and the determination of protein expression profiles generated an unprecedented amount of data, requiring the development of a curated repository to provide easy accesses to this information. In the present article, we review various aspects of the impact of genomics, transcriptomics, and proteomics on black yeast studies. We discuss recent key findings achieved by the use of these technologies and further directions for medical mycology in this area. An important vehicle is the Working Groups on Black Yeasts and Chromoblastomycosis, under the umbrella of ISHAM, which unite the clinicians and a highly diverse population of fundamental scientists to exchange data for joint publications. black yeast, genome, transcriptome, proteome, pathogenesis Introduction Species of the order Chaetothyriales, better known under their popular descriptor ‘black yeasts and filamentous relatives’ (BY), display a wide diversity of lifestyles. In contrast to frequent statements in the literature, they are not commonly found in soil and plants, but their habitats always share a certain degree of hostility: hot, dry,1 toxic aromatic hydrocarbons,2 poor in nutrients,3 or exposed to irradiation.4,5 For this ecology the term ‘polyextremotolerance’ was introduced.6 An ecological explanation for this behavior was an assumption of low competitive ability with the multitude of microbes in more permissive environments.7 Melanization and tolerance of reactive oxygen would allow these fungi to survive phagocytosis in case they would accidentally be introduced into animal tissue, but the human body is not the prime niche of most of these fungi. In addition, the melanized cell wall confers protection against a broad range of environmental stressors, including resistance against UV irradiation, enzymatic lysis and, in some instances, extremes of temperatures.8,9 Once they have entered the host, they may cause recalcitrant infections, in immunocompromised as well as in immunocompetent individuals. But they seem to lack mechanisms to enter the host, as is exemplified by the ubiquitous occurrence, for example, of Exophiala dermatitidis in wet cells of the indoor environment combined with its rarity of infection in otherwise healthy patients.10 This character determines the black yeasts as classical opportunists: able to cause infection, but infection does not contribute to their fitness. Etiology The order Chaetothyriales includes both environmental isolates, not causing disease, and clinically relevant species recognized as agents of severe chromoblastomycosis and phaeohyphomycosis. Recently, the chromoblastomycosis has been added to the World Health Organization (WHO) portfolio of neglected tropical diseases (NTDs). This disease was grouped together with mycetoma and other deep mycoses into a category that receives sources from the NTD Department, as well as notorious exposure on their website and literature, opening new funding opportunities, which are important for actions on prevention, early diagnosis and better therapies (www.who.int). Chromoblastomycosis (CBM), however, a chronic endemic mycosis involving skin and subcutaneous tissues, is puzzling. Without exception, agents of CBM are phylogenetically accommodated in the Chaetothyriales, classified in divergent genera: Cladophialophora, Exophiala, Fonsecaea, Phialophora, and Rhinocladiella, with Cladophialophora carrionii, Fonsecaea pedrosoi, and F. monophora being the prevalent species. Although the precise route of infection remains unclear, the current hypothesis proposes that rural workers acquired the infection by cutaneous inoculation of fungal material from an environmental source. In tissue, the fungi change to large, muriform, thick-walled dematiaceous cells, considered to be the pathogenic form of the fungus.11 Badali et al.12 demonstrated that under appropriate conditions muriform cells are also produced by strictly environmental species of the above genera. Therefore, agents of CBM, as well as their environmental siblings,7 seem to have evolved similar factors that have as yet unknown functions in the environment but enhance accidental, traumatic infection in vertebrate hosts. Some species are remarkably successful as infectious agents: they are prevalent on humans but are difficult to isolate from sites where their strictly saprobic siblings are found. The infective potential thus differs between closely related species, and the concept of opportunism perhaps has to be revised for CBM. Phaeohyphomycosis is characterized by the presence of melanized, septate hyphal elements or sometimes yeast-like cells in tissue. Innate immune response to these elements leads to necrosis, which may become destructive.13 Black yeasts have also been involved infections of cold-blooded animal. Frog, toad, and fish infections have been frequently reported,12 and Vicente et al.14 descried an extensive epizootic caused by the black yeast Fonsecaea brasilienses and which led to massive death of the mangrove-land crab Ucides cordatus (Brachyura: Ocypodidae) along the Brazilian coast. Remarkably, infections in warm-blooded vertebrates other than humans are rare.15,16 Ecology of black fungi Black yeasts (Exophiala species, particularly E. dermatitidis) thrive in man-made habitats, such as dishwashers,17 saunas,18 sinks,19 and bathing facilities.20 Their close vicinity to human hosts would pose a public health risk if the fungi were genuinely pathogenic. But household-acquired infections are either mild21 or are restricted to susceptible patient populations with genetic disorders such as those with cystic fibrosis.22 Nevertheless, the occurrence of severe infections, for example, in patients with primary immunodeficiencies, remains an area of concern. Among the primary immunodeficiency diseases, inborn errors in the gene that codes for caspase recruitment domain-containing protein 9 (CARD9) have been reported in patients with phaeohyphomycosis.23 In addition, acquired conditions, such as human immunodeficiency virus (HIV)-infected patients, as well as patients receiving solid-organ transplants or under immunosuppressive therapies, have also been responsible for disseminated infections. Genome and transcriptome analyses may contribute to understanding of the genes enabling black fungal opportunistic infection in general, and solve questions concerning the possible adaptation of CBM agents toward pathogenicity. Next-generation sequencing (NGS) The application of high-throughput sequencing technologies to elucidate the genetic bases of pathogenicity and niche adaptation in BY started in 2011 when the first whole-genome sequence belonging to a chaetothyrialean fungus, Exophiala dermatitidis, was released as a part of the Broad Institute Fungal Genome Initiative (http://www.broadinstitute.org/annotation/genome/Black_Yeasts/MultiHome.html) and published by Chen et al.20 Since that publication, efforts have led to sequencing and analysis of an additional 37 genomes and transcriptomes, producing an avalanche of data for comparative genomics (Fig. 1, Table 1). Overall, these genomes have been determined using short-read approaches, based on the Illumina, Ion Torrent and/or 454 instruments (Table 1). These NGS sequencing technologies rely on cyclic reversible termination, single nucleotide addition and pyrosequencing, respectively. For a general survey on NGS technologies, please check the review paper by Goodwin et al.24 Although the majority of projects only used a single NGS platform to determine the genome, the combination of different technologies could avoid systemic bias generated by using a single sequencing approach.24 In addition, the use of distinct library preparation methods might prevent bias caused by the PCR amplification, an important source of bias from DNA-seq library preparation protocols. At the transcription level, the dUTP second strand marking method25 has been adopted in at least eight transcriptome studies26 and turned out to be the leading protocol for RNA sequencing of black yeast species. Figure 1. View largeDownload slide Overview of black yeast genome studies between 2014 and 2017. The time line shows important landmarks of black yeast genome sequencing projects, mainly through the Fungal Genome Initiative of the BROAD institute of MIT and Harvard. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Overview of black yeast genome studies between 2014 and 2017. The time line shows important landmarks of black yeast genome sequencing projects, mainly through the Fungal Genome Initiative of the BROAD institute of MIT and Harvard. This Figure is reproduced in color in the online version of Medical Mycology. Table 1. Whole-genome sequencing of black yeast and relative deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/genome). Species  Collection number  Instrument  Sequencing center  Reference  Capronia coronata  CBS 617.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia epimyces  CBS 606.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia semiimmersa  CBS 27337  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  CBS 173.52  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  UM 956  Illumina - HiSeq 2000  University Malaya  Kuan et al.54  Cladophialophora carrionii  dH 23894  454  LNCC  Teixeira et al.26  Cladophialophora carrionii  CBS 160.54  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 834.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 110551  ION Proton Technology  University of Natural Resources and Life Sciences  Sterflinger et al.74  Cladophialophora psammophila  CBS 110553  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora yegresii  CBS 114405  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Coniosporium apollinis  CBS 100218  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala alcalophila  JCM 1751  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala aquamarina  CBS 119918  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala calicioides  JCM 6030  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala dermatitidis  NIH/UT8656  Illumina - HiSeq 2000  Broad Institute  Chen et al.43  Exophiala mesophila  CBS 120910  ION Proton Technology  University of Natural Resources and Life Sciences  Tafer et al.75  Exophiala mesophila  CBS 402.95  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala oligosperma  CBS 725.88  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala sideris  CBS121828  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  CBS 899.68  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  JCM 15939  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala xenobiotica  CBS 118157  Illumina - HiSeq 2000/ION Proton Technology  Broad Institute  Teixeira et al.26  Exophiala xenobiotica  CBS 102455  ION Proton Technology  University of Natural Resources and Life Sciences  N/A  Fonsecaea erecta  CBS 125763  Illumina - HiSeq 2000  Federal University of Paraná  Vicente et al.55  Fonsecaea monophora  CBS 269.37  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Bombassaro et al.76  Fonsecaea multimorphosa  CBS 102226  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Fonsecaea multimorphosa  CBS 980.96  Illumina - MiSeq  Federal University of Paraná  Leao et al.77  Fonsecaea nubica  CBS 269.64  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Costa et al.78  Fonsecaea pedrosoi  CBS 271.37  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Ochroconis mirabilis  UM 578  Illumina - HiSeq 2000  University Malaya  Yew et al.79  Phialophora attae  CBS 131958  ION Proton Technology  Westerdijk Institute  Moreno et al.80  Phialophora europaea  CBS 101466  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Rhinocladiella mackenziei  IHM 22877  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  dH 24460  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  CBS 650.93  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Verruconis gallopava  CBS 437.64  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Species  Collection number  Instrument  Sequencing center  Reference  Capronia coronata  CBS 617.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia epimyces  CBS 606.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Capronia semiimmersa  CBS 27337  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  CBS 173.52  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora bantiana  UM 956  Illumina - HiSeq 2000  University Malaya  Kuan et al.54  Cladophialophora carrionii  dH 23894  454  LNCC  Teixeira et al.26  Cladophialophora carrionii  CBS 160.54  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 834.96  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora immunda  CBS 110551  ION Proton Technology  University of Natural Resources and Life Sciences  Sterflinger et al.74  Cladophialophora psammophila  CBS 110553  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Cladophialophora yegresii  CBS 114405  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Coniosporium apollinis  CBS 100218  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala alcalophila  JCM 1751  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala aquamarina  CBS 119918  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala calicioides  JCM 6030  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala dermatitidis  NIH/UT8656  Illumina - HiSeq 2000  Broad Institute  Chen et al.43  Exophiala mesophila  CBS 120910  ION Proton Technology  University of Natural Resources and Life Sciences  Tafer et al.75  Exophiala mesophila  CBS 402.95  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala oligosperma  CBS 725.88  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala sideris  CBS121828  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  CBS 899.68  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Exophiala spinifera  JCM 15939  Illumina - HiSeq 2500  RIKEN Center for Life Science Technologies  N/A  Exophiala xenobiotica  CBS 118157  Illumina - HiSeq 2000/ION Proton Technology  Broad Institute  Teixeira et al.26  Exophiala xenobiotica  CBS 102455  ION Proton Technology  University of Natural Resources and Life Sciences  N/A  Fonsecaea erecta  CBS 125763  Illumina - HiSeq 2000  Federal University of Paraná  Vicente et al.55  Fonsecaea monophora  CBS 269.37  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Bombassaro et al.76  Fonsecaea multimorphosa  CBS 102226  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Fonsecaea multimorphosa  CBS 980.96  Illumina - MiSeq  Federal University of Paraná  Leao et al.77  Fonsecaea nubica  CBS 269.64  Illumina - MiSeq/ION Proton Technology  Federal University of Paraná  Costa et al.78  Fonsecaea pedrosoi  CBS 271.37  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Ochroconis mirabilis  UM 578  Illumina - HiSeq 2000  University Malaya  Yew et al.79  Phialophora attae  CBS 131958  ION Proton Technology  Westerdijk Institute  Moreno et al.80  Phialophora europaea  CBS 101466  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Rhinocladiella mackenziei  IHM 22877  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  dH 24460  Illumina - MiSeq  Westerdijk Institute  Moreno et al.31  Rhinocladiella mackenziei  CBS 650.93  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  Verruconis gallopava  CBS 437.64  Illumina - HiSeq 2000  Broad Institute  Teixeira et al.26  View Large Genomic patterns in BY With the whole-genome sequence of 27 distinctive species already published, the Chaetothyriales is currently the seventh most sequenced order of the division Pezizomycotina. The ensemble of available genomes comprises species that display a wide diversity of lifestyles, including the human opportunists involved in disseminated and neurotropic infections Cladophialophora bantiana, Fonsecaea monophora, Exophiala dermatitidis, and Rhinocladiella mackenziei; the etiologic agents of chromoblastomycosis Cadophialophora carrionii and Fonsecaea pedrosoi and their environmental counterparts Cl. yegresii and F. erecta, respectively; the species associated with the degradation of toxic hydrocarbons Exophiala oligosperma, E. xenobiotica, E. spinifera, Cl. immunda and Cl. psammophila; the ant-associated Phialophora attae; and several saprobic species occasionally reported causing mild infections. All BY genomes are similar in size, ranging from 25.8 Mb in Capronia coronata to 43 Mb in Cladophialophora immunda CBS 834.96 and a typical genomic feature seems to be the exceptionally high GC content (49%–54.3%), with comparable values only having been reported in Sporothrix. Similar genome sizes have been described in the genus Aspergillus, while reduced genomes are present in the order Onygenales.27 With few exceptions, the largest black yeast genomes belong to the members of the bantiana-clade, and smaller genomes are found in species of the dermatitidis-clade. Interestingly, an association seems apparent between genome size and ecological preferences. A potential explanation for the larger genomes in many species of the bantiana-clade is the striking abundance of the protein families short chain dehydrogenase, CYP P450, aldehyde dehydrogenase, sugar transporters, and membrane transporter proteins.28 It is not a surprise for black yeasts to contain expanded protein families associated to acquisition of nutrients, transporters and environmental stresses, since these families tend to be overrepresented in extremely tolerant fungi such as the halophile Hortaea werneckii.29 Protein family expansion as the result of gene duplication might have generated raw genetic material and consequently enhanced the metabolic plasticity and increased the fitness of black yeasts during the colonization of hostile ecological niches. Another interesting feature is the presence of physically linked genes, forming metabolic clusters, as patterns of selections for reduced accumulation of toxic intermediate compounds (ICs). The order and orientation of genes participating in metabolic pathways has been proposed as an important genomic adaptation against the accumulation of ICs in fungi.30 For example, in Rhinocladiella mackenziei the pathway involved in the conversion of choline to the osmoprotectant glycine betaine produce the toxic IC betaine aldehyde.31 In this fungus, the enzymes choline dehydrogenase and betaine-aldehyde dehydrogenase are adjacent pairs. Such gene organization may synchronize their activity by means of co-expression or prevent independent gene loss that could lead to disturbance in the metabolic cluster.32 The physical linkage of choline metabolism genes has also been described in other fungi.30 Numerous other metabolic gene clusters have been described in BY, many of which are involved in the degradation of xenobiotics, such as toluene and in secondary metabolite biosynthesis.26,33 In contrast to unrelated fungi such as Alternaria brassicicola and Cladosporium sphaerospermum,34 BY genes are not organized in clusters in any of the three pathways for the production of melanin.26 Melanin is the pigment responsible for the dark coloration of BY cells; it is produced by the oxidation of tyrosine in a multistep reaction involving enzymes that are often organized into supergenes or clusters in most fungi. Evolutionary genomics Orthologs and paralogs represent subcategories of homologous genes, which evolved, respectively, by vertical descent (speciation) from a single gene in the last common ancestor, or by gene duplication.35 Although the biological function of these groups of genes, and their products, is not necessarily maintained after such evolutionary processes, ortholog and paralog classification has widely been used to make inferences about the relationship between genes.36 For example, analysis of inparalogs, a subtype of paralogs originated by gene duplication after a speciation event, is important for detecting lineage-specific adaptations and emergence of functional novelties.37 Teixeira et al.26 reported extensive gene duplication processes in several protein families in 23 BY genomes. Among the expanded families, cytochromes p450 (CYP), drug efflux pumps, alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDHs) seem to be broadly distributed across black yeasts, and their substrate promiscuity suggests that they have played critical roles in niche adaptation. Gene duplication of these families might have provided raw material for new open reading frames (ORFs), often with different selective pressures, which could be used to enhance flexibility in acquisition and protection against stressful environments. The duplicated genes could increase fitness in an environment where gene dosage is important. Interestingly, in many opportunistic fungi, such as in Aspergillus fumigatus38 and Candida albicans,39 paralogous genes implicated in infection appear to be located in telomere-proximal regions or even in mobile pathogenicity chromosomes, as verified in the plant pathogen Fusarium oxysporum.40 The location of duplicated genes in BY has not yet been assessed. Similarly, the identification of fast-evolving genes, which are most likely to play a role in host-pathogen interaction, has not yet been addressed in black yeast. Moreno et al.31 reported that in the neurotrope Rhinocladiella mackenziei the paralogous genes accumulate more important mutations, that is, with probably severe impact on their biological function, than orthologous genes. Ancestral state reconstruction analysis determined that the expansions of these enzymes took place during the Cretaceous period < 80 million years ago (Mya),26 likely before the radiation of the families Herpotrichiellaceae and Cyphellophoraceae, which is believed to have occurred in the presence of a new substratum.41 The ecology of black yeast is too complex for a single hypothesis, but some striking aspects may have an intriguing explanation. The preference of many BY for ecological habitats polluted with harmful aromatic hydrocarbons may have been enhanced by the massive gene duplication in ADH, ALDH, drug efflux pumps, and CYP p450 that might have formed the basis for the metabolic versatility observed in modern black yeasts. The ancestor of these BY may have colonized extreme habitats such as hydrocarbon-rich ant- and termite-nests, and in part may have diversified along with these insects.26 Similarly, other important events of genome evolution, such as horizontal gene transfer (HGT), gene rearrangements, and gene loss have been subject to researches to explain central evolutionary processes in BY.33,42 Considered a key source of metabolic gene innovation in fungi, significant cases of HGT took place in black yeasts, particularly involving xenobiotic catabolism,33 inteins,26 and precursors for glycosyltransferase enzymes.43 Events of gene loss are rare in black yeasts, only having been reported in a few protein families in Rhinocladiella mackenziei.31 Ant-association lifestyle and the origin of the polyextremotolerance Phylogenomic studies, in combination with classical phylogenies based on taxon-rich data matrices, have shed light onto the history of life and evolutionary patterns in fungi.44 Phylogenies have been extensively used to explain biological interactions, such as the mutualistic association between fungi and ants.45 These insects are able to produce natural substances with antimicrobial activities that are used to protect their colonies from disease. In many ant species, phenylacetic acid seems to be a major active component against pathogens.46 The list of secreted toxins produced by ants also includes indole-3-acetic acid, 3-hydroxydecanoic acid, and 3-hydroxy acids, in addition of plenty of phenol derivates, such as 3-propylphenol, 3-pentylphenol, 5-propylresorcinol, 5-pentylresorcinol.47,48 As a consequence, only a small number of fungi adapted to tolerate such toxins are able to live in association with these insects. An updated phylogeny, recently published by Vasse et al.,49 revealed that ant-associated black yeasts are extremely frequent and scattered through the phylogeny of the order Chaetothyriales. This interaction was previously reported by Voglmayr et al.50 and Nepel et al.,51 who uncovered at least three distinct clades that accommodated BY from ant-cartons or -domatia. A speculative scenario to explain the ant-BY symbiosis might be found in the harsh and toxic environmental conditions of lichen-colonization, which produce toxic chemicals, and a subsequent environmental jump after manipulation by ants. This hypothesis was firstly proposed by Voglmayr et al.50 using phylogenies of Gueidan et al.52 and gained support with comparative genomic analyses that have recently come to light. Teixeira et al.26 identified a putative route for the catabolism of phenolic compounds via phenylacetic acid and homogentisate and reported a striking abundance of transporters and cytochromes p450 potentially involved in detoxification processes. The authors also predicted that these genetic adaptations were present in the common ancestor of the studied species and were maintained throughout the evolution of the BY. Since the calibrated phylogenetic tree estimated that the radiation of the Chaetothyriales species took place circa 75–50 MYA, during/after the Cretaceous-Paleogene (K-Pg) extinction event, and later than the origin of the ants (estimated in Early Cretaceous to Middle Jurassic, 140–168 MYA), the ant-association as an ancestral trait seems to be a plausible scenario to explain the origin of metabolic plasticity in the black yeasts. Because none of the comparative analysis studies thus far identified striking differences between clinical species and saprobes, we hypothesize here that the entire order has maintained similar ecological preferences, which were acquired during ancestral ant-interactions; species virulence is based on secondary characters, such as ability to survive phagocytosis or to be carried by blood. Free-living lifestyles versus infection While the identification of virulence factors is relatively common in bacterial comparative genomic studies, the prediction of fungal genes involved in infection is challenging.53 None of the recent publications reporting whole genome sequencing of the BY and subsequent comparative genome analysis have discovered unambiguous pathogen-specific genes. The candidates predicted as potential virulence factors, such as enzymes involved in adaptation to new nutrient sources and environmental stresses, are more associated with saprobic lifestyles than with pathogenicity. In part this may be explained by the fact that all the genomes sequenced so far belong to two derived families of Chaetothyriales: Cyphellophoraceae and Herpotrichiellaceae. In order to provide insights into the origin of infectivity it is essential to sequence species that are taxonomically not too remote but have consistently different ecological preferences. Studies thus far unintentionally addressed species with similar genetic make-up, with only gradational differences in, for example, predilection for aromatic hydrocarbons. Species causing disease in warm- versus cold-blooded animals may share traits that permit their growth in the environment, and if this is physiologically stressful this might also allow their survival inside the human body. In an attempt to identify genes that are associated with cerebral phaeohyphomycosis caused in Rhinocladiella mackenziei, Moreno et al.31 proposed a putative route for the degradation of aromatic compounds, which may be environmental contaminants such as styrene and ethylbenzene, but also neurotransmitters that are abundant in the brain. Similarly, Kuan et al.54 performed a genomic comparison of Cladophialophora bantiana, the prevalent cause of brain infection in healthy patients, with other neurotropic fungi and concluded that this fungus has probably evolved as an environmental saprobe. Although these two studies have identified several genes that potentially engaged in pathogenicity, it remains unclear why these species show predilection for the central nervous system. Additionally, other comparisons of opportunistic BY and their environmental counterparts, such as Cl. bantiana versus Cl. psammophila and Cl. carrionii versus Cl. yegresii, did not reveal striking genomic differences or host adaptations that could explain their infective potential.26 The genomes of Cl. carrionii and Cl. yegresii share more than 90% of their proteins, and functional annotation revealed high similarities in carbohydrate-active enzymes and peptidases.26 In line with this, a recent study of survival tests using Galleria mellonella larvae infected with clinical F. pedrosoi, and F. monophora, with saprobic F. erecta showed that the environmental species is equally able to infect and survive in animal host tissue.55 We may state that the terms pathogenic and nonpathogenic have been inaccurately used at the BY species level, causing misleading interpretations. The term ‘opportunism’ is more adequate for most BY, which implies that the fungus is basically saprobic but can survive in animal tissue. Comparative genomic analyses of true human pathogens, such as Coccidioides immitis and C. posadasii against closely related, nonpathogenic relatives, have revealed pathogen-specific adaptations,56 the fungi having retained or acquired enzymes involved in the scavenging of iron and in energy metabolism, while genes associated with plant degradation, such as cellulose, cutinase, pectins as well as genes required for carbohydrate metabolism, were lost. The study concluded that these are true pathogens that are associated with animal hosts.56 In contrast, black yeasts are found in niches that are hostile to microbial competitors, and therefore they exhibit a significant degree of extremotolerance. This general stress tolerance enables them to survive inside living host tissue and cause infection, but as most of them are unable to be transmitted from the host this is not a strategy to enhance their evolutionary fitness. Given the remarkable prevalence of some agents of CBM on human hosts, it may be speculated that some BY have adapted to pathogenicity by being able to escape from the host and transmit acquired mutations to next generations. Black yeasts in the post-genomic era Describing the sequence of a wide variety of black yeast genomes is only a first step to shed light on adaptive processes, including shifts to pathogenicity. The next stage of research is the so-called post-genomic era, which already started for BY, translating knowledge from DNA to transcriptome and proteome analyses. Attempts have been made to elucidate the transcriptional response of BY to stressors such as temperature, pH, radiation, and toxicity.33,43,57,58 The first BY with an assessed transcriptome was Exophiala dermatitidis, often considered as a model for melanized agents of disease.58,59 Robertson et al.58 analyzed gene expression in responses to low-dose ionizing radiation and discussed the importance of the cell wall pigmentation for tolerance; melanin was associated with overexpression of ribosomal biogenesis and transporter genes.58 Several other studies using BY have underlined the overproduction of melanin as a key response to stress. Chen et al.43 compared messenger RNA expression in E. dermatitidis upon alteration of the pH of the growth medium. Three pathways for melanin production were detected: via 1,8-dihydroxynaphthalen (DHN), via 3,4-dihydroxyphenylalanine (DOPA), and via l-tyrosine degradation, and all were activated during pH-stress. Similar results were obtained when Cladophialophora immunda was grown in the presence of toluene as sole carbon and energy source.33 Overall, these results demonstrate the importance of cell wall pigmentation for extremotolerance, providing a barrier against oxidative damage. A role of melanin in skin infection was assessed by Poyntner et al.,60 identifying genes that were transcriptionally altered during the artificial infection of an ex vivo skin model by E. dermatitidis. The transcriptome of an albino strain of Fonsecaea monophora was compared with its melanized parent by Li et al.,61 who identified 2283 differentially expressed genes between the genotypes. Extensive down-regulation of key genes in the DHN pathway was shown in the albino mutant, which was also more susceptible to low pH, high UV radiation, and oxidative stress.62 Down-regulation of melanin-associated genes was also verified in E. dermatitidis under conditions of low temperature (1°C for 1 week), indicating that melanin production changes in response to temperature variation.57 Not only transcriptional studies have been used to better understand the extremophilic character of BY. At proteomic level, Teset et al.63 analyzed the repertoire and abundance of proteins of E. dermatitidis under temperature stress and reported striking changes in its proteome. Long-term exposure to the cold (1°C) reduced metabolic activities of carbon and pyruvate metabolism, but high temperature (45°C) seemed not to induce any stress response, likely reflecting the high level of adaptation of E. dermatitidis to thrive at elevated temperature.63 Down-regulation of genes involved in metabolic processes, including metabolism of amino acids, carbohydrates and glycolysis, are consistent with and complementary to earlier reports in E. dermatitidis transcriptomics.58 In addition, the assessment of the proteins is an important tool to construct phylogenies using a large number of proteins shared by all sequenced black yeasts. As a consequence, geological time-calibrated trees, in addition to specific protein family expansions, provided important insights into the genomic pre-adaptations that might have driven the ecological preferences observed nowadays. Interestingly, species phylogenies based on amino acid sequences, corresponding to multiple single copy orthologous genes, produced similar topologies compared to the previously published phylogenetic trees.64,65 Future directions The employment of ‘omics’ technology, such as genomics, transcriptomics, and proteomics, to assess the genetic information of BY, is now a reality, and an enormous amount of data has already been generated. In addition, the application of other omics technologies, including metagenomics and metabolomics, offers a unique opportunity to interrogate the diversity of this group of fungi and will certainly be soon incorporated into high-throughput analysis using black yeast like-fungi (Fig. 2). A crucial step to keep these projects running is the development of a repository to provide access to this information for comprehensive curated analyses. Similar initiatives have been set up for other fungi, such as Candida66 and Aspergillus.67 In BY a collaborative effort has been made with the launch of a beta-version of a database: the Black Yeast HUB (www.blackyeasthub.com) was developed using GMOD68 and Tripal,69 a combination of database and interactive web pages for manipulating and displaying genome annotations. The current version of the Black Yeast HUB provides the complete genomes of 37 strains and 35 protein sets of chaetothyrialean fungi from the families Herpotrichiellaceae and Cyphellophoraceae. Additional information about gene structure, gene products, phylogenetic analyses, metabolic pathways, gene families and groups of orthologous genes is available for the 35 strains where gene annotations were previously performed. Figure 2. View largeDownload slide Potential applications of “omics” technology in black yeast research. ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; MS, mass spectrometry; qPCR, quantitative polymerase chain reaction; SNP, single nucleotide polymorphism; TLC, thin-layer chromatography. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Potential applications of “omics” technology in black yeast research. ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; MS, mass spectrometry; qPCR, quantitative polymerase chain reaction; SNP, single nucleotide polymorphism; TLC, thin-layer chromatography. This Figure is reproduced in color in the online version of Medical Mycology. With declining costs of next-generation sequencing and the availability of high standard reference genomes, one can expect that in the coming years members belonging to the Trichomeriaceae, Chaetothyriaceae, and Epibryaceae, considered as ‘ancestral’ families of Chaetothyriales, will be sequenced. Inclusion of ancestral species will considerably improve molecular dating of the ordinal tree and possibly confirm raised hypotheses on ant association and aromatic hydrocarbon degradation. The application of post-genomic transcriptomics and proteomics has raised our understanding of stress response in BY to a new level. Although the molecular machinery of pathogenicity is far from being elucidated, important indications have been obtained.60 Other initiatives, such as the use of in vivo models for mimicking the state of infection must be encouraged. The combination of omics techniques and advanced computational analyses cannot only be used to elucidate the origin of pathogenicity, it might also be applied as a diagnostic tool. Smeekens et al. highlighted the importance of omic approaches, such as transcriptomics, to classify and distinguish candidemia from Staphylococcus aureus infection.70 In addition, infection progression could be monitored assessing the transcription profile of specific genes, in which the expression is time-dependent.71 Similarly, proteomics has been proved to be effective to identify clinical pathogens, including Aspergillus species72 as well as candida isolates with reduced caspofungin susceptibility.73 In disseminated infections caused by black yeasts, where the rapid and accurate diagnosis is needed for therapeutic decisions, the discovery of specific biomarkers from omics data is of high importance, from which efficient detection methods, such as in situ hybridization, can be developed. 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Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Apr 1, 2018

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