Origin of Pathogenicity in Clinical Fungi

Understanding pathogenicity and virulence of fungi causing infections in humans is of prime importance in the hospital, predicting clinical course and applying adequate and cost-effective therapy. The clinical world is focused on the major causative agents of disease, particularly Candida, Cryptococcus and Aspergillus. Outside these groups, a large diversity of potentially harmful fungi exist which may be rare, but frequently have the pathogenic potential to be more important than the common clinical fungi. Several are able to cause fatal disease in otherwise perfectly healthy human hosts. Our objective is to reveal the natural ecology and routes of transmission of such fungi, in order to explain their pathology. We carry out a comparative approach, in order to place known virulence factors in perspective. Since most of the clinically significant fungi are opportunistic, a large share of our work is devoted to extremotolerant fungi that possess several factors in common to fungi of clinical significance.

Black yeasts in Chaetothyriales: evolution of human pathogenicity
With the standard application of molecular diagnostics to the black yeasts, that were previously notoriously hard to identify, a new world of fascinating fungi was entered. The ascomycete order Chaetothyriales comprises nearly all melanized fungi that are recurrent on human hosts, and some of them cause extreme, fatal infection in otherwise perfectly healthy humans. The nature of these diseases has always been puzzling, as they seem to differ with the species, but sometimes also with the patient population or with geography. Rhinocladiella mackenziei only occurs in the Middle East; Exophiala spinifera causes severely mutilating, disseminated infections in healthy adolescents only, and brain infections by Exophiala dermatitidis are relatively frequent in East Asia only, although this fungus has a world wide distribution. We sequenced large sets of strains from clinical and non-clinical sources from all over the world, and found indeed that diseases by black yeasts are mild or associated with immune disorders in the U.S.A. and Europe, and frequently take a fatal turn in healthy individuals East Asia. We now have sufficient statistics to prove this endemism is a fact. In the case of Exophiala we suppose that host-factors may play a role, Asians being more susceptible to this type of infection.
            Understanding the origin and routes of transmission of potentially fatal infections is essential for taking preventive measures. The major neurotropic species, E. dermatitidis, was found to be abundantly present in Turkish steam baths, but infection does not necessarily take place by inhalation. An ingestive route has been hypothesized, which is unique in the fungal Kingdom. The fungus may remain asymptomatically in the intestinal tract, and it could cause brain infection via intestinal ulcers and subsequent haematogenous dissemination. Ultimate source of infection seems to be wild berries from the tropical rain forest: the fungus was found there in association with frugivorous animals, such as hornbill birds and flying foxes. This occurrence in rain forests has been proven in Thailand, Nigeria and Brazil. Two multilocus genotypes are observed, which seem to have separated very recently. These genotypes were also recognized with Maldi-tof, and thus differences at the phenetic level are likely. The two genotypes have more or less equal distribution in the natural niche, but their frequences diverge in the human environment: a thermophilic genotype is prevalent in steam baths, whereas a more mesophilic genotype is preponderant in systemic infection: all brain cases analyzed thus far were of a single genotype. Pathogenic speciation seems to occur in Exophiala dermatitidis.
Chromoblastomycosis is a unique clinical syndrome, caused exclusively by members of the Chaetothyriales. The etiologic agents are endemic in semi-arid climates, and thus speciation is probably determined by environmental factors. Cladophialophora carrionii is known to occur in cactus debris. A sympatric sister species, C. yegresii was discovered, which is an endophyte of adjacent cactus plants and produces muriform cells – the invasive form in humans – in cactus spines. Nevertheless clinical cases are exclusively caused by C. carrionii. Differences in virulence were also noted in the plants: C. carrionii was found to be less adapted to growth in cactus. An evolutionary step from plant to human seems to have taken place, and thus transmission from the infected human, by skin scales or from the dead body, must be hypothesized. A considerable phylogenetic distance was noted between the two species which are phenetically very similar, which suggests that the step from plant-association to human virulence and opportunism is an exceptional route.       Large sets of Fonsecaea strains were obtained from different continents, which enabled us to reveal epidemiological patterns. Populations showed considerable structuring.
The Cladophialophora phenotype – i.e., simple or branched acropetal conidial chains without supporting conidiophores – is widely observed in the fungal kingdom. All Cladophialophora phenotypes with infective potential thus far were shown to belong to the order Chaetothyriales. Within this order a number of new species were described, one of which caused chromoblastomycosis in Polynesia. The remaining species seemed to be saprobes, some occurring in slightly osmotic foodstuffs such as juices and tea. Host-specific plant pathogens with this morphology were basal to the Chaetothyriales. Except for the occasional endophytes mentioned above, plant and human pathogenicity seems to be mutually exclusive.
Early diverging species within the Chaetothyriales are mostly involved in mild cutaneous disease and onychomycosis. This phenomenon has thus far been overlooked, because dermatologists tend to discard non-dermatophytes as supposed contaminants. Jointly with clinicians from Germany, Denmark and China we have revealed that a number of chaetothyrialean fungi are consistently found in such clinical samples and are likely to play a role in pathology. New species have been discovered, several of which are limited to human superficial samples. Remarkably, the group also contains meristematic, rock-inhabiting coniosporium-like species, which also predominate among the basal branches of Chaetothyriales. Given the fact that the muriform cell of chromoblastomycosis is a virulence factor, the role of the CDC42 gene (cell division cycle) in pathogenic evolution is being analyzed. Suppressive subtractive hybridization (SSH) techniques are being developed and adapted to black yeasts.
Recent broad-scale phylogenetic analyses have shown that the order Chaetothyriales was sister to the lichen order Verrucariales. The sister relationship between these two ecologically very different orders (the Chaetothyriales, first known as including mostly saprobes and opportunistic animal and human parasites, and the Verrucariales, comprising mostly lichenized saxicolous species) was puzzling. However, investigations of fungal communities colonizing rocks in extreme environments have shown that some slow-growing melanized fungi inhabiting bare rock surfaces belonged to the Chaetothyriales. Therefore, it was hypothesized that the rock-inhabiting habit might be the ecological link between these two orders. Multigene phylogenetic analyses were carried out using Maximum Likelihood and a Bayesian approach in order to confirm the affiliation of 25 of these rock isolates. An ancestral state reconstruction was then undertaken to look at the evolutionary history of the rock-inhabiting habit within Eurotiomycetes, the class of fungi to which Chaetothyriales and Verrucariales belong. Results suggest that the ancestor of the lineage including Verrucariales and Chaetothyriales was likely to be an extremotolerant non-lichenized, rock-inhabiting fungus. Verrucariales, a lichenized lineage with mostly species growing on rocks, seems to have evolved from this non-lichenized rock-inhabiting ancestor, independently from other groups of lichens. Within the Chaetothyriales, potential virulence factors, such as melanization and meristematic growth, might have primary been adaptations for life in extreme habitats in an extremotolerant rock-inhabiting ancestor.
            The Chaetothyriales are also striking for their ability to grow with toxic monoaromatic alkylbenzenes as sole source of carbon. They are consistently isolated from soil polluted with xenobiotics, and are also derived from natural environments such as berries – containing monoaromatic precursors of tannins – by the use of toluene and benzene enrichment techniques. The species encountered usually are sister species of known pathogenic black yeasts, but rarely the pathogens themselves. This suggests that alkylbenzene assimilation is a symplesiomorph enhancing pathogenic evolution, but ecological specialization interferes with the alternative ecology. A similar phenomenon has been observed in the fungal Kingdom with osmotolerance: groups containing osmotolerant and pathogenic species are strongly overlapping, but at the species level the two ecologies are mutually exclusive. The role of hydrocarbon assimilation is being analyzed in more detail, as it also seems to play a role in some other groups, such as Pseudallescheria and Fusarium. Remarkably, these are also the groups with disseminated and neurotropic opportunism, as contrasted with Aspergillus, which is preponderantly pulmonary.
            The fact that members of Chaetothyriales, albeit opportunists, have intrinsic virulence factors is proven by the fact that the order contains a large clade of waterborne species which frequently cause fatal disease in cold-blooded animals such as fish, turtles, sea dragons and toads. Some are even host-specific, causing severe epidemics. A remarkable disease was observed in mangrove crabs along the Brazilian coast, expanding each year since 1995. 
Ochroconis stands out in the fungal Kingdom, with no known teleomorph species at close distance. Although cultural features and microscopic morphology of species are highly characteristic, taxa are extremely diverse genetically, suggesting large phylogenetic distances from one species to the next. Ecologically there is similarity with the Chaetothyriales, the group containing fish-opportunist and a thermophilic species causing animal and human brain disease. We suppose that the phylogenetic position of the genus must be quite basal to the ascomycetes, diversities in introns and spacers suggesting a long evolutionary history with constancy at the expression level, as we also observed in Zygomycetes and some Hemiascomycetes.

Black yeasts in Dothideales: life in the most extreme environments on earth
Black yeasts of the order Dothideales / Capnodiales are heavily melanized and show extensive meristematic conversion in response to extreme conditions of dryness and solar irradiation. Numerous species are found colonizing inert surfaces such as glass, metal and rock. In nature these fungi are extremely common with a bewildering biodiversity, which is still to be described. A particularly interesting ecology is that of rock under extremely cold conditions, such as the Antarctic and the Himalaya. Conditions are so harsh that microorganisms creep into crevices between crystals in order to be subjected to milder conditions, leading to cryptoendolithic communities. Several new genera have been discovered, and many more are still to be described. Morphology is often highly reduced, sometimes expressed in members of the same species, and this has lead to a hypothesis of sudden environmental jumps with fixation in a less versatile but highly resistant phenotype. Adaptation of extremotolerant fungi is a matter of loss rather than of gain. A large clade is uncovered merely consisting of species at a reduced expression level and apparently without sexuality. This is in conflict with the hypothesis that anamorphs are unable to survive over time due to Mueller’s ratchet. We suggest that extremotolerance by reduction is a highly successful strategy, as long as prevalent conditions remain extreme. Adjacent clades also contain halophilic and acidophilic taxa, able to grow with saturated salt and at pH below 1, respectively.
An intensive cooperation with a research group on extremophilic fungi in Slovenia has lead to the description of several new genera and species that are differentially able to survive salty conditions near to the saturation point. The species play different roles during the crystallization process in salterns in subtropical climates. Hortaea werneckii, one of the model organisms, was proven by expression of different genes in the salinity range to have a small fitness depression at 17% salt. Better survival was obtained at lower as well as at higher salinities: the HMGR gene, expressed at low and high salinities, played a crucial role. The species is an unambiguous halophile. The clinical syndrome ‘tinea nigra’, known in adolescents after a beach holiday, is just superficial colonization of salty hands. The species has no invasive potential and should be listed at a lower BioSafety Level.
Fitness depressions are the leading hypothesis of apparently sudden adaptations to the extreme of mesophilic Aureobasidium species. The common fungus Aureobasidium pullulans occurs worldwide with only two (LSU) genotypes. In a very small sampling area in and around glaciers of Spitsbergen, a stunning diversity of Aureobasidium species and populations was found. Through moulins of the glaciers the subglacial ice is reached, which is extremely salty and alkaline. A specific Aureobasidium genotype was found, which was not encountered anywhere else. We hypothesize that this is a highly adapted fungus emerged through accelerated evolution and episodic selection, that becomes extinct immediately when it reaches the milder conditions of normal seawater.

Barcoding, detection and microarray techniques
Clinical laboratories witness a steady increase in the number and diversity of medically important fungi. Morphological species recognition is frequently hampered by the lack of distinctive characters of the strains or the missing expertise of the staff for rare opportunistic fungi. Also DNA-based identification with the aid of public reference databases has its limitations, because of the insufficient taxonomic sampling, the significant proportion of misidentified species in GenBank, and/or the occasional presence of reading errors. We aim to develop confident, dedicated databases for several taxonomic groups of clinically important fungi, such as filamentous basidiomycetes, Zygomycetes, dermatophytes, black yeasts and the Pseudallescheria/Scedosporium complex. This will allow rapid and accurate routine identification based on ITS barcodes. These databases will be publicly available through the CBS website.
The reliability of identification tools strongly relies on comprehensive sampling. The CBS collection contains a large number of type isolates and other well-studied strains that were used to generate the DNA barcodes. In order to assess the intraspecific variability we included strains from different geographic regions, and clinical as well as environmental strains covering a wide range of habitats and substrates. Understanding interspecific divergence ideally includes all species of a clade, and an underlying phylogenetic hypothesis to define the most closely related extant relatives. In addition to the study of species represented in the CBS collection we therefore cooperate with other institutions to fill the gaps in species coverage. The resulting set of barcodes makes it possible to delineate species and to develop “validated barcodes” (= consensus sequences of all barcodes of a certain species). Our databases will allow the user to perform a BLAST analysis for identification of unknown clinical isolates and a Neighbor joining analysis based on binary alignments. In addition to the position in the NJ tree, the alignments of the unknown sequence with the most similar barcodes, the program will also provide an alignment with the “validated barcode” of the target species. In well studied groups such as anthropophilic dermatophytes we anticipate that currently available barcodes cover the existing ITS diversity. However, databases of poorly understudied groups such as the Psathyrellaceae (medically important basidiomycetes) and the black yeasts are likely to be subject to permanent change due to incomplete sampling. For less-known clinical fungi global interaction with clinical networks is mandatory.
The fungal part of the Mouldarray project involved a set of 249 isolates, collected by air sampling in indoor environments in Finland, Sweden, Denmark and the U.K. These strains were isolated in order to verify whether the indoor fungal flora deviates from the expected air contaminants that are generally supposed to be common survivors in indoor climates. The panel of species detected by means of the array should match with those that are actually found during routine sampling in northern temperate climates. rDNA ITS has been selected as the fungal barcoding gene, which therefore should be the basis of all fungal diagnostics. However, in many fungal groups the gene is insufficiently polymorphic. Species should then be distinguished by partial sequences of (introns in) tubulin, actin, calmodulin or other genes. This is the case e.g. in Penicillium, Trichoderma and Cladosporium. For a diagnostic mesoarray based on reversed line-blot assays for rapid detection and identification of filamentous fungi, probes based on ITS as well as a second gene should be available. For the current project we have chosen β-tubulin.
With the recent subdivision of P. boydii into a set of smaller species, it became apparent that each of the newly recognized taxa had a particular predilection, which comes from variations on a basic profile. Central species is Scedosporium apiospermum, which is equally relatively abundant in the nutrient-rich, often polluted environment as it is found in clinical samples. Pseudallescheria boydii inhabits similar environments but is preponderant as an infectious agent, whereas at the other end of the scale S. dehoogii is preponderantly environmental and encountered only occasionally as an agent of mild disease. Given the consistent occurrence of Pseudallescheria species in hydrocarbon-polluted sites we will study relationship of monoaromatic neurotransmitter assimilation and brain invasion for P. boydii, in cooperation with an international consortium. Differential gene and protein expressions will be analyzed between strains growing on neurotransmitters and glucose in vitro, and between strains isolated from brain tissue and other organs of a mouse infection model of P. boydii. Key techniques to be used are Differential-Display Reverse Transcription PCR (DDRT-PCR) and Differential Proteomics techniques.
In groups exhibiting poor morphology, such as the black yeasts, the application of molecular methods leads to the description of a large number of novel species and genera. In contrast, as we have seen earlier in the anthropophilic dermatophytes, large-spored and economically important members of Pleosporales in part tend to be over-classified. A multilocus, AFLP and pathogenicity study of Stemphylium causing disease in pear and other plant hosts showed that some of the major species had to be reduced to synonymy. A model study is being performed in Alternaria, focusing on a species complex with known sexuality and taking a somewhat isolated position in the Pleosporales. In addition to multilocus sequencing, production of secondary metabolites is applied as parameter for distinction.
Several anthropophilic dermatophytes have adapted to the human host several times independently, emerging since the last few thousand years from domesticated animals. Earlier, the origin of major species could be located in Africa, where the highest diversity of genotypes is observed. Anthropophilic species tend to loose their teleomorphs; the species are adapting as clonal offshoots from sexual complexes. A remarkable diversity is found in species associated with wild animals, as was recently proven with a new species specifically associated with badgers. Teleomorphs are commonly produced in zoophilic and geophilic species, and are likely to be environmental. With the mode of transmission being truly human-based, a remarkable change from environmental to human pathogens has been achieved.
Separation of the two currently recognized Coccidioides species has thus far been based on multilocus analysis and microsatellite typing, but no diagnostic criteria were available for clinical practice. We found consistent differences in the rDNA ITS domain. Application to strains from the CBS culture collection proved that C. posadasii had several older synonyms. A proposal for conservation of this name has been submitted in the light of its listing as a US Select Agent for bioterrorism.
Our large ITS database of black yeasts enabled to reconstruct a secondary structure for ITS2. Four domains are generally recognizable in lower as well as in higher organisms, but due to compensatory mutations in stems and occasional absence of domain IV the structure was not always apparent. With the secondary structure at hand, a very small number of base changes in the stems of domain IV enabled reliable recognition of nearly all clinical species of Chaetothyriales. Several of our studies have shown that in these fungi multilocus analyses of all currently used genes are concordant with results of ITS, and sexuality is nearly absent. Thus the domain is an excellent target for the development of specific probes, and is very useful for barcoding. Since species of the order may be too distant to allow confident alignment of ITS, a program for tree reconstruction without alignment was tested in cooperation with the makers of the program.
Ectomycorrhiza refers to association between Basidiomycetes and Ascomycotes fungi with higher plants (e.g. Dipterocarpaceae), which form fungal mantle and intercellular hyphal networks on roots. EcM inoculum fungi persist in the soil after forest change into other land uses, such as rubber agroforests (i.e. smallholder rubber cultivation managed by farmers with low agricultural practices), oil palm plantation and agriculture. A field study showed that inoculation of EcM in the nursery provides a small increase in seedling survival rate but is not essential, since EcM inoculum potential persists in the soil after forest was changed into rubber agroforests. Based on ITS sequences, members of Basidiomycota as well as of Ascomycota (for example, such as Tomentella, Laccaria, unidentified Basidiomycetes, sterile endophytes, Fusarium, Trichoderma and Phoma) are commonly involved in colonization of roots of Shorea seedlings (nursery stage). Further analysis on EcM roots of S. leprosula trees and sporocarps collected in the field is in progress.