Differentiation is accompanied by a progressive loss in transcriptional memory.

BACKGROUND: Cell differentiation requires the integration of two opposite processes, a stabilizing cellular memory, especially at the transcriptional scale, and a burst of gene expression variability which follows the differentiation induction. Therefore, the actual capacity of a cell to undergo phenotypic change during a differentiation process relies upon a modification in this balance which favors change-inducing gene expression variability. However, there are no experimental data providing insight on how fast the transcriptomes of identical cells would diverge on the scale of the very first two cell divisions during the differentiation process. RESULTS: In order to quantitatively address this question, we developed different experimental methods to recover the transcriptomes of related cells, after one and two divisions, while preserving the information about their lineage at the scale of a single cell division. We analyzed the transcriptomes of related cells from two differentiation biological systems (human CD34+ cells and T2EC chicken primary erythrocytic progenitors) using two different single-cell transcriptomics technologies (scRT-qPCR and scRNA-seq). CONCLUSIONS: We identified that the gene transcription profiles of differentiating sister cells are more similar to each other than to those of non-related cells of the same type, sharing the same environment and undergoing similar biological processes. More importantly, we observed greater discrepancies between differentiating sister cells than between self-renewing sister cells. Furthermore, a progressive increase in this divergence from first generation to second generation was observed when comparing differentiating cousin cells to self renewing cousin cells. Our results are in favor of a gradual erasure of transcriptional memory during the differentiation process.

Fungal Colonization of the Airways of Patients with Cystic Fibrosis: the Role of the Environmental Reservoirs.

Filamentous fungi frequently colonize the airways of patients with cystic fibrosis and may cause severe diseases, such as the allergic bronchopulmonary aspergillosis. The most common filamentous fungi capable to chronically colonize the respiratory tract of the patients are Aspergillus fumigatus and Scedosporium species. Defining the treatment strategy may be challenging, the number of available drugs being limited and some of the causative agents being multiresistant microorganisms. The knowledge of the fungal niches in the outdoor and indoor environment is needed for understanding the origin of the contamination of the patients. In light of the abundance of some of the causative molds in compost, agricultural and flower fields, occupational activities related to such environments should be discouraged for patients with cystic fibrosis (CF). In addition, the microbiological monitoring of their indoor environment, including analysis of air and dust on surfaces, is essential to propose preventive measures aiming to reduce the exposure to environmental molds. Nevertheless, some specific niches were also identified in the indoor environment, in relation with humidity which favors the growth of thermotolerant molds. Potted plants were reported as indoor reservoirs for Scedosporium species. Likewise, Exophiala dermatitidis may be spread in the kitchen via dishwashers. However, genotype studies are still required to establish the link between dishwashers and colonization of the airways of CF patients by this black yeast. Moreover, as nothing is known regarding the other filamentous fungi associated with CF, further studies should be conducted to identify other potential specific niches in the habitat.

Evidence for close molecular proximity between reverting and undifferentiated cells.

BACKGROUND: According to Waddington's epigenetic landscape concept, the differentiation process can be illustrated by a cell akin to a ball rolling down from the top of a hill (proliferation state) and crossing furrows before stopping in basins or "attractor states" to reach its stable differentiated state. However, it is now clear that some committed cells can retain a certain degree of plasticity and reacquire phenotypical characteristics of a more pluripotent cell state. In line with this dynamic model, we have previously shown that differentiating cells (chicken erythrocytic progenitors (T2EC)) retain for 24 h the ability to self-renew when transferred back in self-renewal conditions. Despite those intriguing and promising results, the underlying molecular state of those "reverting" cells remains unexplored. The aim of the present study was therefore to molecularly characterize the T2EC reversion process by combining advanced statistical tools to make the most of single-cell transcriptomic data. For this purpose, T2EC, initially maintained in a self-renewal medium (0H), were induced to differentiate for 24H (24H differentiating cells); then, a part of these cells was transferred back to the self-renewal medium (48H reverting cells) and the other part was maintained in the differentiation medium for another 24H (48H differentiating cells). For each time point, cell transcriptomes were generated using scRT-qPCR and scRNAseq. RESULTS: Our results showed a strong overlap between 0H and 48H reverting cells when applying dimensional reduction. Moreover, the statistical comparison of cell distributions and differential expression analysis indicated no significant differences between these two cell groups. Interestingly, gene pattern distributions highlighted that, while 48H reverting cells have gene expression pattern more similar to 0H cells, they are not completely identical, which suggest that for some genes a longer delay may be required for the cells to fully recover. Finally, sparse PLS (sparse partial least square) analysis showed that only the expression of 3 genes discriminates 48H reverting and 0H cells. CONCLUSIONS: Altogether, we show that reverting cells return to an earlier molecular state almost identical to undifferentiated cells and demonstrate a previously undocumented physiological and molecular plasticity during the differentiation process, which most likely results from the dynamic behavior of the underlying molecular network.

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Draft Genome Sequence of the Environmental Fungus Scedosporium dehoogii.

Today, the genus Scedosporium comprises at least ten species with four of them, Scedosporium apiospermum, Scedosporium boydii, Scedosporium aurantiacum and Scedosporium minutisporum capable of colonizing the lungs of patients with cystic fibrosis. Scedosporium dehoogii, which is also common in the soil, has never been reported as causing human pulmonary infections. Here we report the first genome sequence for S. dehoogii, an invaluable resource to understand the genetic bases of pathogenesis in the genus Scedosporium.

WASABI: a dynamic iterative framework for gene regulatory network inference.

BACKGROUND: Inference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations. RESULTS: In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from time-stamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-by-one through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. CONCLUSIONS: Together, these results demonstrate WASABI versatility and ability to tackle some general gene regulatory networks inference issues. It is our hope that WASABI will prove useful in helping biologists to fully exploit the power of time-stamped single-cell data.

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process.

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a "simple" program that all cells execute identically but results from the dynamical behavior of the underlying molecular network.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Scedosporium and Lomentospora: an updated overview of underrated opportunists.

Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase.

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients.

The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex.

PURPOSE: The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. METHODS: Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). RESULTS: Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. CONCLUSIONS: These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF).

Human-impacted areas of France are environmental reservoirs of the Pseudallescheria boydii/Scedosporium apiospermum species complex.

Species of the Pseudallescheria boydii/Scedosporium apiospermum complex (PSC) are emerging fungal pathogens able to chronically colonize the airways of patients with cystic fibrosis (CF). As P. boydii was found more frequently colonizing the lungs of CF patients in France than in other European countries in a previous report, the present study was conducted in order to clarify distribution of PSC species in France and to characterize their natural habitat. The highest densities of PSC isolates were found in human-impacted areas, i.e. agricultural areas, fluids obtained from wastewater treatment plants, playgrounds and industrial areas. PSC was not detected from soil samples collected in forests. Most PSC culture-positive soil samples exhibited a pH range of 6-8. Scedosporium dehoogii, the most abundant species, was detected in all human-impacted area types except vineyards, whereas Scedosporium aurantiacum was mostly found in agricultural areas. Pseudallescheria boydii and S. apiospermum were predominantly isolated from seashores and playgrounds respectively. Pseudallescheria minutispora was found only once from a playground. This study highlights potential sources of contamination of the patients, especially in the CF context.

Scedo-Select III: a new semi-selective culture medium for detection of the Scedosporium apiospermum species complex.

The Scedosporium apiospermum complex is responsible for a large variety of infections in human. Members of this complex have become emerging fungal pathogens with an increasing occurrence in patients with underlying conditions such as immunosuppression or cystic fibrosis. A better knowledge of these fungi and of the sources of contamination of the patients is required and more accurate detection methods from the environment are needed. In this context, a highly selective culture medium was developed in the present study. Thus, various aliphatic, cyclic, or aromatic compounds were tested as the sole carbon source, in combination with some inorganic nitrogen sources and fungicides. The best results were obtained with 4-hydroxy-benzoate combined with ammonium sulfate and the fungicides dichloran and benomyl. This new culture medium called Scedo-Select III was shown to support growth of all species of the S. apiospermum complex. Subsequently, this new culture medium was evaluated successfully on water and soil samples, exhibiting higher sensitivity and selectivity than the previously described SceSel+ culture medium. Therefore, this easy-to-prepare and synthetic semi-selective culture medium may be useful to clarify the ecology of these fungi and to identify their reservoirs in patients' environment.

Distribution of the different species of the Pseudallescheria boydii/Scedosporium apiospermum complex in French patients with cystic fibrosis.

As various new sibling species within the Pseudallescheria boydii/Scedosporium apiospermum complex have been described recently with differences in their susceptibility to antifungals, this study was conducted in order to determine their respective frequency in cystic fibrosis. Results indicated that P. boydii largely predominated (62%), followed by S. apiospermum (24%), Scedosporium aurantiacum (10%) and Pseudallescheria minutispora (4%). Scedosporium dehoogii was not recovered in this study. The multiple correspondence factor analysis highlighted geographical discrepancies within species distribution: P. boydii was rarely encountered in Northern France, while S. apiospermum was less represented in the west of the country. Additionally, we demonstrated that all species encountered in the cystic fibrosis context were capable to chronically colonize the respiratory tract of patients. Molecular typing of a large set of environmental and clinical isolates should be conducted to delineate the epidemiology of each sibling species in the complex.

Lignin-Modifying Enzymes in Scedosporium Species.

Scedosporium species are usually soil saprophytes but some members of the genus such as S. apiospermum and S. aurantiacum have been regularly reported as causing human respiratory infections, particularly in patients with cystic fibrosis (CF). Because of their low sensitivity to almost all available antifungal drugs, a better understanding of the pathogenic mechanisms of these fungi is mandatory. Likewise, identification of the origin of the contamination of patients with CF may be helpful to propose prophylactic measures. In this aim, environmental studies were conducted demonstrating that Scedosporium species are abundant in human-made environments and associated with nutrient-rich substrates. Although their natural habitat remains unknown, there is accumulated evidence to consider them as wood-decaying fungi. This study aimed to demonstrate the ability of these fungi to utilize lignocellulose compounds, especially lignin, as a carbon source. First, the lignolytic properties of Scedosporium species were confirmed by cultural methods, and biochemical assays suggested the involvement of peroxidases and oxidases as lignin-modifying enzymes. Scedosporium genomes were then screened using tBLASTn searches. Fifteen candidate genes were identified, including four peroxidase and seven oxidase genes, and some of them were shown, by real-time PCR experiments, to be overexpressed in lignin-containing medium, thus confirming their involvement in lignin degradation.

Deciphering the Role of PIG1 and DHN-Melanin in Scedosporium apiospermum Conidia.

Scedosporium apiospermum is a saprophytic filamentous fungus involved in human infections, of which the virulence factors that contribute to pathogenesis are still poorly characterized. In particular, little is known about the specific role of dihydroxynaphtalene (DHN)-melanin, located on the external layer of the conidia cell wall. We previously identified a transcription factor, PIG1, which may be involved in DHN-melanin biosynthesis. To elucidate the role of PIG1 and DHN-melanin in S. apiospermum, a CRISPR-Cas9-mediated PIG1 deletion was carried out from two parental strains to evaluate its impact on melanin biosynthesis, conidia cell-wall assembly, and resistance to stress, including the ability to survive macrophage engulfment. ΔPIG1 mutants did not produce melanin and showed a disorganized and thinner cell wall, resulting in a lower survival rate when exposed to oxidizing conditions, or high temperature. The absence of melanin increased the exposure of antigenic patterns on the conidia surface. PIG1 regulates the melanization of S. apiospermum conidia, and is involved in the survival to environmental injuries and to the host immune response, that might participate in virulence. Moreover, a transcriptomic analysis was performed to explain the observed aberrant septate conidia morphology and found differentially expressed genes, underlining the pleiotropic function of PIG1.

An image-guided microfluidic system for single-cell lineage tracking.

Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which was demonstrated to be compatible with downstream single-cell RNA sequencing analysis.

Lower Funneling Pathways in Scedosporium Species.

Lignin, a natural polyaromatic macromolecule, represents an essential component of the lignocellulose biomass. Due to its complexity, the natural degradation of this molecule by microorganisms still remains largely misunderstood. Extracellular oxidative degradation is followed by intracellular metabolic degradation of conserved aromatic intermediate compounds (protocatechuate, catechol, hydroxyquinol, and gentisic acid) that are used as carbon and energy sources. The lower funneling pathways are characterized by the opening of the aromatic ring of these molecules through dioxygenases, leading to degradation products that finally enter into the tricarboxylic acid (TCA) cycle. In order to better understand the adaptation mechanisms of Scedosporium species to their environment, these specific catabolism pathways were studied. Genes encoding ring-cleaving dioxygenases were identified in Scedosporium genomes by sequence homology, and a bioinformatic analysis of the organization of the corresponding gene clusters was performed. In addition, these predictions were confirmed by evaluation of the expression level of the genes of the gentisic acid cluster. When the fungus was cultivated in the presence of lignin or gentisic acid as sole carbon source, experiments revealed that the genes of the gentisic acid cluster were markedly overexpressed in the two Scedosporium species analyzed (Scedosporium apiospermum and Scedosporium aurantiacum). Only the gene encoding a membrane transporter was not overexpressed in the gentisic acid-containing medium. Together, these data suggest the involvement of the lower funneling pathways in Scedosporium adaptation to their environment.