Neddylation orchestrates the complex transcriptional and posttranscriptional program that drives Schwann cell myelination.

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis.

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis ( Mtb ) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR ( virR mut ) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb . We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virR mut . Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

Dynamin-like proteins mediate extracellular vesicle secretion in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) secretes extracellular vesicles (EVs) containing a variety of proteins, lipoproteins, and lipoglycans. While emerging evidence suggests that EVs contribute to tuberculosis pathogenesis, the factors and molecular mechanisms involved in mycobacterial EV production have not been identified. In this study, we use a genetic approach to identify Mtb proteins that mediate vesicle release in response to iron limitation and antibiotic exposure. We uncover a critical role for the isoniazid-induced, dynamin-like proteins, IniA and IniC, in mycobacterial EV biogenesis. Further characterization of a Mtb iniA mutant shows that the production of EVs enables intracellular Mtb to export bacterial components into the extracellular environment to communicate with host cells and potentially modulate the immune response. The findings advance our understanding of the biogenesis and functions of mycobacterial EVs and provide an avenue for targeting vesicle production in vivo.

Immunogenicity of Mycobacterial Extracellular Vesicles Isolated From Host-Related Conditions Informs About Tuberculosis Disease Status.

Tuberculosis (TB) still represents a major global health problem affecting over 10 million people worldwide. The gold-standard procedures for TB diagnosis are culture and nucleic acid amplification techniques. In this context, both lipoarabinomannan (LAM) urine test and rapid molecular tests have been major game changers. However, the low sensitivity of the former and the cost and the prohibitive infrastructure requirements to scale-up in endemic regions of the latter, make the improvement of the TB diagnostic landscape a priority. Most forms of life produce extracellular vesicles (EVs), including bacteria despite differences in bacterial cell envelope architecture. We demonstrated that Mycobacterium tuberculosis (Mtb), the causative agent of TB, produces EVs in vitro and in vivo as part of a sophisticated mechanism to manipulate host cellular physiology and to evade the host immune system. In a previous serology study, we showed that the recognition of several mycobacterial extracellular vesicles (MEV) associated proteins could have diagnostic properties. In this study, we pursued to expand the capabilities of MEVs in the context of TB diagnostics by analyzing the composition of MEVs isolated from Mtb cultures submitted to iron starvation and, testing their immunogenicity against a new cohort of serum samples derived from TB+ patients, latent TB-infected (LTBI) patients and healthy donors. We found that despite the stringent condition imposed by iron starvation, Mtb reduces the number of MEV associated proteins relative to iron sufficient conditions. In addition, TB serology revealed three new MEV antigens with specific biomarker capacity. These results suggest the feasibility of developing a point-of-care (POC) device based on selected MEV-associated proteins.

Secretomes of medically important fungi reflect morphological and phylogenetic diversity.

Secretome represents a main target for understanding the mechanisms of fungal adaptation. In the present study, we focus on the secretomes of fungi associated with infections in humans and other mammals in order to explore relationships between the diverse morphological and phylogenetic groups. Almost all the mammalian pathogenic fungi analyzed have secretome sizes smaller than 1000 proteins and, secreted proteins comprise between 5% and 10% of the total proteome. As expected, the correlation pattern between the secretome size and the total proteome was similar to that described in previous secretome studies of fungi. With regard to the morphological groups, minimum secretome sizes of less than 250 secreted proteins and low values for the fraction of secreted proteins are shown in mammalian pathogenic fungi with reduced proteomes such as microsporidia, atypical fungi and some species of yeasts and yeast-like fungi (Malassezia). On the other hand, filamentous fungi have significantly more secreted proteins and the highest numbers are present in species of filamentous fungi that also are plant or insect pathogens (Fusarium verticilloides, Fusarium oxysporum and Basidiobolus meristosporus). With respect to phylogeny, there are also variations in secretome size across fungal subphyla: Microsporidia, Taphrinomycotina, Ustilagomycotina and Saccharomycotina contain small secretomes; whereas larger secretomes are found in Agaricomycotina, Pezizomycotina, Mucoromycotina and Entomophthoromycotina. Finally, principal component analysis (PCA) was conducted on the complete secretomes. The PCA results revealed that, in general, secretomes of fungi belonging to the same morphological group or subphyla cluster together. In conclusion, our results point out that in medically important fungi there is a relationship between the secretome and the morphological group or phylogenetic classification.

Cross-sectional study of human coding- and non-coding RNAs in progressive stages of Helicobacter pylori infection.

Helicobacter pylori infects 4.4 billion individuals worldwide and is considered the most important etiologic agent for peptic ulcers and gastric cancer. Individual response to H. pylori infection is complex and depends on complex interactions between host and environmental factors. The pathway towards gastric cancer is a sequence of events known as Correa's model of gastric carcinogenesis, a stepwise inflammatory process from normal mucosa to chronic-active gastritis, atrophy, metaplasia and gastric adenocarcinoma. This study examines gastric clinical specimens representing different steps of the Correa pathway with the aim of identifying the expression profiles of coding- and non-coding RNAs that may have a role in Correa's model of gastric carcinogenesis. We screened for differentially expressed genes in gastric biopsies by employing RNAseq, microarrays and qRT-PCR. Here we provide a detailed description of the experiments, methods and results generated. The datasets may help other scientists and clinicians to find new clues to the pathogenesis of H. pylori and the mechanisms of progression of the infection to more severe gastric diseases. Data is available via ArrayExpress.

HuR/ELAVL1 drives malignant peripheral nerve sheath tumor growth and metastasis.

Cancer cells can develop a strong addiction to discrete molecular regulators, which control the aberrant gene expression programs that drive and maintain the cancer phenotype. Here, we report the identification of the RNA-binding protein HuR/ELAVL1 as a central oncogenic driver for malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive sarcomas that originate from cells of the Schwann cell lineage. HuR was found to be highly elevated and bound to a multitude of cancer-associated transcripts in human MPNST samples. Accordingly, genetic and pharmacological inhibition of HuR had potent cytostatic and cytotoxic effects on tumor growth, and strongly suppressed metastatic capacity in vivo. Importantly, we linked the profound tumorigenic function of HuR to its ability to simultaneously regulate multiple essential oncogenic pathways in MPNST cells, including the Wnt/ß-catenin, YAP/TAZ, RB/E2F, and BET pathways, which converge on key transcriptional networks. Given the exceptional dependency of MPNST cells on HuR for survival, proliferation, and dissemination, we propose that HuR represents a promising therapeutic target for MPNST treatment.

Patients with Cholangiocarcinoma Present Specific RNA Profiles in Serum and Urine Extracellular Vesicles Mirroring the Tumor Expression: Novel Liquid Biopsy Biomarkers for Disease Diagnosis.

: Cholangiocarcinoma (CCA) comprises a group of heterogeneous biliary cancers with dismal prognosis. The etiologies of most CCAs are unknown, but primary sclerosing cholangitis (PSC) is a risk factor. Non-invasive diagnosis of CCA is challenging and accurate biomarkers are lacking. We aimed to characterize the transcriptomic profile of serum and urine extracellular vesicles (EVs) from patients with CCA, PSC, ulcerative colitis (UC), and healthy individuals. Serum and urine EVs were isolated by serial ultracentrifugations and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. EVs transcriptome was determined by Illumina gene expression array [messenger RNAs (mRNA) and non-coding RNAs (ncRNAs)]. Differential RNA profiles were found in serum and urine EVs from patients with CCA compared to control groups (disease and healthy), showing high diagnostic capacity. The comparison of the mRNA profiles of serum or urine EVs from patients with CCA with the transcriptome of tumor tissues from two cohorts of patients, CCA cells in vitro, and CCA cells-derived EVs, identified 105 and 39 commonly-altered transcripts, respectively. Gene ontology analysis indicated that most commonly-altered mRNAs participate in carcinogenic steps. Overall, patients with CCA present specific RNA profiles in EVs mirroring the tumor, and constituting novel promising liquid biopsy biomarkers.

Extracellular Vesicles From Liver Progenitor Cells Downregulates Fibroblast Metabolic Activity and Increase the Expression of Immune-Response Related Molecules.

Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.

Signal Integration and Transcriptional Regulation of the Inflammatory Response Mediated by the GM-/M-CSF Signaling Axis in Human Monocytes.

In recent years, the macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) cytokines have been identified as opposing regulators of the inflammatory program. However, the two cytokines are simultaneously present in the inflammatory milieu, and it is not clear how cells integrate these signals. In order to understand the regulatory networks associated with the GM/M-CSF signaling axis, we analyzed DNA methylation in human monocytes. Our results indicate that GM-CSF induces activation of the inflammatory program and extensive DNA methylation changes, while M-CSF-polarized cells are in a less differentiated state. This inflammatory program is mediated via JAK2 associated with the GM-CSF receptor and the downstream extracellular signal-regulated (ERK) signaling. However, PI3K signaling is associated with a negative regulatory loop of the inflammatory program and M-CSF autocrine signaling in GM-CSF-polarized monocytes. Our findings describe the regulatory networks associated with the GM/M-CSF signaling axis and how they contribute to the establishment of the inflammatory program associated with monocyte activation.

Gut microbiome and serum metabolome analyses identify molecular biomarkers and altered glutamate metabolism in fibromyalgia.

BACKGROUND: Fibromyalgia is a complex, relatively unknown disease characterised by chronic, widespread musculoskeletal pain. The gut-brain axis connects the gut microbiome with the brain through the enteric nervous system (ENS); its disruption has been associated with psychiatric and gastrointestinal disorders. To gain an insight into the pathogenesis of fibromyalgia and identify diagnostic biomarkers, we combined different omics techniques to analyse microbiome and serum composition. METHODS: We collected faeces and blood samples to study the microbiome, the serum metabolome and circulating cytokines and miRNAs from a cohort of 105 fibromyalgia patients and 54 age- and environment-matched healthy individuals. We sequenced the V3 and V4 regions of the 16S rDNA gene from faeces samples. UPLC-MS metabolomics and custom multiplex cytokine and miRNA analysis (FirePlex™ technology) were used to examine sera samples. Finally, we combined the different data types to search for potential biomarkers. RESULTS: We found that the diversity of bacteria is reduced in fibromyalgia patients. The abundance of the Bifidobacterium and Eubacterium genera (bacteria participating in the metabolism of neurotransmitters in the host) in these patients was significantly reduced. The serum metabolome analysis revealed altered levels of glutamate and serine, suggesting changes in neurotransmitter metabolism. The combined serum metabolomics and gut microbiome datasets showed a certain degree of correlation, reflecting the effect of the microbiome on metabolic activity. We also examined the microbiome and serum metabolites, cytokines and miRNAs as potential sources of molecular biomarkers of fibromyalgia. CONCLUSIONS: Our results show that the microbiome analysis provides more significant biomarkers than the other techniques employed in the work. Gut microbiome analysis combined with serum metabolomics can shed new light onto the pathogenesis of fibromyalgia. We provide a list of bacteria whose abundance changes in this disease and propose several molecules as potential biomarkers that can be used to evaluate the current diagnostic criteria.

Progressive loss of hybrid histidine kinase genes during the evolution of budding yeasts (Saccharomycotina).

Two-component systems (TCSs) are widely distributed cell signaling pathways used by both prokaryotic and eukaryotic organisms to cope with a wide range of environmental cues. In fungi, TCS signaling routes, that mediate perception of stimuli, correspond to a multi-step phosphorelay between three protein families including hybrid histidine kinases (HHK), histidine phosphotransfer proteins (HPt) and response regulators (RR). The best known of these fungal transduction pathways remains the Sln1(HHK)-Ypd1(HPt)-Ssk1(RR) system that governs the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway for osmo-adaptation in Saccharomyces cerevisiae. Although recent advances have provided a preliminary overview of the distribution of TCS proteins in the kingdom Fungi, underlying mechanisms that drive the remarkable diversity among HHKs and other TCS proteins in different fungal lineages remain unclear. More precisely, evolutionary paths that led to the appearance, transfer, duplication, and loss of the corresponding TCS genes in fungi have never been hitherto addressed. In the present study, we were particularly interested in studying the distribution of TCS modules across the so-called "budding yeasts clade" (Saccharomycotina) by interrogating the genome of 82 species. With the exception of the emergence of an additional RR (named Srr1) in the fungal CTG clade, TCS proteins Ypd1 (HPt), Ssk1 (RR), Skn7 (RR), and Rim15 (RR) are well conserved within the Saccharomycotina. Surprisingly, some species from the basal lineages, especially Lipomyces starkeyi, harbor several filamentous-type HHKs that appear as relict genes that have been likely retained from a common ancestor of Saccharomycotina. Overall, this analysis revealed a progressive diminution of the initial pool of HHK-encoding genes during Saccharomycotina yeast evolution.

Role of Aramchol in steatohepatitis and fibrosis in mice.

Nonalcoholic steatohepatitis (NASH) is the advanced form of nonalcoholic fatty liver disease (NAFLD) which sets the stage for further liver damage. The mechanism for the progression of NASH involves multiple parallel hits including oxidative stress, mitochondrial dysfunction, inflammation and others. Manipulation of any of these pathways may be an approach to prevent NASH development and progression. Aramchol (arachidyl-amido cholanoic acid) is presently in a phase IIb NASH study. The aim of this study was to investigate Aramchol's mechanism of action and its effect on fibrosis using the methionine- and choline-deficient (MCD) diet model of NASH. We collected liver and serum from mice fed a MCD diet containing 0.1% methionine (0.1MCD) for four weeks, which developed steatohepatitis and fibrosis, as well as mice receiving a control diet; the metabolomes and proteomes were determined. 0.1MCD fed mice were given Aramchol (5mg/kg/day for the last 2 weeks); liver samples were analyzed histologically. Aramchol administration reduced features of steatohepatitis and fibrosis in 0.1MCD fed mice. Aramchol downregulated stearoyl-CoA desaturase 1 (SCD1), a key enzyme involved in triglyceride biosynthesis whose loss enhances fatty acid ß-oxidation. Aramchol increased the flux through the transsulfuration pathway, leading to a rise in glutathione (GSH) and GSH/GSSG ratio, the main cellular antioxidant that maintains intracellular redox status. Comparison of serum metabolomic pattern between 0.1MCD fed mice and NAFLD patients showed a substantial overlap. CONCLUSIONS: Aramchol treatment improved steatohepatitis and fibrosis by 1) decreasing SCD1, and 2) increasing the flux through the transsulfuration pathway maintaining cellular redox homeostasis. We also demonstrated that the 0.1MCD model resembles the metabolic phenotype observed in about 50% of NAFLD patients, which supports the potential use of Aramchol in NASH treatment.

Potyvirus virion structure shows conserved protein fold and RNA binding site in ssRNA viruses.

Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å. The atomic model shows a conserved fold for the CPs of flexible filamentous plant viruses, including a universally conserved RNA binding pocket, which is a potential target for antiviral compounds. This conserved fold of the CP is widely distributed in eukaryotic viruses and is also shared by nucleoproteins of enveloped viruses with segmented (-)ssRNA (negative-sense ssRNA) genomes, including influenza viruses.

VerSeDa: vertebrate secretome database.

Based on the current tools, de novo secretome (full set of proteins secreted by an organism) prediction is a time consuming bioinformatic task that requires a multifactorial analysis in order to obtain reliable in silico predictions. Hence, to accelerate this process and offer researchers a reliable repository where secretome information can be obtained for vertebrates and model organisms, we have developed VerSeDa (Vertebrate Secretome Database). This freely available database stores information about proteins that are predicted to be secreted through the classical and non-classical mechanisms, for the wide range of vertebrate species deposited at the NCBI, UCSC and ENSEMBL sites. To our knowledge, VerSeDa is the only state-of-the-art database designed to store secretome data from multiple vertebrate genomes, thus, saving an important amount of time spent in the prediction of protein features that can be retrieved from this repository directly. Database URL: VerSeDa is freely available at http://genomics.cicbiogune.es/VerSeDa/index.php.

SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS: SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS: SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/ß-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS: SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY: Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.

Epigenetic Networks Regulate the Transcriptional Program in Memory and Terminally Differentiated CD8+ T Cells.

Epigenetic mechanisms play a critical role during differentiation of T cells by contributing to the formation of stable and heritable transcriptional patterns. To better understand the mechanisms of memory maintenance in CD8+ T cells, we performed genome-wide analysis of DNA methylation, histone marking (acetylated lysine 9 in histone H3 and trimethylated lysine 9 in histone), and gene-expression profiles in naive, effector memory (EM), and terminally differentiated EM (TEMRA) cells. Our results indicate that DNA demethylation and histone acetylation are coordinated to generate the transcriptional program associated with memory cells. Conversely, EM and TEMRA cells share a very similar epigenetic landscape. Nonetheless, the TEMRA transcriptional program predicts an innate immunity phenotype associated with genes never reported in these cells, including several mediators of NK cell activation (VAV3 and LYN) and a large array of NK receptors (e.g., KIR2DL3, KIR2DL4, KIR2DL1, KIR3DL1, KIR2DS5). In addition, we identified up to 161 genes that encode transcriptional regulators, some of unknown function in CD8+ T cells, and that were differentially expressed in the course of differentiation. Overall, these results provide new insights into the regulatory networks involved in memory CD8+ T cell maintenance and T cell terminal differentiation.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Comparative and transcriptional analysis of the predicted secretome in the lignocellulose-degrading basidiomycete fungus Pleurotus ostreatus.

Fungi interact with their environment by secreting proteins to obtain nutrients, elicit responses and modify their surroundings. Because the set of proteins secreted by a fungus is related to its lifestyle, it should be possible to use it as a tool to predict fungal lifestyle. To test this hypothesis, we bioinformatically identified 538 and 554 secretable proteins in the monokaryotic strains PC9 and PC15 of the white rot basidiomycete Pleurotus ostreatus. Functional annotation revealed unknown functions (37.2%), glycosyl hydrolases (26.5%) and redox enzymes (11.5%) as the main groups in the two strains. When these results were combined with RNA-seq analyses, we found that the relative importance of each group was different in different strains and culture conditions and the relevance of the unknown function proteins was enhanced. Only a few genes were actively expressed in a given culture condition in expanded multigene families, suggesting that family expansi on could increase adaptive opportunities rather than activity under a specific culture condition. Finally, we used the set of P. ostreatus secreted proteins as a query to search their counterparts in other fungal genomes and found that the secretome profiles cluster the tested basidiomycetes into lifestyle rather than phylogenetic groups.