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.

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.

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.

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.

PECAS: prokaryotic and eukaryotic classical analysis of secretome.

Full sets of proteins that are transported to the extracellular space, called secretomes, have been studied for a variety of organisms to understand their potential role in crucial metabolic pathways and complex health conditions. However, there is a lack of tools for integrative classical analysis of secretomes that consider all the data sources available nowadays. Thus, PECAS (Prokaryotic and Eukaryotic Classical Analysis of Secretome) has been developed to provide a well-established prediction pipeline on secreted proteins for prokaryote and eukaryote species.

Genomewide analysis of phytochrome proteins in the phylum Basidiomycota.

Phytochromes are photoreceptor proteins involved in the detection of the red and far-red regions of the visible light spectrum. Fungal phytochromes are hybrid histidine kinases with a conserved domain architecture composed of an N-terminal photosensory module and a C-terminal regulatory output module that includes the histidine kinase and response regulator receiver domains. In this study, we have analyzed the distribution, domain architecture, and phylogenetic analysis of phytochrome proteins in 47 published genome sequences among the phylum Basidiomycota. Genome analysis revealed that almost every genome of basidiomycetes contained at least one gene encoding a phytochrome protein. Domain architecture of fungal phytochromes was completely conserved in the identified phytochromes of basidiomycetes, and phylogenetic analysis clustered these proteins into clades related with the phylogenetic classification of this fungal phylum.

Highly expressed captured genes and cross-kingdom domains present in Helitrons create novel diversity in Pleurotus ostreatus and other fungi.

BACKGROUND: Helitrons are class-II eukaryotic transposons that transpose via a rolling circle mechanism. Due to their ability to capture and mobilize gene fragments, they play an important role in the evolution of their host genomes. We have used a bioinformatics approach for the identification of helitrons in two Pleurotus ostreatus genomes using de novo detection and homology-based searching. We have analyzed the presence of helitron-captured genes as well as the expansion of helitron-specific helicases in fungi and performed a phylogenetic analysis of their conserved domains with other representative eukaryotic species. RESULTS: Our results show the presence of two helitron families in P. ostreatus that disrupt gene colinearity and cause a lack of synteny between their genomes. Both putative autonomous and non-autonomous helitrons were transcriptionally active, and some of them carried highly expressed captured genes of unknown origin and function. In addition, both families contained eukaryotic, bacterial and viral domains within the helitron's boundaries. A phylogenetic reconstruction of RepHel helicases using the Helitron-like and PIF1-like helicase conserved domains revealed a polyphyletic origin for eukaryotic helitrons. CONCLUSION: P. ostreatus helitrons display features similar to other eukaryotic helitrons and do not tend to capture host genes or gene fragments. The occurrence of genes probably captured from other hosts inside the helitrons boundaries pose the hypothesis that an ancient horizontal transfer mechanism could have taken place. The viral domains found in some of these genes and the polyphyletic origin of RepHel helicases in the eukaryotic kingdom suggests that virus could have played a role in a putative lateral transfer of helitrons within the eukaryotic kingdom. The high similarity of some helitrons, along with the transcriptional activity of its RepHel helicases indicates that these elements are still active in the genome of P. ostreatus.

Dual-histidine kinases in basidiomycete fungi.

Dual-histidine kinases (HKs) are complex hybrid HKs containing in a single polypeptide two HK transmitter modules (T) and two-response regulator received domains (R) that are combined in a TRTR geometry. In fungi, this protein family is limited to some particular species of the phylum Basidiomycota and absent in the other phyla. This study extends the investigation of dual-HKs to 80 fully sequenced genomes of basidiomycetes, analyzing their distribution, domain architecture and phylogenetic relationships. Moreover, similarly to dual-HKs of basidiomycetes, several species of bacteria were found that contain hybrid HKs with a TRTR domain architecture encoded in a single gene.

Comparative analysis of secretomes in basidiomycete fungi.

Lignocellulose, the major reservoir of organic carbon on Earth, is recalcitrant to turnover and resistant to microbial and enzymatic attack because of the protective action of lignin and the crystalline structure of cellulose. Basidiomycetes are the only organisms known to degrade lignocellulose using two alternative degradation strategies: white rot basidiomycetes break down the lignin moiety extensively before attacking the cellulose, whereas brown rot basidiomycetes cause limited lignin alteration while primarily degrading cellulose. These strategies depend on complex portfolios of extracellular enzymes whose production and export are environment and substrate dependent. More than 265 fungal genomes (more than 90 corresponding to basidiomycetes) have been sequenced. These sequences can be bioinformatically screened to identify presumptive secreted proteins that, however, differ from the experimental data, stressing the importance of using complementary approaches for analyzing these complex systems. The study of fungal secretomes from the scope of their different lignin degradation strategies and lifestyles would facilitate their use in the treatment of lignocellulose as carbon feedstock for biofuel production and further biorefinery processes. In this article, we carry out a comparative review of the analytical tools and strategies used for studying basidiomycetes' secretomes and compare the available results from these two points of view. BIOLOGICAL SIGNIFICANCE: In this article, we review the tools and strategies used in the analysis of basidiomycetes' secretomes and we compare the secretome data available for this type of fungi with a special emphasis on the enzymes involved in lignocellulose degradation. This comparative study provides a basis for distinguishing different lifestyles (saprotrophs, symbionts and parasites) on the basis of their respective secretomes.

SECRETOOL: integrated secretome analysis tool for fungi.

The secretome (full set of secreted proteins) has been studied in multiple fungal genomes to elucidate the potential role of those protein collections involved in a number of metabolic processes from host infection to wood degradation. Being aminoacid composition a key factor to recognize secretory proteins, SECRETOOL comprises a group of web tools that enable secretome predictions out of aminoacid sequence files, up to complete fungal proteomes, in one step. SECRETOOL is freely available on the web at http://genomics.cicbiogune.es/SECRETOOL/Secretool.php .

FUNGALOXPHOS: an integrated database for oxidative phosphorylation in fungi.

The oxidative phosphorylation (OXPHOS) system is the main energy-producing pathway in aerobic organisms. Here we present FUNGALOXPHOS, a web based platform that stores OXPHOS proteins encoded in fungal nuclear genomes and that incorporates tools for the extraction, classification and bioinformatic screening of all the putative nuclear encoded fungal OXPHOS proteins. FUNGALOXPHOS includes local, parsing and remote tools that allow exploring the properties of OXPHOS proteins in fungal genomes. FUNGALOXPHOS is freely available on the web at http://bioinformatics.unavarra.es:1000/FUNGALOXPHOS_CSS/main.html.

Two-component signal transduction in Agaricus bisporus: a comparative genomic analysis with other basidiomycetes through the web-based tool BASID2CS.

Two-component systems (TCSs) are signal transduction mechanisms present in many eukaryotes, including fungi that play essential roles in the regulation of several cellular functions and responses. In this study, we carry out a genomic analysis of the TCS proteins in two varieties of the white button mushroom Agaricus bisporus. The genomes of both A. bisporus varieties contain eight genes coding for TCS proteins, which include four hybrid Histidine Kinases (HKs), a single histidine-containing phosphotransfer (HPt) protein and three Response Regulators (RRs). Comparison of the TCS proteins among A. bisporus and the sequenced basidiomycetes showed a conserved core complement of five TCS proteins including the Tco1/Nik1 hybrid HK, HPt protein and Ssk1, Skn7 and Rim15-like RRs. In addition, Dual-HKs, unusual hybrid HKs with 2 HK and 2 RR domains, are absent in A. bisporus and are limited to various species of basidiomycetes. Differential expression analysis showed no significant up- or down-regulation of the Agaricus TCS genes in the conditions/tissue analyzed with the exception of the Skn7-like RR gene (Agabi_varbisH97_2|198669) that is significantly up-regulated on compost compared to cultured mycelia. Furthermore, the pipeline web server BASID2CS (http://bioinformatics.unavarra.es:1000/B2CS/BASID2CS.htm) has been specifically designed for the identification, classification and functional annotation of putative TCS proteins from any predicted proteome of basidiomycetes using a combination of several bioinformatic approaches.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

The plant cell wall-decomposing machinery underlies the functional diversity of forest fungi.

Brown rot decay removes cellulose and hemicellulose from wood--residual lignin contributing up to 30% of forest soil carbon--and is derived from an ancestral white rot saprotrophy in which both lignin and cellulose are decomposed. Comparative and functional genomics of the "dry rot" fungus Serpula lacrymans, derived from forest ancestors, demonstrated that the evolution of both ectomycorrhizal biotrophy and brown rot saprotrophy were accompanied by reductions and losses in specific protein families, suggesting adaptation to an intercellular interaction with plant tissue. Transcriptome and proteome analysis also identified differences in wood decomposition in S. lacrymans relative to the brown rot Postia placenta. Furthermore, fungal nutritional mode diversification suggests that the boreal forest biome originated via genetic coevolution of above- and below-ground biota.

Differences in two-component signal transduction proteins among the genus Brucella: implications for host preference and pathogenesis.

Two-component systems (TCSs) are the predominant bacterial signal transduction mechanisms. Species of the genus Brucella are genetically highly related and differ mainly in mammalian host adaptation and pathogenesis. In this study, TCS proteins encoded in the available genome sequences of Brucella species have been identified using bioinformatic methods. All the Brucella species share an identical set of TCS proteins, and the number of TCS proteins in the closely related opportunistic human pathogen Ochrobactrum anthropi was higher than in Brucella species as expected from its lifestyle. O. anthropi lacks orthologs of the Brucella TCSs NodVW, TceSR and TcfSR, suggesting that these TCS proteins could be necessary for the adaptation of Brucella as an intracellular pathogen. This genomic analysis revealed the presence of a differential distribution of TCS pseudogenes among Brucella species. Moreover, there were also differences in TCS pseudogenes between strains belonging to the same Brucella species, and in particular between B. suis biovars 1 and 2.

Genomic analysis of two-component signal transduction proteins in basidiomycetes.

Two-component system (TCS) proteins are components of complex signal transduction pathways in fungi, and play essential roles in the regulation of several cellular functions and responses. Species of basidiomycetes have a marked variation in their specific physiological traits, morphological complexity and lifestyles. In this study, we have used the available complete genomes of basidiomycetes to carry out a thorough identification and an extensive comparative analysis of the TCS proteins in this fungal phylum. In comparison with ascomycetes, basidiomycetes exhibit an intermediate number of TCS proteins. Several TCS proteins are highly conserved among all the basidiomycetes, and other TCS proteins appear to be specific to particular species of basidiomycetes. Moreover, some species appear to have developed a unique histidine kinase group with unusual domain architecture, the Dual-histidine kinases. The presence of differential sets of TCS proteins between basidiomycete species might reflect their adaptation to diverse environmental niches.

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion.

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H(2)O(2). These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H(2)O(2) react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

Comparative genomics of the oxidative phosphorylation system in fungi.

In this study, we have carried out an in silico analysis of the available mitochondrial and nuclear genomes of fungi in order to identify the oxidative phosphorylation (OXPHOS) proteome, the complete set of proteins that perform the OXPHOS in mitochondria. The presence of OXPHOS proteins has been investigated in 27 nuclear and 52 mitochondrial genomes of fungi. Comparative genomics reveals a high conservation of the OXPHOS system within each fungal phyla, and notable differences between the OXPHOS proteomes of the fungal phyla. The most striking differences concerned Complexes I and V. The absence of Complex I has been previously described in various species of Ascomycota and Microsporidia, and the NDUFB4 and NURM accessory subunits of Complex I appear to be specific of fungi belonging to the subphylum Pezizomycotina. In addition, the Complex V essential subunit ATP14 appears to be specific of two subphyla of Ascomycota: the Saccharomycotina and Pezizomycotina.

Comparative genomic analysis of two-component regulatory proteins in Pseudomonas syringae.

BACKGROUND: Pseudomonas syringae is a widespread bacterial plant pathogen, and strains of P. syringae may be assigned to different pathovars based on host specificity among different plant species. The genomes of P. syringae pv. syringae (Psy) B728a, pv. tomato (Pto) DC3000 and pv. phaseolicola (Pph) 1448A have been recently sequenced providing a major resource for comparative genomic analysis. A mechanism commonly found in bacteria for signal transduction is the two-component system (TCS), which typically consists of a sensor histidine kinase (HK) and a response regulator (RR). P. syringae requires a complex array of TCS proteins to cope with diverse plant hosts, host responses, and environmental conditions. RESULTS: Based on the genomic data, pattern searches with Hidden Markov Model (HMM) profiles have been used to identify putative HKs and RRs. The genomes of Psy B728a, Pto DC3000 and Pph 1448A were found to contain a large number of genes encoding TCS proteins, and a core of complete TCS proteins were shared between these genomes: 30 putative TCS clusters, 11 orphan HKs, 33 orphan RRs, and 16 hybrid HKs. A close analysis of the distribution of genes encoding TCS proteins revealed important differences in TCS proteins among the three P. syringae pathovars. CONCLUSION: In this article we present a thorough analysis of the identification and distribution of TCS proteins among the sequenced genomes of P. syringae. We have identified differences in TCS proteins among the three P. syringae pathovars that may contribute to their diverse host ranges and association with plant hosts. The identification and analysis of the repertoire of TCS proteins in the genomes of P. syringae pathovars constitute a basis for future functional genomic studies of the signal transduction pathways in this important bacterial phytopathogen.