Structural, Evolutionary, and Functional Analysis of the Protein O-Mannosyltransferase Family in Pathogenic Fungi.
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ABSTRACT:
SUBMITTER: Pejenaute-Ochoa MD
PROVIDER: S-EPMC8147084 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Caleosin/peroxygenases, CLO/PXG, (designated PF05042 in Pfam) are a group of genes/proteins with anomalous distributions in eukaryotic taxa. We have previously characterised CLO/PXGs in the Viridiplantae. The aim of this study was to investigate the evolution and functions of the CLO/PXGs in the Fungi and other non-plant clades and to elucidate the overall origin of this gene family. RESULTS:CLO/PXG-like genes are distributed across the full range of fungal groups from the basal clades, Cryptomycota and Microsporidia, to the largest and most complex Dikarya species. However, the genes were only present in 243 out of 844 analysed fungal genomes. CLO/PXG-like genes have been retained in many pathogenic or parasitic fungi that have undergone considerable genomic and structural simplification, indicating that they have important functions in these species. Structural and functional analyses demonstrate that CLO/PXGs are multifunctional proteins closely related to similar proteins found in all major taxa of the Chlorophyte Division of the Viridiplantae. Transcriptome and physiological data show that fungal CLO/PXG-like genes have complex patterns of developmental and tissue-specific expression and are upregulated in response to a range of biotic and abiotic stresses as well as participating in key metabolic and developmental processes such as lipid metabolism, signalling, reproduction and pathogenesis. Biochemical data also reveal that the Aspergillus flavus CLO/PXG has specific functions in sporulation and aflatoxin production as well as playing roles in lipid droplet function. CONCLUSIONS:In contrast to plants, CLO/PXGs only occur in about 30% of sequenced fungal genomes but are present in all major taxa. Fungal CLO/PXGs have similar but not identical roles to those in plants, including stress-related oxylipin signalling, lipid metabolism, reproduction and pathogenesis. While the presence of CLO/PXG orthologs in all plant genomes sequenced to date would suggest that they have core housekeeping functions in plants, the selective loss of CLO/PXGs in many fungal genomes suggests more restricted functions in fungi as accessory genes useful in particular environments or niches. We suggest an ancient origin of CLO/PXG-like genes in the 'last eukaryotic common ancestor' (LECA) and their subsequent loss in ancestors of the Metazoa, after the latter had diverged from the ancestral fungal lineage.
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Project description:The fungal genus Colletotrichum contains hemibiotrophic phytopathogens being highly variable in host and tissue specificities. We sequenced a C. fructicola genome (1104-7) derived from an isolate of apple in China and compared it with the reference genome (Nara_gc5) derived from an isolate of strawberry in Japan. Mauve alignment and BlastN search identified 0.62 Mb lineage-specific (LS) genomic regions in 1104-7 with a length criterion of 10 kb. Genes located within LS regions evolved more dynamically, and a strongly elevated proportion of genes were closely related to non-Colletotrichum sequences. Two LS regions, containing nine genes in total, showed features of fungus-to-fungus horizontal transfer supported by both gene order collinearity and gene phylogeny patterns. We further compared the gene content variations among 13 Colletotrichum and 11 non-Colletotrichum genomes by gene function annotation, OrthoMCL grouping and CAFE analysis. The results provided a global evolutionary picture of Colletotrichum gene families, and identified a number of strong duplication/loss events at key phylogenetic nodes, such as the contraction of the detoxification-related RTA1 family in the monocot-specializing graminicola complex and the expansions of several ammonia production-related families in the fruit-infecting gloeosporioides complex. We have also identified the acquirement of a RbsD/FucU fucose transporter from bacterium by the Colletotrichum ancestor. In sum, this study summarized the pathogenic evolutionary features of Colletotrichum fungi at multiple taxonomic levels and highlights the concept that the pathogenic successes of Colletotrichum fungi require shared as well as lineage-specific virulence factors.
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Project description:The PIRSF protein classification system (http://pir.georgetown.edu/pirsf/) reflects evolutionary relationships of full-length proteins and domains. The primary PIRSF classification unit is the homeomorphic family, whose members are both homologous (evolved from a common ancestor) and homeomorphic (sharing full-length sequence similarity and a common domain architecture). PIRSF families are curated systematically based on literature review and integrative sequence and functional analysis, including sequence and structure similarity, domain architecture, functional association, genome context, and phyletic pattern. The results of classification and expert annotation are summarized in PIRSF family reports with graphical viewers for taxonomic distribution, domain architecture, family hierarchy, and multiple alignment and phylogenetic tree. The PIRSF system provides a comprehensive resource for bioinformatics analysis and comparative studies of protein function and evolution. Domain or fold-based searches allow identification of evolutionarily related protein families sharing domains or structural folds. Functional convergence and functional divergence are revealed by the relationships between protein classification and curated family functions. The taxonomic distribution allows the identification of lineage-specific or broadly conserved protein families and can reveal horizontal gene transfer. Here we demonstrate, with illustrative examples, how to use the web-based PIRSF system as a tool for functional and evolutionary studies of protein families.
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Project description:Crop diseases caused by pathogens critically affect global food security and plant ecology. Pathogens are well adapted to their host plants and have developed sophisticated mechanisms allowing successful colonization. Plants in turn have taken measures to counteract pathogen attacks resulting in an evolutionary arms race. Recent studies provided mechanistic insights into how two plant Kiwellin proteins from Zea mays mitigate the activity of the chorismate mutase Cmu1, a virulence factor secreted by the fungal pathogen Ustilago maydis during maize infection. Formerly identified as human allergens in kiwifruit, the biological function of Kiwellins is apparently linked to plant defense. We combined the analysis of proteome data with structural predictions to obtain a holistic overview of the Kiwellin protein family, that is subdivided into proteins with and without a N-terminal kissper domain. We found that Kiwellins are evolutionarily conserved in various plant species. At median five Kiwellin paralogs are encoded in each plant genome. Structural predictions revealed that Barwin-like proteins and Kiwellins cannot be discriminated purely at the sequence level. Our data shows that Kiwellins emerged in land plants (embryophyta) and are not present in fungi as suggested earlier. They evolved via three major duplication events that lead to clearly distinguishable subfamilies. We introduce a systematic Kiwellin nomenclature based on a detailed evolutionary reconstruction of this protein family. A meta-analysis of publicly available transcriptome data demonstrated that Kiwellins can be differentially regulated upon the interaction of plants with pathogens but also with symbionts. Furthermore, significant differences in Kiwellin expression levels dependent on tissues and cultivars were observed. In summary, our study sheds light on the evolution and regulation of a large protein family and provides a framework for a more detailed understanding of the molecular functions of Kiwellins.
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Project description:A primary level of control for nuclear factor kappa B (NF-κB) is effected through its interactions with the inhibitor protein, inhibitor of kappa B (IκB). Several lines of evidence confirm the existence of multiple forms of IκB that appear to regulate NF-κB by distinct mechanisms. Therefore, we performed a comprehensive bioinformatics analysis to understand the evolutionary history and intrinsic functional diversity of IκB family members. Phylogenetic relationships were constructed to trace the evolution of the IκB family genes. Our phylogenetic analysis revealed 10 IκB subfamily members that clustered into 5 major clades. Since the ankyrin (ANK) domain appears to be more ancient than the Rel homology domain (RHD), our phylogenetic analysis suggests that some undefined ancestral set of ANK repeats acquired an RHD before any duplication and was later duplicated and then diverged into the different IκB subfamilies. Functional analysis identified several functionally divergent sites in the ANK repeat domains (ARDs) and revealed that this region has undergone strong purifying selection, suggesting its functional importance in IκB genes. Structural analysis showed that the major variations in the number of ANK repeats and high conformational changes in the finger loop ARD region contribute to the differing binding partner specificities, thereby leading to distinct IκB functions. In summary, our study has provided useful information about the phylogeny and structural and functional divergence of the IκB family. Additionally, we identified a number of amino acid sites that contribute to the predicted functional divergence of these proteins.
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Project description:Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by β-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×108M-1s-1). Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria.
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Project description:The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of ?-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.
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Project description:Protein evolution is constrained by structure and function, creating patterns in residue conservation that are routinely exploited to predict structure and other features. Similar constraints should affect variation across individuals, but it is only with the growth of human population sequencing that this has been tested at scale. Now, human population constraint has established applications in pathogenicity prediction, but it has not yet been explored for structural inference. Here, we map 2.4 million population variants to 5885 protein families and quantify residue-level constraint with a new Missense Enrichment Score (MES). Analysis of 61,214 structures from the PDB spanning 3661 families shows that missense depleted sites are enriched in buried residues or those involved in small-molecule or protein binding. MES is complementary to evolutionary conservation and a combined analysis allows a new classification of residues according to a conservation plane. This approach finds functional residues that are evolutionarily diverse, which can be related to specificity, as well as family-wide conserved sites that are critical for folding or function. We also find a possible contrast between lethal and non-lethal pathogenic sites, and a surprising clinical variant hot spot at a subset of missense enriched positions.
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Project description:Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.
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Project description:Plant-pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are virulence factors secreted by fungi, which fulfil diverse functions to support the infection process. Most of these proteins are highly specialized, with structural and biochemical information often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homologue Uvi2 from Ustilago hordei. Both proteins adopt a double-Ψβ-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure-function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and might synergize with cell wall-degrading and decorating proteins during maize infection. The architecture of Cpl1 harbouring four surface-exposed loop regions supports the idea that it might play a role in the spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions that might explain a diverging function.
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