Secreted Glycoside Hydrolase Proteins as Effectors and Invasion Patterns of Plant-Associated Fungi and Oomycetes.

During host colonization, plant-associated microbes, including fungi and oomycetes, deliver a collection of glycoside hydrolases (GHs) to their cell surfaces and surrounding extracellular environments. The number and type of GHs secreted by each organism is typically associated with their lifestyle or mode of nutrient acquisition. Secreted GHs of plant-associated fungi and oomycetes serve a number of different functions, with many of them acting as virulence factors (effectors) to promote microbial host colonization. Specific functions involve, for example, nutrient acquisition, the detoxification of antimicrobial compounds, the manipulation of plant microbiota, and the suppression or prevention of plant immune responses. In contrast, secreted GHs of plant-associated fungi and oomycetes can also activate the plant immune system, either by acting as microbe-associated molecular patterns (MAMPs), or through the release of damage-associated molecular patterns (DAMPs) as a consequence of their enzymatic activity. In this review, we highlight the critical roles that secreted GHs from plant-associated fungi and oomycetes play in plant-microbe interactions, provide an overview of existing knowledge gaps and summarize future directions.Published onlin

The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics

The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families. These families are created based on experimentally characterized proteins and are populated by sequences from public databases with significant similarity. Protein biochemical information is continuously curated based on the available literature and structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification (i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties. This resource has been available for over 10 years to the scientific community, contributing to information dissemination and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/

Stereochemical course of the hydrolysis reaction catalyzed by chitinases Al and D from Bacillus circulans WL-12

AbstractChitinases A1 and D were purified from the periplasmic proteins produced by Escherichia coli HB101 harbouring recombinant plasmids carrying respectively the chiA and chiD genes of Bacillus circulans WL-12. HPLC analysis indicated that during the hydrolysis of chitotriose, both chitinases initially produce N-acetylglucosamine and only one anomer of chitobiose. 1H NMR spectroscopy of the hydrolysis of chitotetraitol showed that this anomer corresponds to β-chitobiose, demonstrating that chitinases Al and D act by a molecular mechanism that retains the anomeric configuration. This mechanism is similar to that of lysozymes although both chitinases belong to a family of proteins sharing no demonstrable amino acid sequence similarity with lysozymes

Refining and mining the phylogeny of Glycoside Hydrolase Family 74 via structure-function analysis

Sustained interest in the use of carbohydrates from plant cell walls, coupled with the advancement of high-throughput (meta)genomic sequencing, has led to the discovery of an overwhelming number of predicted carbohydrate-active enzymes (CAZymes) in the last decade. The CAZy database provides a powerful framework for the study of CAZymes, including Glycoside Hydrolases (GHs), by enabling the prediction of key enzyme features such as 3-D fold, catalytic residues, catalytic mechanism, and – with certain limitations – substrate specificity. Refined phylogenetic analyses contribute to increasing the accuracy of predictions by further clustering proteins into sub-families (1, 2). However, reliable prediction of substrate specificity for newly discovered GHs remains a challenge due to a general lack of in-depth biochemical and structural characterization across the existing phylogenetic diversity. Glycoside Hydrolase family 74 (GH74) comprises endo-glucanases, many of which have predominant activity toward xyloglucan, a highly branched plant cell wall matrix glycan. To better delineate overall substrate specificity, backbone cleavage position, and endo-dissociative vs. endo-processive hydrolytic modes, a broad-based structure-function analysis of GH74 guided by molecular phylogeny was performed. Seven sub-families were discerned, which grouped nearly 40% of the current \u3e300 GH74 sequences in the public CAZy database. Thirty one GH74 members were targeted for further investigation based on their phylogenetic position and unique primary structural features identified during manual curation. The biochemical characterization of 18 recombinant GH74s revealed key sequence features governing xyloglucan backbone cleavage sites and highlighted clear phylogenetic differences between endo-dissociative and endo-processive enzymes. Commensurate with previous studies (3), site-directed mutagenesis of key active-site tryptophan residues defined their essential contributions to processivity on the soluble polysaccharide substrate. Six new GH74 tertiary structures (apo and/or in complex with xylogluco-oligosaccharides) were determined that further resolved the contribution of active-site loops in modulating the size of oligosaccharide products released by individual subfamily members. Refining the correlation between phylogeny and enzyme structure-function properties in GH74 significantly enhances the prediction of catalytic ability, highlights key steps in the evolution of function in the family, and ultimately informs applications in biomass conversion. 1. Stam MR, Danchin EGJ, Rancurel C, Coutinho PM, Henrissat B. 2006. Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins. Protein Engineering Design & Selection 19:555-562. 2. Aspeborg H, Coutinho PM, Wang Y, Brumer H, Henrissat B. 2012. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). Bmc Evolutionary Biology 12. 3. Matsuzawa T, Saito Y, Yaoi K. 2014. Key amino acid residues for the endo-processive activity of GH74 xyloglucanase. FEBS Lett 588:1731-8

Comparative genomics between Trichomonas tenax and Trichomonas vaginalis: CAZymes and candidate virulence factors

Introduction: The oral trichomonad Trichomonas tenax is increasingly 82 appreciated as a likely contributor to periodontitis, a chronic inflammatory 83 disease induced by dysbiotic microbiota, in humans and domestic animals and 84 is strongly associated with its worst prognosis. Our current understanding of 85 the molecular basis of T. tenax interactions with host cells and the microbiota 86 of the oral cavity are still rather limited. One laboratory strain of T. tenax (Hs- 87 4:NIH/ATCC 30207) can be grown axenically and two draft genome assemblies 88 have been published for that strain, although the structural and functional 89 annotation of these genomes is not available. 90Methods: GenSAS and Galaxy were used to annotate two publicly available 91 draft genomes for T. tenax, with a focus on protein-coding genes. A custom 92 pipeline was used to annotate the CAZymes for T. tenax and the human 93 sexually transmitted parasite Trichomonas vaginalis, the most well-characterized 94 trichomonad. A combination of bioinformatics analyses was used to screen for 95 homologs of T. vaginalis virulence and colonization factors within the T. tenax 96 annotated proteins. 97Results: Our annotation of the two T. tenax draft genome sequences and their 98 comparison with T. vaginalis proteins provide evidence for several candidate 99 virulence factors. These include candidate surface proteins, secreted proteins 100 and enzymes mediating potential interactions with host cells and/or members 101 of the oral microbiota. The CAZymes annotation identified a broad range of 102 glycoside hydrolase (GH) families, with the majority of these being shared 103between the two Trichomonas species. 104 105 Discussion: The presence of candidate T. tenax virulence genes supports the 106hypothesis that this species is associated with periodontitis through direct and 107 indirect mechanisms. Notably, several GH proteins could represent potential new 108 virulence factors for both Trichomonas species. These data support a model 109 where T. tenax interactions with host cells and members of the oral microbiota 110 could synergistically contribute to the damaging inflammation characteristic of 111 periodontitis, supporting a causal link between T. tenax and periodontitis

101 Dothideomycetes genomes: A test case for predicting lifestyles and emergence of pathogens.

Dothideomycetes is the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetes and they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomycetes has significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetes introducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetes phylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species