FunOMIC: Pipeline with built-in fungal taxonomic and functional databases for human mycobiome profiling

Fungal databases; Taxonomy and functions; Inter-kingdom interactionsBases de datos de hongos; Taxonomía y funciones; Interacciones entre reinosBases de dades de fongs; Taxonomia i funcions; Interaccions entre regnesWhile analysis of the bacterial microbiome has become routine, that of the fungal microbiome is still hampered by the lack of robust databases and bioinformatic pipelines. Here, we present FunOMIC, a pipeline with built-in taxonomic (1.6 million marker genes) and functional (3.4 million non-redundant fungal proteins) databases for the identification of fungi. Applied to more than 2,600 human metagenomic samples, the tool revealed fungal species associated with geography, body sites, and diseases. Correlation network analysis provided new insights into inter-kingdom interactions. With this pipeline and two of the most comprehensive fungal databases, we foresee a fast-growing resource for mycobiome studies.This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Action, Innovative Training Network [grant number 812969] and by the Instituto de Salud Carlos III /FEDER, a government agency (Grant No: PI17/00614; PI20/00130)

Shotgun metagenomics reveals interkingdom association between intestinal bacteria and fungi involving competition for nutrients

Comprehensive database; Diet; MicrobiomeBase de datos integral; Dieta; MicrobiomaBase de dades integral; Dieta; MicrobiomaBackground The accuracy of internal-transcribed-spacer (ITS) and shotgun metagenomics has not been robustly evaluated, and the effect of diet on the composition and function of the bacterial and fungal gut microbiome in a longitudinal setting has been poorly investigated. Here we compared two approaches to study the fungal community (ITS and shotgun metagenomics), proposed an enrichment protocol to perform a reliable mycobiome analysis using a comprehensive in-house fungal database, and correlated dietary data with both bacterial and fungal communities. Results We found that shotgun DNA sequencing after a new enrichment protocol combined with the most comprehensive and novel fungal databases provided a cost-effective approach to perform gut mycobiome profiling at the species level and to integrate bacterial and fungal community analyses in fecal samples. The mycobiome was significantly more variable than the bacterial community at the compositional and functional levels. Notably, we showed that microbial diversity, composition, and functions were associated with habitual diet composition instead of driven by global dietary changes. Our study indicates a potential competitive inter-kingdom interaction between bacteria and fungi for food foraging. Conclusion Together, our present work proposes an efficient workflow to study the human gut microbiome integrating robustly fungal, bacterial, and dietary data. These findings will further advance our knowledge of the interaction between gut bacteria and fungi and pave the way for future investigations in human mycobiome.This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Action, Innovative Training Network [grant number 812969]

Intercontinental Gut Microbiome Variances in IBD

The development of biomarkers for inflammatory bowel disease (IBD) diagnosis would be relevant in a generalized context. However, intercontinental investigation on these microbial biomarkers remains scarce. We examined taxonomic microbiome variations in IBD using published DNA shotgun metagenomic data. For this purpose, we used sequenced data from our previous Spanish Crohn's disease (CD) and ulcerative colitis (UC) cohort, downloaded sequence data from a Chinese CD cohort, and downloaded taxonomic and functional profiling tables from a USA CD and UC cohort. At the global level, geographical location and disease phenotype were the main explanatory covariates of microbiome variations. In healthy controls (HC) and UC, geography turned out to be the most important factor, while disease intestinal location was the most important one in CD. Disease severity correlated with lower alpha-diversity in UC but not in CD. Across geography, alpha-diversity was significantly different independently of health status, except for CD. Despite recruitment from different countries and with different disease severity scores, CD patients may harbor a very similar microbial taxonomic profile. Our study pointed out that geographic location, disease activity status, and other environmental factors are important contributing factors in microbiota changes in IBD. We therefore strongly recommend taking these factors into consideration for future IBD studies to obtain globally valid and reproducible biomarker

The levamisole sensitive nicotinic acetylcholine receptor of the potato cyst nematode Globodera pallida

The potato cyst nematode Globodera pallida costs the UK potato industry over £50 million per annum. In order to invade a host plant, the infective J2 stage must hatch from eggs within the soil and migrate towards the root system. Orthologues of Caenorhabditis elegans genes involved in neurotransmission were identified in the G. pallida and G. rostochiensis genome assemblies. The complement of cys loop ligand gated ion channel genes was distinct compared to C. elegans and other parasitic nematodes. Orthologues of genes encoding subunits which comprise the C. elegans levamisole sensitive nicotinic acetylcholine receptor (cel-lev 1, cel-lev 8, cel-unc 29, cel-unc 63 and cel-unc 38) were searched for, and cel-lev 1 and cel-lev 8 orthologues were absent in both Globodera spp. Two orthologues were identified for cel-unc 29 and cel-unc 38. This suggested that the composition of the G. pallida L nAChR may differ. The use of C. elegans as a heterologous system to study the expression pattern of G. pallida nAChR genes was explored. GFP expressing lines were created using promoter regions of gpa acr 2 and gpa unc 63. Expression was observed in the ventral nerve cord and nerve ring for pgpa-acr 2. Expression of pgpa-unc 63 was variable, but was found in the head and tail region and along the ventral side of the body. The impact of this distinct complement of cys loop subunits on anthelmintic sensitivity was demonstrated by the increased resistance of both G. pallida and G. rostochiensis J2s to levamisole. The EC50 of G. pallida and G. rostochiensis was 19.7 mM and 5.6 mM respectively, compared to the EC50 of 9 µM for C. elegans, representing a 500 – 2000 fold increase in levamisole resistance. This increased resistance to levamisole was associated with an orthologue of cel-unc 38 identified in G. pallida, gpa unc 38.1. Rescue of C. elegans unc 38(x20) mutants with gpa unc 38.1 restores normal movement suggesting a functional reconstitution of the L nAChR, but full sensitivity to levamisole is not restored. Gpa unc 38.1 was expressed with the remaining four subunits from C. elegans in Xenopus oocytes to produce a chimeric receptor. The EC50 of the response to acetylcholine and levamisole of the chimeric receptor and the native receptor was comparable and had similar opening responses to different agonists. Chimeric genes were created to analyse key motifs in gpa unc 38.1 that may affect receptor function and levamisole sensitivity. Gpa unc 38.1 was necessary for structural reformation of the receptor, but not acetylcholine binding. Removal or addition of a loop B glutamate residue, previously associated with levamisole sensitivity of Cel UNC 38, did not affect levamisole sensitivity of Cel-UNC 38 or Gpa UNC 38.1. An amino acid change (I>M) in TM2 of Cel-UNC 38 increased levamisole sensitivity and basal thrashing rate. The reciprocal change (M>I) in Gpa UNC 38.1 comprised basal thrashing rescue. The basis of increased levamisole resistance of gpa unc 38.1 was not identified, as all gpa unc 38.1 chimeric genes retained a higher resistance to levamisole than cel-unc 38. This works reveals that the nAChRs of plant parasitic nematodes have distinct pharmacological characteristics

Gut microbial dysbiosis in patients with Cushing’s disease in long-term remission: Relationship with cardiometabolic risk

Cushing's disease; Cardiovascular risk; Gut microbiotaEnfermedad de Cushing; Riesgo cardiovascular; Microbiota intestinalMalaltia de Cushing; Risc cardiovascular; Microbiota intestinalBackground: Patients with Cushing’s disease (CD) in remission maintain an increased cardiovascular risk. Impaired characteristics of gut microbiome (dysbiosis) have been associated with several cardiometabolic risk factors. Methods: Twenty-eight female non-diabetic patients with CD in remission with a mean ± SD) age of 51 ± 9 years, mean ( ± SD) BMI, 26 ± 4, median (IQR) duration of remission, 11(4) years and 24 gender-, age, BMI–matched controls were included. The V4 region of the bacterial 16S rDNA was PCR amplified and sequenced to analyse microbial alpha diversity (Chao 1 index, observed number of species, Shannon index) and beta diversity analysis through the Principal Coordinates Analysis (PCoA) of weighted and unweighted UniFrac distances. Inter-group difference in microbiome composition was analysed using MaAsLin2. Results: The Chao 1 index was lower in CD as compared with controls (Kruskal-Wallis test, q = 0.002), indicating lower microbial richness in the former. Beta diversity analysis showed that faecal samples from CS patients clustered together and separated from the controls (Adonis test, p<0.05). Collinsella, a genus form of the Actinobacteria phylum was present in CD patients only, whereas Sutterella, a genus from Proteobacteria phylum, was scarcely detectable/undetectable in CD patients as well as Lachnospira, a genus of the Lachnospiraceae family of the Firmicutes phylum. In CS, the Chao 1 index was associated with fibrinogen levels and inversely correlated with both triglyceride concentrations and the HOMA-IR index (p<0.05). Conclusions: Patients with CS in remission have gut microbial dysbiosis which may be one of the mechanisms whereby cardiometabolic dysfunctions persist after “cure”

Topoisomerase II regulates yeast genes with singular chromatin architectures

Eukaryotic topoisomerase II (topo II) is the essential decatenase of newly replicated chromosomes and the main relaxase of nucleosomal DNA. Apart from these general tasks, topo II participates in more specialized functions. In mammals, topo IIa interacts with specific RNA polymerases and chromatin-remodeling complexes, whereas topo IIb regulates developmental genes in conjunction with chromatin remodeling and heterochromatin transitions. Here we show that in budding yeast, topo II regulates the expression of specific gene subsets. To uncover this, we carried out a genomic transcription run-on shortly after the thermal inactivation of topo II. We identified a modest number of genes not involved in the general stress response but strictly dependent on topo II. These genes present distinctive functional and structural traits in comparison with the genome average. Yeast topo II is a positive regulator of genes with well-defined promoter architecture that associates to chromatin remodeling complexes; it is a negative regulator of genes extremely hypo-acetylated with complex promoters and undefined nucleosome positioning, many of which are involved in polyamine transport. These findings indicate that yeast topo II operates on singular chromatin architectures to activate or repress DNA transcription and that this activity produces functional responses to ensure chromatin stability

Unveiling Candida albicans intestinal carriage in healthy volunteers: the role of micro- and mycobiota, diet, host genetics and immune response

Candida albicans; Colonization resistance; MetagenomicsCandida albicans; Resistència a la colonització; MetagenòmicaCandida albicans; Resistencia a la colonización; MetagenómicaCandida albicans is a commensal yeast present in the gut of most healthy individuals but with highly variable concentrations. However, little is known about the host factors that influence colonization densities. We investigated how microbiota, host lifestyle factors, and genetics could shape C. albicans intestinal carriage in 695 healthy individuals from the Milieu Intérieur cohort. C. albicans intestinal carriage was detected in 82.9% of the subjects using quantitative PCR. Using linear mixed models and multiway-ANOVA, we explored C. albicans intestinal levels with regard to gut microbiota composition and lifestyle factors including diet. By analyzing shotgun metagenomics data and C. albicans qPCR data, we showed that Intestinimonas butyriciproducens was the only gut microbiota species whose relative abundance was negatively correlated with C. albicans concentration. Diet is also linked to C. albicans growth, with eating between meals and a low-sodium diet being associated with higher C. albicans levels. Furthermore, by Genome-Wide Association Study, we identified 26 single nucleotide polymorphisms suggestively associated with C. albicans colonization. In addition, we found that the intestinal levels of C. albicans might influence the host immune response, specifically in response to fungal challenge. We analyzed the transcriptional levels of 546 immune genes and the concentration of 13 cytokines after whole blood stimulation with C. albicans cells and showed positive associations between the extent of C. albicans intestinal levels and NLRP3 expression, as well as secreted IL-2 and CXCL5 concentrations. Taken together, these findings open the way for potential new interventional strategies to curb C. albicans intestinal overgrowth.This work was supported by a grant from Agence Nationale de la Recherche (FunComPath ANR-14-IFEC-0004), the French Government’s Investissement d’Avenir program (Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases [ANR10-LABX-62-IBEID], and [ANR-10-LABX-69-01]), the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie action, Innovative Training Network (FunHoMic; Grant No. 812969), and the European Union’s Horizon 2020 Research and Innovation Program (HDM-FUN, Grant No. 847507). AWW and the Rowett Institute (University of Aberdeen) received core funding support from the Scottish Government’s Rural and Environmental Sciences and Analytical Services (RESAS)

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives

Candida; Antifungal immunity; MicrobiotaCándida; Inmunidad antimicótica; MicrobiotaCàndida; Immunitat antifúngica; MicrobiotaCandida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients

Fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 reduces bacterial translocation in rats treated with carbon tetrachloride

Citocines; Malalties del fetgeCitocinas, Enfermedades del hígadoCytokines; Liver diseasesProbiotics can prevent pathological bacterial translocation by modulating intestinal microbiota and improving the gut barrier. The aim was to evaluate the effect of a fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 on bacterial translocation in rats with carbon tetrachloride (CCl4)-induced cirrhosis. Sprague-Dawley rats treated with CCl4 were randomized into a probiotic group that received fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 in drinking water or a water group that received water only. Laparotomy was performed one week after ascites development. We evaluated bacterial translocation, intestinal microbiota, the intestinal barrier and cytokines in mesenteric lymph nodes and serum. Bacterial translocation decreased and gut dysbiosis improved in the probiotic group compared to the water group. The ileal β-defensin-1 concentration was higher and ileal malondialdehyde levels were lower in the probiotic group than in water group. There were no differences between groups in serum cytokines but TNF-α levels in mesenteric lymph nodes were lower in the probiotic group than in the water group. Fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 decreases bacterial translocation, gut dysbiosis and ileal oxidative damage and increases ileal β-defensin-1 expression in rats treated with CCl4, suggesting an improvement in the intestinal barrier integrity.This study was partially funded by Danone Research (Palaiseau, Cedex, France), and grants PI08/0262 and PI14/00680 from the Instituto de Salud Carlos III, Madrid, Spain, and cofinanced by Fondos FEDER (Fondo Europeo de Desarrollo Regional), “Una manera de hacer Europa”, European Union, and CERCA Programme, Generalitat de Catalunya. The investigational product was provided by Danone Research. Silvia Vidal was supported by Fondo de Investigaciones Sanitarias (FIS) and is a participant in the Program for Stabilization of Investigators of the Direcció d’Estrategia i Coordinació del Departament de Salut, Generalitat de Catalunya. Pau Sancho-Bru is funded by Instituto de Salud Carlos III, Miguel Servet (CP11/00071)

A single faecal microbiota transplantation modulates the microbiome and improves clinical manifestations in a rat model of colitis

Inflammatory bowel disease; Faecal microbiota transplantation; Rat model of colitisEnfermedad inflamatoria intestinal; Trasplante de microbiota fecal; Modelo de colitis en ratasMalaltia inflamatòria intestinal; Trasplantament de microbiota fecal; Model de colitis en ratesBackground: Faecal microbiota transplantation (FMT) is a novel potential therapy for inflammatory bowel diseases, but it is poorly characterised. Methods: We evaluated the performance of the mouse and rat as a pre-clinical model for human microbiota engraftment. We then characterised the effect of a single human stool transfer (HST) on a humanised model of DSS-induced colitis. Colonic and faecal microbial communities were analysed using the 16S rRNA approach and clinical manifestations were assessed in a longitudinal setting. Findings: The microbial community of rats showed greater similarity to that of humans, while the microbiome of mice showed less similarity to that of humans. Moreover, rats captured more human microbial species than mice after a single HST. Using the rat model, we showed that HST compensated faecal dysbiosis by restoring alpha-diversity and by increasing the relative abundance of health-related microbial genera. To some extent, HST also modulated the microbial composition of colonic tissue. These faecal and colonic microbial communities alterations led to a relative restoration of colon length, and a significant decrease in both epithelium damage and disease severity. Remarkably, stopping inflammation by removing DSS before HST caused a faster and greater recovery of both microbiome and clinical manifestation features. Interpretation: Our results indicate that the rat outperforms the mouse as a model for human microbiota engraftment and show that the efficacy of HST can be enhanced when inflammation stimulation is withdrawn. Finally, our findings support a new therapeutic strategy based on the use FMT combined with anti inflammatory drugs.Study funded by the Instituto de Salud Carlos III/FEDER (PI17/00614), a government agency. The funder had no role in study design, data collection, data analysis, interpretation or writing of the report