Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile.

Despite decades of research, the structure and assembly of the nuclear pore complex (NPC), which is composed of ∼30 nucleoporins (Nups), remain elusive. Here, we report the genome of the thermophilic fungus Chaetomium thermophilum (ct) and identify the complete repertoire of Nups therein. The thermophilic proteins show improved properties for structural and biochemical studies compared to their mesophilic counterparts, and purified ctNups enabled the reconstitution of the inner pore ring module that spans the width of the NPC from the anchoring membrane to the central transport channel. This module is composed of two large Nups, Nup192 and Nup170, which are flexibly bridged by short linear motifs made up of linker Nups, Nic96 and Nup53. This assembly illustrates how Nup interactions can generate structural plasticity within the NPC scaffold. Our findings therefore demonstrate the utility of the genome of a thermophilic eukaryote for studying complex molecular machines.

Opportunities and barriers in omics-based biomarker discovery for steatotic liver diseases.

The rising prevalence of liver diseases related to obesity and excessive use of alcohol is fuelling an increasing demand for accurate biomarkers aimed at community screening, diagnosis of steatohepatitis and significant fibrosis, monitoring, prognostication and prediction of treatment efficacy. Breakthroughs in omics methodologies and the power of bioinformatics have created an excellent opportunity to apply technological advances to clinical needs, for instance in the development of precision biomarkers for personalised medicine. Via omics technologies, biological processes from the genes to circulating protein, as well as the microbiome - including bacteria, viruses and fungi, can be investigated on an axis. However, there are important barriers to omics-based biomarker discovery and validation, including the use of semi-quantitative measurements from untargeted platforms, which may exhibit high analytical, inter- and intra-individual variance. Standardising methods and the need to validate them across diverse populations presents a challenge, partly due to disease complexity and the dynamic nature of biomarker expression at different disease stages. Lack of validity causes lost opportunities when studies fail to provide the knowledge needed for regulatory approvals, all of which contributes to a delayed translation of these discoveries into clinical practice. While no omics-based biomarkers have matured to clinical implementation, the extent of data generated has enabled the hypothesis-free discovery of a plethora of candidate biomarkers that warrant further validation. To explore the many opportunities of omics technologies, hepatologists need detailed knowledge of commonalities and differences between the various omics layers, and both the barriers to and advantages of these approaches.

Human gut microbes impact host serum metabolome and insulin sensitivity.

Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus are identified as the main species driving the association between biosynthesis of BCAAs and insulin resistance, and in mice we demonstrate that P. copri can induce insulin resistance, aggravate glucose intolerance and augment circulating levels of BCAAs. Our findings suggest that microbial targets may have the potential to diminish insulin resistance and reduce the incidence of common metabolic and cardiovascular disorders.

Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota.

In recent years, several associations between common chronic human disorders and altered gut microbiome composition and function have been reported. In most of these reports, treatment regimens were not controlled for and conclusions could thus be confounded by the effects of various drugs on the microbiota, which may obscure microbial causes, protective factors or diagnostically relevant signals. Our study addresses disease and drug signatures in the human gut microbiome of type 2 diabetes mellitus (T2D). Two previous quantitative gut metagenomics studies of T2D patients that were unstratified for treatment yielded divergent conclusions regarding its associated gut microbial dysbiosis. Here we show, using 784 available human gut metagenomes, how antidiabetic medication confounds these results, and analyse in detail the effects of the most widely used antidiabetic drug metformin. We provide support for microbial mediation of the therapeutic effects of metformin through short-chain fatty acid production, as well as for potential microbiota-mediated mechanisms behind known intestinal adverse effects in the form of a relative increase in abundance of Escherichia species. Controlling for metformin treatment, we report a unified signature of gut microbiome shifts in T2D with a depletion of butyrate-producing taxa. These in turn cause functional microbiome shifts, in part alleviated by metformin-induced changes. Overall, the present study emphasizes the need to disentangle gut microbiota signatures of specific human diseases from those of medication.

Perturbation of gut microbiota decreases susceptibility but does not modulate ongoing autoimmune neurological disease.

The gut microbiota regulates the host immune and nervous systems and plays an important role in the pathogenesis of autoimmune neurological disease multiple sclerosis (MS). There are considerable efforts currently being undertaken to develop therapies for MS based on the modulation of microbiota. Evidence from experimental models suggests that the manipulation of microbiota through diet or antibiotics prior to the disease development limits disease susceptibility. However, it is currently unclear if microbiota manipulation therapies would also have an impact on ongoing neurological disease. Here, we examined the effect of antibiotic-based microbiota modulation in spontaneous experimental autoimmune encephalomyelitis (EAE) mouse models of MS before and after the onset of autoimmune disease. Prophylactic antibiotic treatment led to a significant reduction of susceptibility to spontaneous EAE. In contrast, antibiotic treatment after the onset of spontaneous EAE did not show a significant amelioration. These results reveal that the perturbation of gut bacteria alters disease susceptibility but has minimal impact on the ongoing neurological disease.

Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice.

Remodeling of the gut microbiota is implicated in various metabolic and inflammatory diseases of the gastrointestinal tract. We hypothesized that the gut microbiota affects the DNA methylation profile of intestinal epithelial cells (IECs) which could, in turn, alter intestinal function. In this study, we used mass spectrometry and methylated DNA capture to respectively investigate global and genome-wide DNA methylation of intestinal epithelial cells from germ-free (GF) and conventionally raised mice. In colonic IECs from GF mice, DNA was markedly hypermethylated. This was associated with a dramatic loss of ten-eleven-translocation activity, a lower DNA methyltransferase activity and lower circulating levels of the 1-carbon metabolite, folate. At the gene level, we found an enrichment for differentially methylated regions proximal to genes regulating the cytotoxicity of NK cells (false-discovery rate < 8.9E-6), notably genes regulating the cross-talk between NK cells and target cells, such as members of the NK group 2 member D ligand superfamily Raet. This distinct epigenetic signature was associated with a marked decrease in Raet1 expression and a loss of CD56+/CD45+ cells in the intestine of GF mice. Thus, our results indicate that altered activity of methylation-modifying enzymes in GF mice influences the IEC epigenome and modulates the crosstalk between IECs and NK cells. Epigenetic reprogramming of IECs may modulate intestinal function in diseases associated with altered gut microbiota.-Poupeau, A., Garde, C., Sulek, K., Citirikkaya, K., Treebak, J. T., Arumugam, M., Simar, D., Olofsson, L. E., Bäckhed, F., Barrès, R. Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice.

Genomic variation landscape of the human gut microbiome.

Whereas large-scale efforts have rapidly advanced the understanding and practical impact of human genomic variation, the practical impact of variation is largely unexplored in the human microbiome. We therefore developed a framework for metagenomic variation analysis and applied it to 252 faecal metagenomes of 207 individuals from Europe and North America. Using 7.4 billion reads aligned to 101 reference species, we detected 10.3 million single nucleotide polymorphisms (SNPs), 107,991 short insertions/deletions, and 1,051 structural variants. The average ratio of non-synonymous to synonymous polymorphism rates of 0.11 was more variable between gut microbial species than across human hosts. Subjects sampled at varying time intervals exhibited individuality and temporal stability of SNP variation patterns, despite considerable composition changes of their gut microbiota. This indicates that individual-specific strains are not easily replaced and that an individual might have a unique metagenomic genotype, which may be exploitable for personalized diet or drug intake.

Richness of human gut microbiome correlates with metabolic markers.

We are facing a global metabolic health crisis provoked by an obesity epidemic. Here we report the human gut microbial composition in a population sample of 123 non-obese and 169 obese Danish individuals. We find two groups of individuals that differ by the number of gut microbial genes and thus gut bacterial richness. They contain known and previously unknown bacterial species at different proportions; individuals with a low bacterial richness (23% of the population) are characterized by more marked overall adiposity, insulin resistance and dyslipidaemia and a more pronounced inflammatory phenotype when compared with high bacterial richness individuals. The obese individuals among the lower bacterial richness group also gain more weight over time. Only a few bacterial species are sufficient to distinguish between individuals with high and low bacterial richness, and even between lean and obese participants. Our classifications based on variation in the gut microbiome identify subsets of individuals in the general white adult population who may be at increased risk of progressing to adiposity-associated co-morbidities.

Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer.

OBJECTIVE: To evaluate the potential for diagnosing colorectal cancer (CRC) from faecal metagenomes. DESIGN: We performed metagenome-wide association studies on faecal samples from 74 patients with CRC and 54 controls from China, and validated the results in 16 patients and 24 controls from Denmark. We further validated the biomarkers in two published cohorts from France and Austria. Finally, we employed targeted quantitative PCR (qPCR) assays to evaluate diagnostic potential of selected biomarkers in an independent Chinese cohort of 47 patients and 109 controls. RESULTS: Besides confirming known associations of Fusobacterium nucleatum and Peptostreptococcus stomatis with CRC, we found significant associations with several species, including Parvimonas micra and Solobacterium moorei. We identified 20 microbial gene markers that differentiated CRC and control microbiomes, and validated 4 markers in the Danish cohort. In the French and Austrian cohorts, these four genes distinguished CRC metagenomes from controls with areas under the receiver-operating curve (AUC) of 0.72 and 0.77, respectively. qPCR measurements of two of these genes accurately classified patients with CRC in the independent Chinese cohort with AUC=0.84 and OR of 23. These genes were enriched in early-stage (I-II) patient microbiomes, highlighting the potential for using faecal metagenomic biomarkers for early diagnosis of CRC. CONCLUSIONS: We present the first metagenomic profiling study of CRC faecal microbiomes to discover and validate microbial biomarkers in ethnically different cohorts, and to independently validate selected biomarkers using an affordable clinically relevant technology. Our study thus takes a step further towards affordable non-invasive early diagnostic biomarkers for CRC from faecal samples.

Enterotypes of the human gut microbiome.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

Country-specific antibiotic use practices impact the human gut resistome.

Despite increasing concerns over inappropriate use of antibiotics in medicine and food production, population-level resistance transfer into the human gut microbiota has not been demonstrated beyond individual case studies. To determine the "antibiotic resistance potential" for entire microbial communities, we employ metagenomic data and quantify the totality of known resistance genes in each community (its resistome) for 68 classes and subclasses of antibiotics. In 252 fecal metagenomes from three countries, we show that the most abundant resistance determinants are those for antibiotics also used in animals and for antibiotics that have been available longer. Resistance genes are also more abundant in samples from Spain, Italy, and France than from Denmark, the United States, or Japan. Where comparable country-level data on antibiotic use in both humans and animals are available, differences in these statistics match the observed resistance potential differences. The results are robust over time as the antibiotic resistance determinants of individuals persist in the human gut flora for at least a year.

Metagenomic species profiling using universal phylogenetic marker genes.

To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.

A human gut microbial gene catalogue established by metagenomic sequencing.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Genome-wide association study identifies host genetic variants influencing oral microbiota diversity and metabolic health.

The microbial communities of the oral cavity are important elements of oral and systemic health. With emerging evidence highlighting the heritability of oral bacterial microbiota, this study aimed to identify host genome variants that influence oral microbial traits. Using data from 16S rRNA gene amplicon sequencing, we performed genome-wide association studies with univariate and multivariate traits of the salivary microbiota from 610 unrelated adults from the Danish ADDITION-PRO cohort. We identified six single nucleotide polymorphisms (SNPs) in human genomes that showed associations with abundance of bacterial taxa at different taxonomical tiers (P < 5 × 10-8). Notably, SNP rs17793860 surpassed our study-wide significance threshold (P < 1.19 × 10-9). Additionally, rs4530093 was linked to bacterial beta diversity (P < 5 × 10-8). Out of these seven SNPs identified, six exerted effects on metabolic traits, including glycated hemoglobin A1c, triglyceride and high-density lipoprotein cholesterol levels, the risk of type 2 diabetes and stroke. Our findings highlight the impact of specific host SNPs on the composition and diversity of the oral bacterial community. Importantly, our results indicate an intricate interplay between host genetics, the oral microbiota, and metabolic health. We emphasize the need for integrative approaches considering genetic, microbial, and metabolic factors.

Consistent mutational paths predict eukaryotic thermostability.

BACKGROUND: Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. RESULTS: Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations. CONCLUSIONS: The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.