Elena S. Kostryukova

Human gut microbiota community structures in urban and rural populations in Russia

The microbial community of the human gut has a crucial role in sustaining host homeostasis. High-throughput DNA sequencing has delineated the structural and functional configurations of gut metagenomes in world populations. The microbiota of the Russian population is of particular interest to researchers, because Russia encompasses a uniquely wide range of environmental conditions and ethnogeographical cohorts. Here we conduct a shotgun metagenomic analysis of gut microbiota samples from 96 healthy Russian adult subjects, which reveals novel microbial community structures. The communities from several rural regions display similarities within each region and are dominated by the bacterial taxa associated with the healthy gut. Functional analysis shows that the metabolic pathways exhibiting differential abundance in the novel types are primarily associated with the trade-off between the Bacteroidetes and Firmicutes phyla. The specific signatures of the Russian gut microbiota are likely linked to the host diet, cultural habits and socioeconomic status.

Genome-Wide Mycobacterium tuberculosis Variation (GMTV) Database: A New Tool for Integrating Sequence Variations and Epidemiology

BackgroundTuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome.DescriptionHere we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes.ConclusionsImplementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Significance Biocompatible microfluidic double water-in-oil-in-water emulsion (MDE) enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. The MDE–FACS platform we developed enabled highly sensitive single-cell selection of predesigned activity and exploration of pairwise interactions between target and effector cells without interference from other microbiota species. Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE–FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

Gut microbiota and diet in patients with different glucose tolerance

Type 2 diabetes (T2D) is a serious disease. The gut microbiota (GM) has recently been identified as a new potential risk factor in addition to well-known diabetes risk factors. To investigate the GM composition in association with the dietary patterns in patients with different glucose tolerance, we analyzed 92 patients: with normal glucose tolerance (n=48), prediabetes (preD, n=24), and T2D (n=20). Metagenomic analysis was performed using 16S rRNA sequencing. The diet has been studied by a frequency method with a quantitative evaluation of food intake using a computer program. Microbiota in the samples was predominantly represented by Firmicutes, in a less degree by Bacteroidetes. Blautia was a dominant genus in all samples. The representation of Blautia, Serratia was lower in preD than in T2D patients, and even lower in those with normal glucose tolerance. After the clustering of the samples into groups according to the percentage of protein, fat, carbohydrates in the diet, the representation of the Bacteroides turned to be lower and Prevotella abundance turned to be higher in carbohydrate cluster. There were more patients with insulin resistance, T2D in the fat–protein cluster. Using the Calinski–Harabasz index identified the samples with more similar diets. It was discovered that half of the patients with a high-fat diet had normal tolerance, the others had T2D. The regression analysis showed that these T2D patients also had a higher representation of Blautia. Our study provides the further evidence concerning the structural modulation of the GM in the T2DM pathogenesis depending on the dietary patterns.

Genomic Sequencing and Biological Characteristics of a Novel Escherichia Coli Bacteriophage 9g, a Putative Representative of a New Siphoviridae Genus

Bacteriophage 9g was isolated from horse feces using Escherichia coli C600 as a host strain. Phage 9g has a slightly elongated capsid 62 × 76 nm in diameter and a non-contractile tail about 185 nm long. The complete genome sequence of this bacteriophage consists of 56,703 bp encoding 70 predicted open reading frames. The closest relative of phage 9g is phage PhiJL001 infecting marine alpha-proteobacterium associated with Ircinia strobilina sponge, sharing with phage 9g 51% of amino acid identity in the main capsid protein sequence. The DNA of 9g is resistant to most restriction endonucleases tested, indicating the presence of hypermodified bases. The gene cluster encoding a biosynthesis pathway similar to biosynthesis of the unusual nucleoside queuosine was detected in the phage 9g genome. The genomic map organization is somewhat similar to the typical temperate phage gene layout but no integrase gene was detected. Phage 9g efficiently forms stable associations with its host that continues to produce the phage over multiple passages, but the phage can be easily eliminated via viricide treatment indicating that no true lysogens are formed. Since the sequence, genomic organization and biological properties of bacteriophage 9g are clearly distinct from other known Enterobacteriaceae phages, we propose to consider it as the representative of a novel genus of the Siphoviridae family.

Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-D-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis gene locus

Acinetobacter baumannii 1053 is the type strain for the maintenance of specific bacteriophage AP22, which infects a fairly broad range of A. baumannii strains circulating in Russian clinics and hospitals. A capsular polysaccharide (CPS) was isolated from cells of strain 1053 and studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit was established: -->4)-β-D-ManpNAcA-(1-->4)-β-D-ManpNAcA-(1-->3)-α-D-FucpNAc-(1--> where ManNAcA and FucNAc indicate 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2,6-dideoxygalactose, respectively. A polysaccharide having the same repeating unit but a shorter chain was isolated by the phenol-water extraction of bacterial cells. Sequencing of the CPS biosynthesis gene locus showed that A. baumannii 1053 belongs to a new group designated KL91. The gene functions assigned putatively by a comparison with available databases were in agreement with the CPS structure established.

No Evidence of a Common DNA Variant Profile Specific to World Class Endurance Athletes

There are strong genetic components to cardiorespiratory fitness and its response to exercise training. It would be useful to understand the differences in the genomic profile of highly trained endurance athletes of world class caliber and sedentary controls. An international consortium (GAMES) was established in order to compare elite endurance athletes and ethnicity-matched controls in a case-control study design. Genome-wide association studies were undertaken on two cohorts of elite endurance athletes and controls (GENATHLETE and Japanese endurance runners), from which a panel of 45 promising markers was identified. These markers were tested for replication in seven additional cohorts of endurance athletes and controls: from Australia, Ethiopia, Japan, Kenya, Poland, Russia and Spain. The study is based on a total of 1520 endurance athletes (835 who took part in endurance events in World Championships and/or Olympic Games) and 2760 controls. We hypothesized that world-class athletes are likely to be characterized by an even higher concentration of endurance performance alleles and we performed separate analyses on this subsample. The meta-analysis of all available studies revealed one statistically significant marker (rs558129 at GALNTL6 locus, p = 0.0002), even after correcting for multiple testing. As shown by the low heterogeneity index (I2 = 0), all eight cohorts showed the same direction of association with rs558129, even though p-values varied across the individual studies. In summary, this study did not identify a panel of genomic variants common to these elite endurance athlete groups. Since GAMES was underpowered to identify alleles with small effect sizes, some of the suggestive leads identified should be explored in expanded comparisons of world-class endurance athletes and sedentary controls and in tightly controlled exercise training studies. Such studies have the potential to illuminate the biology not only of world class endurance performance but also of compromised cardiac functions and cardiometabolic diseases.

Specific pools of endogenous peptides are present in gametophore, protonema, and protoplast cells of the moss Physcomitrella patens.

BackgroundProtein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process.ResultsWe thoroughly analyzed native peptide pools of Physcomitrella patens moss in two developmental stages as well as in protoplasts. Peptidomic analysis was supplemented by transcriptional profiling and quantitative analysis of precursor proteins. In total, over 20,000 unique endogenous peptides, ranging in size from 5 to 78 amino acid residues, were identified. We showed that in both the protonema and protoplast states, plastid proteins served as the main source of peptides and that their major fraction formed outside of chloroplasts. However, in general, the composition of peptide pools was very different between these cell types. In gametophores, stress-related proteins, e.g., late embryogenesis abundant proteins, were among the most productive precursors. The Driselase-mediated protonema conversion to protoplasts led to a peptide generation “burst”, with a several-fold increase in the number of components in the latter. Degradation of plastid proteins in protoplasts was accompanied by suppression of photosynthetic activity.ConclusionWe suggest that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism. We assume that the peptide “burst” is a form of biotic stress response that might produce peptides with antimicrobial activity from originally functional proteins. Potential functions of peptides in different developmental stages are discussed.

Unusual Large-Scale Chromosomal Rearrangements in Mycobacterium tuberculosis Beijing B0/W148 Cluster Isolates

The Mycobacterium tuberculosis (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.