Biology subjects

Ghimire, S.

Publications and source records attributed to Ghimire, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A predominately pulmonary activation of complement in a mouse model of severe COVID-19

Evidence from in vitro studies and observational human disease data suggest the complement system plays a significant role in SARS-CoV-2 pathogenesis, although how complement dysregulation develops in patients with severe COVID-19 is unknown. Here, using a mouse-adapted SARS-CoV-2 virus (SARS2-N501YMA30) and a mouse model of severe COVID-19, we identify significant serologic and pulmonary complement activation following infection. We observed C3 activation in airway and alveolar epithelia, and in pulmonary vascular endothelia. Our evidence suggests that while the alternative pathway is the primary route of complement activation, components of both the alternative and classical pathways are produced locally by respiratory epithelial cells following infection, and increased in primary cultures of human airway epithelia in response to cytokine exposure. This locally generated complement response appears to precede and subsequently drive lung injury and inflammation. Results from this mouse model recapitulate findings in humans, which suggest sex-specific variance in complement activation, with predilection for increased C3 activity in males, a finding that may correlate with more severe disease. Our findings indicate that complement activation is a defining feature of severe COVID-19 in mice and lay the foundation for further investigation into the role of complement in COVID-19.

immunology↗

bioRxiv2024

A Caenorhabditis elegans based system for high-throughput functional phenotyping of human gut microbiota

The conventional bottom-up approach to probing the human gut microbiomes link with hosts in germ-free models is hampered by considerable costs and time. To address this, our study introduces the nematode Caenorhabditis elegans as an innovative high-throughput model for exploring the gut microbiomes impact on functional phenotypes. Traditionally, C. elegans studies have used continuous feeding for bacterial administration, a method that is unsuitable for anaerobes. For the first time, we have standardized a protocol for colonizing C. elegans with human gut anaerobes. By screening a microbial culturomics library representing 70% of the gut microbiomes functional capacity, we showed successful colonization for 46% of the library. Functional phenotyping revealed that 5 of 10 strains, previously identified in vitro as inhibiting C. difficile, also inhibited in vivo. Validation of a selected strain in a germ-free mouse model confirmed colonization resistance and an immune response consistent with findings in C. elegans, underscoring the models translational potential.

microbiology↗

bioRxiv2023

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is primarily expressed in the vasculature in vivo and that its depletion leads to vascular abnormalities in vivo and loss of barrier integrity in cultured endothelial cells. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct, high-affinity interaction with CCM2. Modeling and functional testing of TLNRD1 and CCM2 mutants reveal that their interaction is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the 4-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Notably, a CCM2 pathogenic mutation linked to vascular dementia in patients maps to the interface and disrupts the interaction. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity by controlling TLNRD1 bundling activity.

cell biology↗

bioRxiv2023

Enhancing Recovery from Gut Microbiome Dysbiosis and Alleviating DSS-Induced Colitis in Mice with a Consortium of Rare Short-Chain Fatty Acid-Producing Bacteria

The human gut microbiota is a complex community comprising hundreds of species, with a few present in high abundance and the vast majority in low abundance. The biological functions and effects of these low-abundant species on their hosts are not yet fully understood. In this study, we assembled a bacterial consortium (SC-4) consisting of B. paravirosa, C. comes, M. indica, and A. butyriciproducens, which are low-abundant, short-chain fatty acid (SCFA)-producing bacteria isolated from healthy human gut, and tested its effect on host health using germfree and human microbiota-associated colitis mouse models. The selection also favored these four bacteria being reduced in abundance in either Ulcerative Colitis (UC) or Crohns disease (CD) metagenome samples. Our findings demonstrate that SC-4 can colonize germ-free (GF) mice, increasing mucin thickness by activating MUC-1 and MUC-2 genes, thereby protecting GF mice from Dextran Sodium Sulfate (DSS)-induced colitis. Moreover, SC-4 aided in the recovery of human microbiota-associated mice from DSS-induced colitis, and intriguingly, its administration enhanced the alpha diversity of the gut microbiome, shifting the community composition closer to control levels. The results showed enhanced phenotypes across all measures when the mice were supplemented with Inulin as a dietary fiber source alongside SC-4 administration. We also showed a functional redundancy existing in the gut microbiome, resulting in the low abundant SCFA producers acting as a form of insurance, which in turn accelerates recovery from the dysbiotic state upon the administration of SC-4. SC-4 colonization also upregulated iNOS gene expression, further supporting its ability to produce an increasing number of goblet cells. Collectively, our results provide evidence that low-abundant SCFA-producing species in the gut may offer a novel therapeutic approach to IBD.

microbiology↗

bioRxiv2023

Synaptogyrin regulates neuronal activity dependent autophagy to degrade synaptic vesicle components and pathological Tau

Synapses are specialized neuronal compartments essential for brain communication. Neuronal communication mostly relies on the adequate supply and renovation of synaptic vesicles that fuse with the plasma membrane and release neurotransmitters in response to action potentials. Autophagy is an evolutionary conserved cellular mechanism essential for homeostasis that can be locally regulated in the neuronal synapse. However, the precise mechanisms controlling synaptic autophagy, especially during neuronal communication and pathological scenarios, remain elusive. Here, we report that neuronal activity and amino-acid deprivation regulate synaptic autophagy via distinct molecular mechanisms. We show that Synaptogyrin, a highly abundant presynaptic protein found in synaptic vesicles, is a novel negative regulator of synaptic autophagy in response to neuronal activity without affecting autophagy induction via amino-acid deprivation. We demonstrate that loss of Synaptogyrin modifies the localization of the autophagy protein Atg9 and boosts autophagosome formation at the synapse. Furthermore, activation of synaptic autophagy by loss of Synaptogyrin, but not by amino-acid deprivation, leads to the degradation of synaptic vesicle components via autophagy. Reducing the levels of Synaptogyrin results in the degradation of synaptic Tau via autophagy and restores autophagy dysfunction observed in a Drosophila Tau model of Frontotemporal Dementia (FTD). Our data provide novel and valuable information to understand how autophagy is regulated at the synapse in response to neuronal activity and how this process participates in neuronal (dys)function.

neuroscience↗

bioRxiv2023

The appearance of sugI mixed loci in three individuals during treatment for MDR-TB, supports the involvement of sugI in Mycobacterium tuberculosis d-cycloserine resistance in vivo.

To study the adaptation of multi-drug resistant Mycobacterium tuberculosis (MDR-TB) during treatment patients diagnosed with MDR-TB were recruited into an observational study. Clinical data and M. tuberculosis DNA at diagnosis and between seven days and two months of MDR-TB treatment were collected. The drugs prescribed were recorded. Interpretable WGS data from 118 isolates from 54 participants was obtained (11 in Belarus and 43 in Moldova) and screened for the presence of unfixed single nucleotide polymorphisms (mixed SNPs / loci). This study was performed shortly after the publication of the 2019 WHO consolidated guidelines on drug-resistant tuberculosis treatment. Existing drug supplies and procurement in one country after the switch to the all oral MDR-TB regimen in addition to patient factors, influenced the selection of and exposure to drugs. Confidently mixed SNPs were identified in samples from multiple participants in only five genes (gyrA, pncA, Rv1129c, Rv1148c, and sugI). All other genes with confidently mixed SNPs were identified in isolates from only a single individual. A significant proportion of the participants (52 of 54 participants) received d-cycloserine as part of their initial treatment, most participants who initially received d-cycloserine did not receive bedaquiline in their initial regimen (all at one site). Three different mixed SNPs were identified in sugI gene from a follow up isolate from three participants (P7A, P7T, and Q6stop). Mutations in sugI have previously been reported in spontaneous in vitro d-cycloserine resistant mutants. Alterations in the sugI gene may indicate a sub optimal d-cycloserine containing regimen and potentially be of clinical significance with respect to adaptation to d-cycloserine. Monitoring the accumulation of low frequency escape mutants may help identify regimens insufficiently powerful to block the accumulation of antimicrobial resistance mutants and identify drug(s) at risk of resistance selection.

microbiology↗

bioRxiv2022

Repurposing of Drugs against mutated strain of Eurasian Avian like H1N1 (EA H1N1) Swine flu, Genotype-4 (G4) virus

Mutation, reassortment and recombination have led to the evolution and the emergence of more pathogenic and new subtypes of influenza virus. The surge of highly mutated viruses has prompted the need of coherent solution for the so called "medical holocaust" viral outbreaks. The genotype 4 of EAH1N1 strain has been circulating in the swine population as a dominant genotype, exhibiting even human to human transmission. This has risen the possibility of causing another global health threat as a lethal viral outbreak in the future. The Computer Aided Drug Discovery (CADD) could be a prudent mechanism to develop new drug candidates against such disease for its mitigation. In this regard, the computational in silico methods had been envisaged in this research for the prediction of lead compounds against the selected proteins of EA H1N1 G4 strain, namely Haemagglutinin (HA) and Polymerase acidic protein(PA). The research focused on the selection of the target viral protein and molecular docking for the identification of putative ligands. It was followed by the identification of the probable mutations and assessment of effectiveness of identified drugs against their respective targets. Total of 3 compounds Enalapril, Enalaprilat and Ivabradine have been identified as a potential inhibitor of HA and PA protein that were prioritized on the basis of preference index parameter and binding energy of compound with the respective target. Besides, the probable mutations in each target protein in future were predicted and all these 3 top hits were found to be effective against mutated variant of these proteins. Thus, Enalapril, Enalaprilat and Ivabradine could be the lead compounds to explore further as multi target inhibiting drugs against wild and mutant variant of target proteins.

bioinformatics↗

bioRxiv2022

Fast4DReg: Fast registration of 4D microscopy datasets

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal quantification of biological processes. While multiple methods and tools exist to correct images post-acquisition, performing drift correction of large 3D videos using open-source solutions remains challenging and time-consuming. Here we present a new tool developed for ImageJ/Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using 2D cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg performs better than other state-of-the-art open-source drift correction tools and significantly outperforms them in speed (5x to 60x). We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis. Fast4DReg is available on GitHub.

bioinformatics↗

bioRxiv2022

Myosin-X recruits lamellipodin to filopodia tips

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. Yet, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped RAPH1 interaction with adhesome components to its talinbinding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises an area with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not involved in regulating integrin activity in filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

cell biology↗

bioRxiv2022

Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model.

A complex microbial community in the gut generally prevent the colonization of enteric pathogens such as Salmonella. Because of the high complexity, several species or combination of species in the gut can confer colonization resistance. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica Typhimurium. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium reduced Salmonella colonization, intestinal tissue damage and inflammation in both models. Cell free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition or reinforcement of host immunity. Out of ten species, three species in Mix10 did not colonize while three species constituted more than 70% of the community. Two of these species represents previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host. ImportanceSalmonella colonization in chicken and human infections originating from Salmonella- contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

microbiology↗

bioRxiv2022

IL-17A controls CNS autoimmunity by regulating gut microbiota and inducing regulatory T cells

A disrupted equilibrium among gut microbiota, IL-17A-producing CD4 T-cells (Th17), and regulatory CD4 T-cells (Treg) have been linked with the pathobiology of multiple sclerosis (MS). While gut microbiota can regulate both Treg and Th17 cells, the impact of IL-17A on this gut-immune connection remains unclear. Utilizing HLA-DR3 transgenic mouse model of MS, we show that IL-17A deficiency (HLA-DR3.IL17A-/-) resulted in milder disease characterized by increased Tregs and expansion of Treg-promoting gut microbes, including Prevotella. Cohousing HLA-DR3 mice with HLA-DR3.IL17A-/- transferred the milder disease phenotype and associated microbiota changes to DR3 mice, highlighting the dominant role of gut microbiota in Treg induction and disease amelioration. DR3.IL17A-/- mice also showed a higher abundance of functional pathways linked with short-chain fatty acid synthesis and elevated IL-10 in dendritic cells. Enrichment of the Treg-promoting PPAR signaling pathway expression in the colon of HLA-DR3.IL17A-/- mice and following Prevotella administration in HLA-DR3 mice underscores the importance of gut microbiota in IL-17A-mediated immune regulation. Thus, our study uncovers a previously unappreciated role for IL-17A in shaping gut microbiota and immune regulation, with far-reaching implications for MS treatment. One-Sentence SummaryIL-17A modulates Treg and gut microbiota to control EAE

immunology↗

bioRxiv2021

A model screening pipeline for bile acid converting anti-Clostridioides difficile bacteria reveals unique biotherapeutic potential of Peptacetobacter hiranonis

Clostridioides difficile is an antibiotic-resistant bacterium that causes serious, toxin-mediated enteric disease in humans and animals. Gut dysbiosis and resultant alterations in the intestinal bile acid profile play an important role in the pathogenesis of C. difficile infection (CDI). Restoration of the gut microbiota and re-establishment of bacterial bile acid metabolism using fecal microbiota transplantation (FMT) has been established as a promising strategy against this disease, although this method has several limitations. Thus, a more defined and precise microbiota-based approach using bacteria that biotransform primary bile acids into secondary bile acids could effectively overcome these limitations and control CDI. Therefore, a screening pipeline was developed to isolate bile acid converting bacteria from fecal samples. Dogs were selected as a model CDI-resistant microbiota donor for this pipeline, which yielded a novel Peptacetobacter hiranonis strain that possesses unique anti-C. difficile properties, and both bile acid deconjugation and 7- dehydroxylating activities to perform bile acid conversion. The screening pipeline included a set of in vitro tests along with a precision in vivo gut colonization and bile acid conversion test using altered Schadler flora (ASF) colonized mice. In addition, this pipeline also provided essential information on the growth requirements for screening and cultivating the candidate bacterium, its survival in a CDI predisposing environment, and potential pathogenicity. The model pipeline documented here yielded multiple bile acid converting bacteria, including a P. hiranonis isolate with unique anti-C. difficile biotherapeutic potential, which can be further tested in subsequent preclinical and human clinical trials.

microbiology↗

bioRxiv2021

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

ObjectiveMice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin secreting beta cells, causing diabetes. In human, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. MethodsWe generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. In addition, we searched whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. ResultsCUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1268 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements frequently overlapping within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA, RFX or PBX transcription factors. Moreover, we found that 22% of the genes downregulated in NEUROG3-/- hESC-derived PEPs are bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 targets include transcription factors known to have important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself. Remarkably, we uncovered that NEUROG3 binds transcriptional regulator genes with enriched expression in human fetal pancreatic alpha (e.g., IRX1, IRX2), beta (e.g., NKX6-1, SMAD9, ISX, TFCP2L1) and delta cells (ERBB4) suggesting that NEUROG3 could control islets subtype programs. Moreover, NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8 and PCSK1). In addition, we unveiled a panel of ncRNA potentially regulated by NEUROG3. Lastly, we identified several T2DM risk SNPs within NEUROG3 peaks suggesting a possible developmental role of NEUROG3 in T2DM susceptibility. ConclusionMapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation. HighlightsO_LINEUROG3 CUT&RUN analysis revealed 1268 target genes in human pancreatic endocrine progenitors (PEPs) C_LIO_LINEUROG3 binding sites overlap with active chromatin regions in PEPs. C_LIO_LI1/5 of the genes downregulated in NEUROG3-/- hESC-derived PEPs are bound by NEUROG3. C_LIO_LINEUROG3 targets islet specific TFs and regulators of insulin secretion. C_LIO_LISeveral T2DM risk allelles lie within NEUROG3 bound regions. C_LI

genomics↗

bioRxiv2021

Performance of a scalable extraction-free RNA-seq method

RNA sequencing enables high-contents/high-complexity measurements in small molecule screens performed on biological samples. Whereas the costs of DNA sequencing and the complexity of RNA-seq library preparation and analysis have decreased consistently, RNA extraction remains a significant bottleneck for RNA-seq of hundreds of samples in parallel. Direct use of cell lysate for RNA-seq library prep is common in single cell RNA-seq but not in bulk RNA-seq protocols. Recently published protocols suggest that direct lysis is compatible with simplified RNA-seq library prep. Here, we evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combine a low-cost direct lysis buffer compatible with cDNA synthesis ("in-lysate cDNA synthesis") with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show that whereas variable RNA inputs for each sample in the in-lysate cDNA synthesis protocol result in variable sequencing depth, this had minimal effect on data quality, measurement of gene expression patterns, or generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep allows generation of high-quality data when compared to library prep with extracted RNA, or when compared to Illumina TruSeq. Our data show that small molecule screens using RNA-seq are feasible at low reagent and time costs.

genomics↗

bioRxiv2020

A Critical Role of Hepatic GABA in The Metabolic Dysfunction and Hyperphagia of Obesity

Hepatic lipid accumulation is a hallmark of type II diabetes (T2D) and associated with hyperinsulinemia, insulin resistance, and hyperphagia. Hepatic synthesis of GABA, catalyzed by GABA-transaminase (GABA-T), is upregulated in obese mice. To assess the role of hepatic GABA production in obesity-induced metabolic and energy dysregulation, we treated mice with two pharmacologic GABA-T inhibitors and knocked down hepatic GABA-T expression using an antisense oligonucleotide. Hepatic GABA-T inhibition and knockdown decreased basal hyperinsulinemia and hyperglycemia, and improved glucose intolerance. GABA-T knockdown improved insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps in obese mice. Hepatic GABA-T knockdown also decreased food intake and induced weight loss without altering energy expenditure in obese mice. Data from people with obesity support the notion that hepatic GABA production and transport are associated with serum insulin, HOMA-IR, T2D, and BMI. These results support a key role for hepatocyte GABA production in the dysfunctional glucoregulation and feeding behavior associated with obesity.

physiology↗

bioRxiv2020

Rice Bran and Quercetin Produce a Positive Synergistic Effect on Human Gut Microbiota, Elevate the Level of Propionate, and Reduce the Population of Enterobacteriaceae family when Determined using a Bioreactor Model.

Diet is one of the prominent determinants of gut microbiota composition significantly impacting human health. Recent studies with dietary supplements such as rice bran and quercetin have been shown to provide a beneficial impact on the host by positively influencing the gut microbiota. However, the specific bacterial species impacted when rice bran or quercetin is present in the diet is not well understood. Therefore, in this study, we used a minibioreactor array system as a model to determine the effect of quercetin and rice bran individually, as well as in combination, on gut microbiota without the confounding host factors. We found that rice bran exerts higher shift in gut microbiome composition when compared to quercetin. At the species level, Acidaminococcus intestini was the only significantly enriched taxa when quercetin was supplemented, while 15 species were enriched in rice bran supplementation and 13 were enriched when quercetin and rice bran were supplemented in combination. When comparing the short chain fatty acid production, quercetin supplementation significantly enriched isobutyrate production while propionate dominated the quercetin and rice bran combined group. Higher levels of propionate were highly correlated to the lower abundance of the potentially pathogenic Enterobacteriaceae family. These findings suggest that the combination of rice bran and quercetin serve to enrich beneficial bacteria and reduce potential opportunistic pathogens. However, further in vivo studies are necessary to determine the synergistic effect of rice bran and quercetin on host health and immunity. ImportanceRice bran and quercetin are dietary components that shape host health by interacting with the gut microbiome. Both these substrates have been reported to provide nutritional and immunological benefits individually. However, considering the complexity of the human diet, it is useful to determine how the combination of food ingredients such as rice bran and quercetin influences the human gut microbiota. Our study provides insights into how these ingredients influence microbiome composition alone and in combination in vitro. This will allow us to identify which species in the gut microbiome are responsible for biotransformation of these dietary ingredients.. Such information is helpful for the development of synbiotics to improve gut health and immunity.

microbiology↗

bioRxiv2019

Description of Ruminococcus catenae SW178 sp. nov., a new anaerobic species isolated from feral chicken

A Gram-positive, obligately anaerobic coccobacillus, with the white raised circular colony was isolated from the cecum of feral chickens in Brookings, South Dakota, USA. The 16S rRNA gene sequence analysis suggested that the closest species to strain SW178 was Ruminococcus torques ATCC 27756T (96.94% similarity) that belongs to the family Lachnospiraceae. The genome of strain SW178 is 3.18 Mbp with G+C content of 46.9 mol%. Based on the phylogenetic and phenotypic comparison, we propose that strain SW178 be assigned to the genus Ruminococcus as a novel species, for which the name Ruminococcus catenae is proposed. The type strain is SW178 (= CCOS 1886 T, =DSM 109242T).

microbiology↗

bioRxiv2019

Identifying Clostridioides difficile-inhibiting gut commensals using culturomics, phenotyping, and combinatorial community assembly

A major function of the gut microbiota is to provide colonization resistance, wherein pathogens are inhibited or suppressed below infectious level. However, the fraction of gut microbiota required for colonization resistance remains unclear. We used culturomics to isolate a gut microbiota culture collection comprising 1590 isolates belonging to 102 species. Estimated by metagenomic sequencing of fecal samples used for culture, this culture collection represents 50.73% of taxonomic diversity and 70% functional capacity. Using whole genome sequencing we characterized species representatives from this collection, and predicted their phenotypic traits, further characterizing isolates by defining nutrient utilization profile and short chain fatty acid (SCFA) production. When screened using a co-culture assay, 66 species in our culture collection inhibited C. difficile. Several phenotypes, particularly, growth rate, production of SCFAs, and the utilization of mannitol, sorbitol or succinate correlated with C. difficile inhibition. We used a combinatorial community assembly approach to formulate defined bacterial mixes inhibitory to C. difficile. When 256 combinations were tested, we found both species composition and blend size to be important in inhibition. Our results show that the interaction of bacteria with each other in a mix and with other members of gut commensals must be investigated for designing defined bacterial mixes for inhibiting C. difficile in vivo.\n\nIMPORTANCEAntibiotic treatment causes instability of gut microbiota and the loss of colonization resistance, allowing pathogens such as C. difficile to colonize, causing recurrent infection and mortality. Although fecal microbiome transplantation has shown to be an effective treatment for C. difficile infection (CDI), a more desirable approach would be the use of a defined mix of inhibitory gut bacteria. C. difficile-inhibiting species and bacterial combinations we identify herein improve our understanding of the ecological interactions controlling colonization resistance against C. difficile, and could aid the design of defined bacteriotherapy as a non-antibiotic alternative against CDI.

microbiology↗