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Cell Host & Microbe Sep 2009The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes...
The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B(12)). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems.
Topics: Animals; Bacteroides; Gastrointestinal Tract; Germ-Free Life; Humans; Mice; Phylogeny; Symbiosis
PubMed: 19748469
DOI: 10.1016/j.chom.2009.08.003 -
Nature Protocols Aug 2021The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model...
The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.
Topics: Bacteroides thetaiotaomicron; Coculture Techniques; Colon; Epithelial Cells; Humans; Intestinal Mucosa; Organoids
PubMed: 34183870
DOI: 10.1038/s41596-021-00562-w -
MBio Oct 2022The role of the gut microbiota in the pathogenesis of Shigella flexneri remains largely unknown. To understand the impact of the gut microbiota on S. flexneri virulence,...
The role of the gut microbiota in the pathogenesis of Shigella flexneri remains largely unknown. To understand the impact of the gut microbiota on S. flexneri virulence, we examined the effect of interspecies interactions with Bacteroides thetaiotaomicron, a prominent member of the gut microbiota, on S. flexneri invasion. When grown in B. thetaiotaomicron-conditioned medium, S. flexneri showed reduced invasion of human epithelial cells. This decrease in invasiveness of S. flexneri resulted from a reduction in the level of the S. flexneri master virulence regulator VirF. Reduction of VirF corresponded with a decrease in expression of a secondary virulence regulator, , as well as expression of S. flexneri virulence genes required for invasion, intracellular motility, and spread. Repression of S. flexneri virulence factors by B. thetaiotaomicron-conditioned medium was not caused by either a secreted metabolite or secreted protein but rather was due to the presence of B. thetaiotaomicron outer membrane vesicles (OMVs) in the conditioned medium. The addition of purified B. thetaiotaomicron OMVs to S. flexneri growth medium recapitulated the inhibitory effects of B. thetaiotaomicron-conditioned medium on invasion, virulence gene expression, and virulence protein levels. Total lipids extracted from either B. thetaiotaomicron cells or B. thetaiotaomicron OMVs also recapitulated the effects of B. thetaiotaomicron-conditioned medium on expression of the S. flexneri virulence factor IpaC, indicating that B. thetaiotaomicron OMV lipids, rather than a cargo contained in the vesicles, are the active factor responsible for the inhibition of S. flexneri virulence. Shigella flexneri is the causative agent of bacillary dysentery in humans. spp. are one of the leading causes of diarrheal morbidity and mortality, especially among children in low- and middle-income countries. The rise of antimicrobial resistance combined with the lack of an effective vaccine for heightens the importance of studies aimed at better understanding previously uncharacterized aspects of pathogenesis. Here, we show that conditioned growth medium from the commensal bacterium Bacteroides thetaiotaomicron represses the invasion of S. flexneri. This repression is due to the presence of B. thetaiotaomicron outer membrane vesicles. These findings establish a role for interspecies interactions with a prominent member of the gut microbiota in modulating the virulence of S. flexneri and identify a novel function of outer membrane vesicles in interbacterial signaling between members of the gut microbiota and an enteric pathogen.
Topics: Child; Humans; Shigella flexneri; Virulence; Bacteroides thetaiotaomicron; Virulence Factors; Anti-Infective Agents; Lipids
PubMed: 36102517
DOI: 10.1128/mbio.02360-22 -
Cell Reports Aug 2023To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess the temporal dynamics of its fitness over the course of...
To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess the temporal dynamics of its fitness over the course of colonization. Here, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron (Bt), into the guts of germ-free mice to determine whether and how the genetic requirements for colonization shift over time. Combining a high-throughput functional genetics assay and transcriptomics, we find that gene usage changes drastically during the first days of colonization, shifting from high expression of amino acid biosynthesis genes to broad upregulation of diverse polysaccharide utilization loci. Within the first week, metabolism becomes centered around utilization of a predominant dietary oligosaccharide, and these changes are largely sustained through 6 weeks of colonization. Spontaneous mutations in wild-type Bt also evolve around this locus. These findings highlight the importance of considering temporal colonization dynamics in developing more effective microbiome-based therapies.
Topics: Humans; Animals; Mice; Bacteroides thetaiotaomicron; Acclimatization; Biological Assay; Gene Expression Profiling; Microbiota
PubMed: 37598339
DOI: 10.1016/j.celrep.2023.113009 -
American Journal of Physiology.... May 2024Fecal microbiota transplantation (FMT) is a promising therapy for inflammatory bowel disease (IBD) via rectifying gut microbiota. The aim of this study was to identify a...
Fecal microbiota transplantation (FMT) is a promising therapy for inflammatory bowel disease (IBD) via rectifying gut microbiota. The aim of this study was to identify a mechanism of how specific bacteria-associated immune response contributes to alleviated colitis. Forty donors were divided into high () and low () groups according to the diversity and the abundance of and by 16S rRNA sequencing. FMT was performed on dextran sulfate sodium (DSS)-induced colitis in mice. Mice with colitis showed significant improvement in intestinal injury and immune imbalance after FMT with ( < 0.05). and were identified as targeted strains in donor feces by real-time PCR and droplet digital PCR. Mice with colitis were treated with mono- or dual-bacterial gavage therapy. Dual-bacterial therapy significantly ameliorated intestinal injury compared with mono-bacterial therapy ( < 0.05). Dual-bacterial therapy increased the M2/M1 macrophage polarization and improved the Th17/Treg imbalance and elevated IL-10 production by Tregs compared with the DSS group ( < 0.05). Metabolomics showed increased abundance of lecithin in the glycerophospholipid metabolism pathway. In conclusion, , as the key bacteria in donor feces, alleviate colitis in mice. The mechanism may involve increasing lecithin and regulating IL-10 production of intestinal Tregs. We demonstrate that donors with high abundance of and ameliorate dextran sulfate sodium (DSS)-induced colitis in mice by fecal microbiota transplantation (FMT). The combination therapy of and is superior to mono-bacterial therapy in ameliorating colitis in mice, of which mechanism may involve promoting lecithin and inducing IL-10 production of intestinal Tregs.
Topics: Animals; Fecal Microbiota Transplantation; Colitis; Faecalibacterium prausnitzii; Mice; Gastrointestinal Microbiome; Male; Humans; Bacteroides thetaiotaomicron; Dextran Sulfate; Mice, Inbred C57BL; Interleukin-10; Adult; Female; Feces; Disease Models, Animal; Middle Aged
PubMed: 38502145
DOI: 10.1152/ajpgi.00303.2023 -
The ISME Journal Nov 2023Bacterial growth often alters the environment, which in turn can impact interspecies interactions among bacteria. Here, we used an in vitro batch system containing mucin...
Bacterial growth often alters the environment, which in turn can impact interspecies interactions among bacteria. Here, we used an in vitro batch system containing mucin beads to emulate the dynamic host environment and to study its impact on the interactions between two abundant and prevalent human gut bacteria, the primary fermenter Bacteroides thetaiotaomicron and the butyrate producer Roseburia intestinalis. By combining machine learning and flow cytometry, we found that the number of viable B. thetaiotaomicron cells decreases with glucose consumption due to acid production, while R. intestinalis survives post-glucose depletion by entering a slow growth mode. Both species attach to mucin beads, but only viable cell counts of B. thetaiotaomicron increase significantly. The number of viable co-culture cells varies significantly over time compared to those of monocultures. A combination of targeted metabolomics and RNA-seq showed that the slow growth mode of R. intestinalis represents a diauxic shift towards acetate and lactate consumption, whereas B. thetaiotaomicron survives glucose depletion and low pH by foraging on mucin sugars. In addition, most of the mucin monosaccharides we tested inhibited the growth of R. intestinalis but not B. thetaiotaomicron. We encoded these causal relationships in a kinetic model, which reproduced the observed dynamics. In summary, we explored how R. intestinalis and B. thetaiotaomicron respond to nutrient scarcity and how this affects their dynamics. We highlight the importance of understanding bacterial metabolic strategies to effectively modulate microbial dynamics in changing conditions.
Topics: Humans; Bacteroides thetaiotaomicron; Bacteroides; Mucins; Glucose
PubMed: 37670028
DOI: 10.1038/s41396-023-01501-1 -
Microorganisms Sep 2022A decrease in populations of and is observed during the development of colitis and fungal overgrowth, while restoration of these populations reduces inflammatory...
A decrease in populations of and is observed during the development of colitis and fungal overgrowth, while restoration of these populations reduces inflammatory parameters and fungal overgrowth in mice. This study investigated the effect of two fatty acids from and on macrophages and Caco-2 cells, as well as their impact on the inflammatory immune response and on overgrowth in a murine model of dextran sulfate sodium (DSS)-induced colitis. Oleic acid (OA) and palmitic acid (PA) from and were detected during their interaction with epithelial cells from colon samples. OA alone or OA combined with PA (FAs) reduced the expression of proinflammatory mediators in intestinal epithelial Caco-2 cells challenged with DSS. OA alone or FAs increased FFAR1, FFAR2, AMPK, and IL-10 expression in macrophages. Additionally, OA alone or FAs decreased COX-2, TNFα, IL-6, and IL-12 expression in LPS-stimulated macrophages. In the DSS murine model, oral administration of FAs reduced inflammatory parameters, decreased and populations, and eliminated from the gut. Overall, these findings provide evidence that OA combined with PA exhibits anti-inflammatory and antifungal properties.
PubMed: 36144406
DOI: 10.3390/microorganisms10091803 -
Gut Microbes 2024Gut microbiota plays an essential role in the progression of nonalcoholic fatty liver disease (NAFLD), making the gut-liver axis a potential therapeutic strategy....
Gut microbiota plays an essential role in the progression of nonalcoholic fatty liver disease (NAFLD), making the gut-liver axis a potential therapeutic strategy. Bacteroides genus, the enriched gut symbionts, has shown promise in treating fatty liver. However, further investigation is needed to identify specific beneficial Bacteroides strains for metabolic disorders in NAFLD and elucidate their underlying mechanisms. In this study, we observed a positive correlation between the abundance of () and the alleviation of metabolic syndrome in the early and end stages of NAFLD. Administration of to HFD-fed mice for 12 weeks reduced body weight and fat accumulation, decreased hyperlipidemia and insulin resistance, and prevented hepatic steatohepatitis and liver injury. Notably, did not affect these indicators in low-fat diet (LFD)-fed mice and exhibited good safety. Mechanistically, regulated gut microbial composition, characterized by a decreased Firmicutes/Bacteroidetes ratio in HFD-Fed mice. It also increased gut-liver folate levels and hepatic metabolites, alleviating metabolic dysfunction. Additionally, treatment with increased the proportion of polyunsaturated fatty acid in the mouse liver, offering a widely reported benefit for NAFLD improvement. In conclusion, this study provides evidence that ameliorates NAFLD by regulating gut microbial composition, enhancing gut-liver folate and unsaturated fatty acid metabolism, highlighting the therapeutic role of as a potential probiotic for NAFLD.
Topics: Mice; Animals; Non-alcoholic Fatty Liver Disease; Bacteroides thetaiotaomicron; Gastrointestinal Microbiome; Diet, High-Fat; Liver; Mice, Inbred C57BL
PubMed: 38277137
DOI: 10.1080/19490976.2024.2304159 -
Proceedings of the National Academy of... Feb 2022is a gut symbiont that inhabits the mucus layer and adheres to and metabolizes food particles, contributing to gut physiology and maturation. Although adhesion and...
is a gut symbiont that inhabits the mucus layer and adheres to and metabolizes food particles, contributing to gut physiology and maturation. Although adhesion and biofilm formation could be key features for stress resistance and gut colonization, little is known about the determinants of biofilm formation. We previously showed that the reference strain VPI-5482 is a poor in vitro biofilm former. Here, we demonstrated that bile, a gut-relevant environmental cue, triggers the formation of biofilm in many isolates and common gut Bacteroidales species. We determined that bile-dependent biofilm formation involves the production of the DNase BT3563 or its homologs, degrading extracellular DNA (eDNA) in several strains. Our study therefore shows that, although biofilm matrix eDNA provides a biofilm-promoting scaffold in many studied Firmicutes and Proteobacteria, BT3563-mediated eDNA degradation is required to form biofilm in the presence of bile.
Topics: Bacterial Proteins; Bacteroides thetaiotaomicron; Bile; Biofilms; DNA, Bacterial; Deoxyribonucleases; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic
PubMed: 35145026
DOI: 10.1073/pnas.2111228119 -
Frontiers in Immunology 2021Inhibition of allergic airway diseases (AAD) by immunomodulation of the adaptive immune system through restoration of the enteric dysbiosis is an emerging therapeutic...
Inhibition of allergic airway diseases (AAD) by immunomodulation of the adaptive immune system through restoration of the enteric dysbiosis is an emerging therapeutic strategy. Patients with allergic rhinitis (n = 6) and healthy controls (n = 6) were enrolled, and gut microbiome composition analysis was performed by 16S rDNA sequencing. We also established an ovalbumin (OVA)-induced allergic airway inflammation murine model. Dysbiosis of the gut flora was observed in both AAD patients and the mice, with the decrease of the biodiversity and the quantity of the Bacteroidetes phylum. Oral application of ameliorated the symptoms of OVA-induced airway hyperresponsiveness (AHR) and attenuated the airway inflammation in mice. In addition, nasal lavage fluid (NALF) and bronchoalveolar lavage fluid (BALF) from AAD mice orally administered with showed reduced numbers of immune cells, and diminished secretion of T helper (Th)-2 cytokines (IL-4, IL-5, and IL-13) compared with the corresponding control mice, whereas the levels of Th1 cytokineIFN-γ was not changed in both the groups. When was co-administered with metronidazole in AAD mice, the immunomodulatory effect was weakened and the allergic inflammatory response was aggravated. The ratios of CD4Foxp3 cells, CD4ICOS T cells, CD4ICOS Foxp3 regulatory T cells, and IL-10-expressing CD4Foxp3 cells were increased in lymphocytes of spleen, mesenteric, and cervical lymph nodes of AAD mice administrated with . Therefore, our data indicate that oral administration of effectively inhibited the development of AAD in murine model; inhibition was mediated by the activation of Tregs and inhibition of Th2 response without promoting a Th1 response.
Topics: Allergens; Animals; Bacteroides thetaiotaomicron; Cells, Cultured; Cytokines; Gastrointestinal Microbiome; Humans; Hypersensitivity; Immunomodulation; Inducible T-Cell Co-Stimulator Protein; Inflammation; Mice; Mice, Inbred BALB C; Models, Animal; Ovalbumin; Respiratory Hypersensitivity; Respiratory System; T-Lymphocytes, Regulatory; Th2 Cells
PubMed: 33815374
DOI: 10.3389/fimmu.2021.620943