An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity.

Plasticity in gene expression allows bacteria to adapt to diverse environments. This is particularly relevant in the dynamic niche of the human intestinal tract; however, transcriptional networks remain largely unknown for gut-resident bacteria. Here we apply differential RNA sequencing (RNA-seq) and conventional RNA-seq to the model gut bacterium Bacteroides thetaiotaomicron to map transcriptional units and profile their expression levels across 15 in vivo-relevant growth conditions. We infer stress- and carbon source-specific transcriptional regulons and expand the annotation of small RNAs (sRNAs). Integrating this expression atlas with published transposon mutant fitness data, we predict conditionally important sRNAs. These include MasB, which downregulates tetracycline tolerance. Using MS2 affinity purification and RNA-seq, we identify a putative MasB target and assess its role in the context of the MasB-associated phenotype. These data-publicly available through the Theta-Base web browser ( http://micromix.helmholtz-hiri.de/bacteroides/ )-constitute a valuable resource for the microbiome community.

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Bacteroides thetaiotaomicron metabolic activity decreases with polysaccharide molecular weight.

The human colon hosts hundreds of commensal bacterial species, many of which ferment complex dietary carbohydrates. To transform these fibers into metabolically accessible compounds, microbes often express a series of dedicated enzymes homologous to the starch utilization system (Sus) encoded in polysaccharide utilization loci (PULs). The genome of Bacteroides thetaiotaomicron (Bt), a common member of the human gut microbiota, encodes nearly 100 PULs, conferring a strong metabolic versatility. While the structures and functions of individual enzymes within the PULs have been investigated, little is known about how polysaccharide complexity impacts the function of Sus-like systems. We here show that the activity of Sus-like systems depends on polysaccharide size, ultimately impacting bacterial growth. We demonstrate the effect of size-dependent metabolism in the context of dextran metabolism driven by the specific utilization system PUL48. We find that as the molecular weight of dextran increases, Bt growth rate decreases and lag time increases. At the enzymatic level, the dextranase BT3087, a glycoside hydrolase (GH) belonging to the GH family 66, is the main GH for dextran utilization, and BT3087 and BT3088 contribute to Bt dextran metabolism in a size-dependent manner. Finally, we show that the polysaccharide size-dependent metabolism of Bt impacts its metabolic output in a way that modulates the composition of a producer-consumer community it forms with Bacteroides fragilis. Altogether, our results expose an overlooked aspect of Bt metabolism that can impact the composition and diversity of microbiota. IMPORTANCE: Polysaccharides are complex molecules that are commonly found in our diet. While humans lack the ability to degrade many polysaccharides, their intestinal microbiota contain bacterial commensals that are versatile polysaccharide utilizers. The gut commensal Bacteroides thetaiotaomicron dedicates roughly 20% of their genomes to the expression of polysaccharide utilization loci for the broad range utilization of polysaccharides. Although it is known that different polysaccharide utilization loci are dedicated to the degradation of specific polysaccharides with unique glycosidic linkages and monosaccharide compositions, it is often overlooked that specific polysaccharides may also exist in various molecular weights. These different physical attributes may impact their processability by starch utilization system-like systems, leading to differing growth rates and nutrient-sharing properties at the community level. Therefore, understanding how molecular weight impacts utilization by gut microbe may lead to the potential design of novel precision prebiotics.

Gut microbiota-based discovery of Houttuyniae Herba as a novel prebiotic of Bacteroides thetaiotaomicron with anti-colitis activity.

Ulcerative colitis (UC) represents an inflammatory disease characterized by fluctuations in severity, posing substantial challenges in treatment. The gut microbiota plays a pivotal role in the pathogenesis of UC. This study sought to identify drugs specifically targeting the gut microbiota to mitigate UC. We initiated a meta-analysis on gut microbiota in UC patients to identify UC-associated bacterial strains. Subsequently, we screened 164 dietary herbal medicines in vitro to identify potential prebiotics for the UC-associated bacterium, Bacteroides thetaiotaomicron. The DSS-induced colitis mouse model was utilized to evaluate the anti-colitis efficacy of the identified dietary herbal medicine. Full-length 16 S rRNA amplicon sequencing was employed to observe changes in gut microbiota following dietary herbal medicine intervention. The relative abundance of Bacteroides was notably diminished in UC patients compared to their healthy counterparts. B. thetaiotaomicron exhibited an inverse relationship with UC symptoms, indicating its potential as an anti-colitis agent. In vitro assessments revealed that H. Herba significantly bolstered the proliferation of B. thetaiotaomicron. Further experiments showed that treating DSS-induced mice with an aqueous extract of H. Herba considerably alleviated colitis indicators such as weight loss, colon shortening, disease activity score (DAI), and systemic inflammation. Microbial analysis revealed B. thetaiotaomicron as the sole bacterium substantially augmented by H. Herba in vivo. Overall H. Herba emerges as a promising prebiotic for B. thetaiotaomicron, offering significant anti-colitis benefits. Employing a gut microbiota-centric approach proves valuable in the quest for drug discovery.This study provides a new paradigm for drug discovery that targets the gut microbiota to treat UC.

Differential temporal release and lipoprotein loading in B. thetaiotaomicron bacterial extracellular vesicles.

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs.

Bacteroides thetaiotaomicron ameliorates mouse hepatic steatosis through regulating gut microbial composition, gut-liver folate and unsaturated fatty acids metabolism.

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 Bacteroides thetaiotaomicron (B. theta) and the alleviation of metabolic syndrome in the early and end stages of NAFLD. Administration of B. theta 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, B. theta did not affect these indicators in low-fat diet (LFD)-fed mice and exhibited good safety. Mechanistically, B. theta 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 B. theta 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 B. theta ameliorates NAFLD by regulating gut microbial composition, enhancing gut-liver folate and unsaturated fatty acid metabolism, highlighting the therapeutic role of B. theta as a potential probiotic for NAFLD.

Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Bacteroides Species.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron.

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.

peu-MIR2916-p3-enriched garlic exosomes ameliorate murine colitis by reshaping gut microbiota, especially by boosting the anti-colitic Bacteroides thetaiotaomicron.

Plant-derived exosome-like nanoparticles (ELNs) have drawn considerable attention for oral treatment of colonic diseases. However, the roles of ELNs derived from garlic on colitis remain unclear. Here, we demonstrate that garlic ELNs (GELNs), with desirable particle sizes (79.60 nm) and trafficking large amounts of functional proteins and microRNAs, stably roam in the gut and confer protection against ulcerative colitis (UC). In mice with DSS-induced colitis, orally administered GELNs effectively ameliorated bloody diarrhea, normalized the production of proinflammatory cytokines, and prevented colonic barrier impairment. Mechanistically, GELNs were taken up by gut microbes and reshaped DSS-induced gut microbiota dysbiosis, in which Bacteroides was the dominant respondent genus upon GELNs treatment. Notably, GELNs-enriched peu-MIR2916-p3 specifically promoted the growth of Bacteroides thetaiotaomicron, an intestinal symbiotic bacterium with palliative effects on colitis. Our findings provide new insights into the medicinal application of GELNs and highlight their potential as natural nanotherapeutic agents for preventing and treating UC.

Loss of Bacteroides thetaiotaomicron bile acid-altering enzymes impacts bacterial fitness and the global metabolic transcriptome.

IMPORTANCE: Recent work on bile salt hydrolases (BSHs) in Gram-negative bacteria, such as Bacteroides, has primarily focused on how they can impact host physiology. However, the benefits bile acid metabolism confers to the bacterium that performs it are not well understood. In this study, we set out to define if and how Bacteroides thetaiotaomicron (B. theta) uses its BSHs and hydroxysteroid dehydrogenase to modify bile acids to provide a fitness advantage for itself in vitro and in vivo. Genes encoding bile acid-altering enzymes were able to impact how B. theta responds to nutrient limitation in the presence of bile acids, specifically carbohydrate metabolism, affecting many polysaccharide utilization loci. This suggests that B. theta may be able to shift its metabolism, specifically its ability to target different complex glycans including host mucin, when it comes into contact with specific bile acids in the gut.

Bacteroides thetaiotaomicron and its inactivated bacteria ameliorate colitis by inhibiting macrophage activation.

BACKGROUND: Studies have demonstrated that Bacteroides thetaiotaomicron (BT) has protective effect against colon inflammation in murine models. Macrophages play an important role in gut immunity. However, the specific mechanisms of BT on macrophage are still unelucidated. Thus, our study investigates the anti-inflammatory effect of BT and its heat-treated inactivated bacteria on experimental colitis and macrophages. METHODS: A dextran sulfate sodium (DSS)-induced acute colitis model with male C57BL/6 mice, BT (ATCC29148) strain, THP1 cell lines were used in this study. Live and heat-treated inactivated BT (IBT) solution (1 × 10^9cfu/ml) were intragastrically gavaged daily for 14 days. Colonic inflammation was determined by the disease activity index (DAI) score, colon length, histological score, and inflammatory factors. THP1 cells were induced towards M1, then treated with different concentrations of inactivated BT solution and p38 inhibitor. Western blotting, immunohistochemistry, immunofluorescence and qRT-PCR were performed to assess the levels of inflammatory cytokines and molecules of MAPK pathway including IL-6, TNF-α, IL-1ß, IL-22, p38 and phosphor-p38 expressions. Moreover, 16S rRNA sequencing of colitis murine fecal samples was applied to investigate the influence of supplementation of BT to the gut microbiota homeostasis. RESULTS: Both live and heat-treated inactivated BT decreased the DAI and histological scores as well as levels of inflammatory factors, particularly IL-6 while increasing IL-22 of DSS-induced colitis murine models. The cell experiments showed that inactivated BT downregulates IL-6 expression in THP1 via inhibiting p38 phosphorylation and affecting M1 polarization. Moreover, the 16S rRNA sequencing results showed that BT and IBT gavage could increase beta-diversity of gut flora in DSS-induced colitis mice. Furthermore, the significance test for differences between the groups showed that BT could increase Faecalebaculum, Lactobacillus and Bacteroides, while decreasing Akkermansia. CONCLUSION: In summary, our findings imply that BT and its heat-treated inactivated bacteria exert a protective effect by suppressing macrophage-induced IL-6 through the inhibition of p38 MAPK pathway and ameliorating intestinal gut dysbiosis in experimental colitis.

A mathematical model of Bacteroides thetaiotaomicron, Methanobrevibacter smithii, and Eubacterium rectale interactions in the human gut.

The human gut microbiota is a complex ecosystem that affects a range of human physiology. In order to explore the dynamics of the human gut microbiota, we used a system of ordinary differential equations to model mathematically the biomass of three microorganism populations: Bacteroides thetaiotaomicron, Eubacterium rectale, and Methanobrevibacter smithii. Additionally, we modeled the concentrations of relevant nutrients necessary to sustain these populations over time. Our model highlights the interactions and the competition among these three species. These three microorganisms were specifically chosen due to the system's end product, butyrate, which is a short chain fatty acid that aids in developing and maintaining the intestinal barrier in the human gut. The basis of our mathematical model assumes the gut is structured such that bacteria and nutrients exit the gut at a rate proportional to its volume, the rate of volumetric flow, and the biomass or concentration of the particular population or nutrient. We performed global sensitivity analyses using Sobol' sensitivities to estimate the relative importance of model parameters on simulation results.

Multiple TonB homologs are important for carbohydrate utilization by Bacteroides thetaiotaomicron.

IMPORTANCE: The human gut microbiota, including Bacteroides, is required for the degradation of otherwise undigestible polysaccharides. The gut microbiota uses polysaccharides as an energy source, and fermentation products such as short-chain fatty acids are beneficial to the human host. This use of polysaccharides is dependent on the proper pairing of a TonB protein with polysaccharide-specific TonB-dependent transporters; however, the formation of these protein complexes is poorly understood. In this study, we examine the role of 11 predicted TonB homologs in polysaccharide uptake. We show that two proteins, TonB4 and TonB6, may be functionally redundant. This may allow for the development of drugs targeting Bacteroides species containing only a TonB4 homolog with limited impact on species encoding the redundant TonB6.

Inducible CRISPR-targeted "knockdown" of human gut Bacteroides in gnotobiotic mice discloses glycan utilization strategies.

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic Bacteroidaceae species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these "alternate consumers" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in Phocaeicola vulgatus, a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.

Starvation responses impact interaction dynamics of human gut bacteria Bacteroides thetaiotaomicron and Roseburia intestinalis.

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.

Dynamic genetic adaptation of Bacteroides thetaiotaomicron during murine gut colonization.

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.

The NQR Complex Regulates the Immunomodulatory Function of Bacteroides thetaiotaomicron.

The gut microbiome and intestinal immune system are engaged in a dynamic interplay that provides myriad benefits to host health. However, the microbiome can also elicit damaging inflammatory responses, and thus establishing harmonious immune-microbiome interactions is essential to maintain homeostasis. Gut microbes actively coordinate the induction of anti-inflammatory responses that establish these mutualistic interactions. Despite this, the microbial pathways that govern this dialogue remain poorly understood. We investigated the mechanisms through which the gut symbiont Bacteroides thetaiotaomicron exerts its immunomodulatory functions on murine- and human-derived cells. Our data reveal that B. thetaiotaomicron stimulates production of the cytokine IL-10 via secreted factors that are packaged into outer membrane vesicles, in a TLR2- and MyD88-dependent manner. Using a transposon mutagenesis-based screen, we identified a key role for the B. thetaiotaomicron-encoded NADH:ubiquinone oxidoreductase (NQR) complex, which regenerates NAD+ during respiration, in this process. Finally, we found that disruption of NQR reduces the capacity of B. thetaiotaomicron to induce IL-10 by impairing biogenesis of outer membrane vesicles. These data identify a microbial pathway with a previously unappreciated role in gut microbe-mediated immunomodulation that may be targeted to manipulate the capacity of the microbiome to shape host immunity.

A pectic polysaccharide isolated from Achyranthes bidentata is metabolized by human gut Bacteroides spp.

Achyranthes bidentata (A. bidentata) is a famous traditional Chinese medicine (TGM) for treatment osteoporosis. Polysaccharides, a major factor for shaping the gut microbiota, are the primary ingredients of A. bidentata. However, bioactivity of A. bidentata polysaccharide on human gut microbiota (HGM) remains unknown. Here, a homogeneous pectic polysaccharide A23-1 with average molecular weight of 93.085 kDa was extracted and purified from A. bidentata. And A23-1 was compsed of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 7.26: 0.76: 5.12: 2.54: 23.51: 60.81. GC-MS, partial acid hydrolysis and NMR results indicated the backbone of A23-1 was composed of 1, 2, 4-Rhap and 1, 4-GlapA, while the branches were composed of galactose, arabinose, glucose and glucuronic acid. Further, A23-1 was found to be degraded into monosaccharides and fragments. Taking Bacteroides thetaiotaomicron (BT) as a model, we suggested three polysaccharide utilization loci (PULs) might be involved in the A23-1 degradation. Degraded products generated by BO might not support the growth of probiotics. Besides, acetate and propionate as the main end products were generated by Bacteroides spp. and probiotics utilizing A23-1. These findings suggested A23-1 was possible one of food sources of human gut Bacteroides spp.

Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes.

Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.