Intratumoral microbiome of adenoid cystic carcinomas and comparison with other head and neck cancers.

Adenoid cystic carcinoma (ACC) is a rare, usually slow-growing yet aggressive head and neck malignancy. Despite its clinical significance, our understanding of the cellular evolution and microenvironment in ACC remains limited. We investigated the intratumoral microbiomes of 50 ACC tumor tissues and 33 adjacent normal tissues using 16S rRNA gene sequencing. This allowed us to characterize the bacterial communities within the ACC and explore potential associations between the bacterial community structure, patient clinical characteristics, and tumor molecular features obtained through RNA sequencing. The bacterial composition in the ACC was significantly different from that in adjacent normal salivary tissue, and the ACC exhibited diverse levels of species richness. We identified two main microbial subtypes within the ACC: oral-like and gut-like. Oral-like microbiomes, characterized by increased diversity and abundance of Neisseria, Leptotrichia, Actinomyces, Streptococcus, Rothia, and Veillonella (commonly found in healthy oral cavities), were associated with a less aggressive ACC-II molecular subtype and improved patient outcomes. Notably, we identified the same oral genera in oral cancer and head and neck squamous cell carcinomas. In both cancers, they were part of shared oral communities associated with a more diverse microbiome, less aggressive tumor phenotype, and better survival that reveal the genera as potential pancancer biomarkers for favorable microbiomes in ACC and other head and neck cancers. Conversely, gut-like intratumoral microbiomes, which feature low diversity and colonization by gut mucus layer-degrading species, such as Bacteroides, Akkermansia, Blautia, Bifidobacterium, and Enterococcus, were associated with poorer outcomes. Elevated levels of Bacteroides thetaiotaomicron were independently associated with significantly worse survival and positively correlated with tumor cell biosynthesis of glycan-based cell membrane components.

Beneficial metabolic effects of PAHSAs depend on the gut microbiota in diet-induced obese mice but not in chow-fed mice.

Dietary lipids play an essential role in regulating the function of the gut microbiota and gastrointestinal tract, and these luminal interactions contribute to mediating host metabolism. Palmitic Acid Hydroxy Stearic Acids (PAHSAs) are a family of lipids with antidiabetic and anti-inflammatory properties, but whether the gut microbiota contributes to their beneficial effects on host metabolism is unknown. Here, we report that treating chow-fed female and male germ-free (GF) mice with PAHSAs improves glucose tolerance, but these effects are lost upon high fat diet (HFD) feeding. However, transfer of feces from PAHSA-treated, but not vehicle-treated, chow-fed conventional mice increases insulin sensitivity in HFD-fed GF mice. Thus, the gut microbiota is necessary for, and can transmit, the insulin-sensitizing effects of PAHSAs in HFD-fed GF male mice. Analyses of the cecal metagenome and lipidome of PAHSA-treated mice identified multiple lipid species that associate with the gut commensal Bacteroides thetaiotaomicron (Bt) and with insulin sensitivity resulting from PAHSA treatment. Supplementing live, and to some degree, heat-killed Bt to HFD-fed female mice prevented weight gain, reduced adiposity, improved glucose tolerance, fortified the colonic mucus barrier and reduced systemic inflammation compared to HFD-fed controls. These effects were not observed in HFD-fed male mice. Furthermore, ovariectomy partially reversed the beneficial Bt effects on host metabolism, indicating a role for sex hormones in mediating the Bt probiotic effects. Altogether, these studies highlight the fact that PAHSAs can modulate the gut microbiota and that the microbiota is necessary for the beneficial metabolic effects of PAHSAs in HFD-fed mice.

Exploring potential polysaccharide utilization loci involved in the degradation of typical marine seaweed polysaccharides by Bacteroides thetaiotaomicron.

Introduction: Research on the mechanism of marine polysaccharide utilization by Bacteroides thetaiotaomicron has drawn substantial attention in recent years. Derived from marine algae, the marine algae polysaccharides could serve as prebiotics to facilitate intestinal microecological balance and alleviate colonic diseases. Bacteroides thetaiotaomicron, considered the most efficient degrader of polysaccharides, relates to its capacity to degrade an extensive spectrum of complex polysaccharides. Polysaccharide utilization loci (PULs), a specialized organization of a collection of genes-encoded enzymes engaged in the breakdown and utilization of polysaccharides, make it possible for Bacteroides thetaiotaomicron to metabolize various polysaccharides. However, there is still a paucity of comprehensive studies on the procedure of polysaccharide degradation by Bacteroides thetaiotaomicron. Methods: In the current study, the degradation of four kinds of marine algae polysaccharides, including sodium alginate, fucoidan, laminarin, and Pyropia haitanensis polysaccharides, and the underlying mechanism by Bacteroides thetaiotaomicron G4 were investigated. Pure culture of Bacteroides thetaiotaomicron G4 in a substrate supplemented with these polysaccharides were performed. The change of OD600, total carbohydrate contents, and molecular weight during this fermentation were determined. Genomic sequencing and bioinformatic analysis were further performed to elucidate the mechanisms involved. Specifically, Gene Ontology (GO) annotation, Clusters of Orthologous Groups (COG) annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were utilized to identify potential target genes and pathways. Results: Underlying target genes and pathways were recognized by employing bioinformatic analysis. Several PULs were found that are anticipated to participate in the breakdown of these four polysaccharides. These findings may help to understand the interactions between these marine seaweed polysaccharides and gut microorganisms. Discussion: The elucidation of polysaccharide degradation mechanisms by Bacteroides thetaiotaomicron provides valuable insights into the utilization of marine polysaccharides as prebiotics and their potential impact on gut health. Further studies are warranted to explore the specific roles of individual PULs and their contributions to polysaccharide metabolism in the gut microbiota.

Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii served as key components of fecal microbiota transplantation to alleviate colitis.

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 (donor H) and low (donor L) groups according to the diversity and the abundance of Bacteroides and Faecalibacterium 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 group donor H (P < 0.05). Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii 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 (P < 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 (P < 0.05). Metabolomics showed increased abundance of lecithin in the glycerophospholipid metabolism pathway. In conclusion, B. thetaiotaomicron and F. prausnitzii, 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.NEW & NOTEWORTHY We demonstrate that donors with high abundance of Bacteroides and Faecalibacterium ameliorate dextran sulfate sodium (DSS)-induced colitis in mice by fecal microbiota transplantation (FMT). The combination therapy of Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii 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.

Bile-induced biofilm formation in Bacteroides thetaiotaomicron requires magnesium efflux by an RND pump.

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle, and it was recently shown that B. thetaiotaomicron biofilm formation is promoted by the presence of bile. This process also requires a B. thetaiotaomicron extracellular DNase, which is not, however, regulated by bile. Here, we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC [for Bile Induced Pump A (BT3337), B (BT3338), and C (BT3339)], is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of extracellular DNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore, identified a new molecular determinant of B. thetaiotaomicron biofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCEBacteroides thetaiotaomicron is a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown that B. thetaiotaomicron exposure to intestinal bile promotes biofilm formation. Here, we reveal that a specific B. thetaiotaomicron membrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study, therefore, provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events.

CRISPRi-Driven Genetic Screening for Designing Novel Microbial Phenotypes.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has enabled rapid advances in genomic engineering and transcriptional regulation. Specifically, CRISPR interference (CRISPRi) system has been used to systematically investigate the gene functions of microbial strains in a high-throughput manner. This method involves growth profiling using cells that have been transformed with the deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) libraries that target individual genes. The fitness scores of each gene are calculated by measuring the abundance of individual sgRNAs during cell growth and represent gene essentiality. In this chapter, a process is described for functional genetic screening using CRISPRi at the whole-genome scale, starting from the synthesis of sgRNA libraries, construction of CRISPRi libraries, and identification of essential genes through growth profiling. The commensal bacterium Bacteroides thetaiotaomicron was used to implement the protocol. This method is expected to be applicable to a broader range of microorganisms to explore the novel phenotypic characteristics of microorganisms.

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.

Vitamin B12 analogues from gut microbes and diet differentially impact commensal propionate producers of the human gut.

To produce the health-associated metabolite propionate, gut microbes require vitamin B12 as a cofactor to convert succinate to propionate. B12 is sourced in the human gut from the unabsorbed dietary fraction and in situ microbial production. However, experimental data for B12 production by gut microbes is scarce, especially on their produced B12-analogues. Further, the promotion of propionate production by microbially-produced and dietary B12 is not yet fully understood. Here, we demonstrated B12 production in 6 out of 8 in silico predicted B12-producing bacteria from the human gut. Next, we showed in vitro that B12 produced by Blautia hydrogenotrophica, Marvinbryantia formatexigens, and Blautia producta promoted succinate to propionate conversion of two prevalent B12-auxotrophic gut bacteria, Akkermansia muciniphila and Bacteroides thetaiotaomicron. Finally, we examined the propiogenic effect of commercially available B12-analogues present in the human diet (cyano-B12, adenosyl-B12 and hydroxy-B12) at two doses. The low dose resulted in partial conversion of succinate to propionate for A. muciniphila when grown with adenosyl-B12 (14.6 ± 2.4 mM succinate and 18.7 ± 0.6 mM propionate) and hydroxy-B12 (13.0 ± 1.1 mM and 21.9 ± 1.2 mM), in comparison to cyano-B12 (0.7 ± 0.1 mM and 34.1 ± 0.1 mM). Higher doses of adenosyl-B12 and hydroxy-B12 resulted in significantly more conversion of succinate to propionate in both propionate-producing species, compared to the low dose. B12 analogues have different potential to impact the propionate metabolism of prevalent propionate producers in the gut. These results could contribute to strategies for managing gut disorders associated with decreased propionate production.

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.

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.

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.

Bacteroides methylmalonyl-CoA mutase produces propionate that promotes intestinal goblet cell differentiation and homeostasis.

Propionate is a short-chain fatty acid that is generated upon microbiome-mediated fiber fermentation in the intestine. By modulating immune and metabolic pathways, propionate exerts many health benefits. Key bacterial species, such as Bacteroides thetaiotaomicron, generate propionate, but the biochemical pathways and specific functions remain undetermined. We identified a gene operon-encoding methylmalonyl-CoA mutase (MCM) that contributes to propionate biosynthesis in B. thetaiotaomicron. Colonization of germ-free mice with wild-type or MCM-deficient strains as well as in vitro examination demonstrated that MCM-mediated propionate production promotes goblet cell differentiation and mucus-related gene expression. Intestinal organoids lacking the propionate receptor, GPR41, showed reduced goblet cell differentiation upon MCM-mediated propionate production. Furthermore, although wild-type B. thetaiotaomicron alleviated DSS-induced intestinal inflammation, this effect was abolished in mice receiving the MCM-deficient strain but restored upon propionate supplementation. These data emphasize the critical role of MCM-mediated propionate biosynthesis in goblet cell differentiation, offering potential pathways to ameliorate colitis.

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.

Corrigendum: The selective advantage of the lac operon for Escherichia coli is conditional on diet and microbiota composition.

[This corrects the article DOI: 10.3389/fmicb.2021.709259.].

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.

Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice.

IMPORTANCE: Evaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate-induced colitis, that colitis erases biogeographic patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy.

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.

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

Bacteroides thetaiotaomicron (B. theta) is a Gram-negative gut bacterium that encodes enzymes that alter the bile acid pool in the gut. Primary bile acids are synthesized by the host liver and are modified by gut bacteria. B. theta encodes two bile salt hydrolases (BSHs), as well as a hydroxysteroid dehydrogenase (HSDH). We hypothesize that B. theta modifies the bile acid pool in the gut to provide a fitness advantage for itself. To investigate each gene's role, different combinations of genes encoding bile acid altering enzymes (bshA, bshB, and hsdhA) were knocked out by allelic exchange, including a triple KO. Bacterial growth and membrane integrity assays were done in the presence and absence of bile acids. To explore if B. theta's response to nutrient limitation changes due to the presence of bile acid altering enzymes, RNASeq analysis of WT and triple KO strains in the presence and absence of bile acids was done. WT B. theta is more sensitive to deconjugated bile acids (CA, CDCA, and DCA) compared to the triple KO, which also decreased membrane integrity. The presence of bshB is detrimental to growth in conjugated forms of CDCA and DCA. RNA-Seq analysis also showed bile acid exposure impacts multiple metabolic pathways in B. theta, but DCA significantly increases expression of many genes in carbohydrate metabolism, specifically those in polysaccharide utilization loci or PULs, in nutrient limited conditions. This study suggests that bile acids B. theta encounters in the gut may signal the bacteria to increase or decrease its utilization of carbohydrates. Further study looking at the interactions between bacteria, bile acids, and the host may inform rationally designed probiotics and diets to ameliorate inflammation and disease. Importance: Recent work on 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 is not well understood. In this study we set out to define if and how B. theta uses its BSHs and HSDH 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 (PULs). 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. This work will aid in our understanding of how to rationally manipulate the bile acid pool and the microbiota to exploit carbohydrate metabolism in the context of inflammation and other GI diseases.

Iron Deficiency Modulates Metabolic Landscape of Bacteroidetes Promoting Its Resilience during Inflammation.

Bacteria have to persist under low iron conditions in order to adapt to the nutritional immunity of a host. Since the knowledge of iron stimulon of Bacteroidetes is sparse, we examined oral (Porphyromonas gingivalis and Prevotella intermedia) and gut (Bacteroides thataiotaomicron) representatives for their ability to adapt to iron deplete and iron replete conditions. Our transcriptomics and comparative genomics analysis show that many iron-regulated mechanisms are conserved within the phylum. They include genes upregulated in low iron, as follows: fldA (flavodoxin), hmu (hemin uptake operon), and loci encoding ABC transporters. Downregulated genes were frd (ferredoxin), rbr (rubrerythrin), sdh (succinate dehydrogenase/fumarate reductase), vor (oxoglutarate oxidoreductase/dehydrogenase), and pfor (pyruvate:ferredoxin/flavodoxin oxidoreductase). Some genus-specific mechanisms, such as the sus of B. thetaiotaomicron coding for carbohydrate metabolism and the xusABC coding for xenosiderophore utilization were also identified. While all bacteria tested in our study had the nrfAH operon coding for nitrite reduction and were able to reduce nitrite levels present in culture media, the expression of the operon was iron dependent only in B. thetaiotaomicron. It is noteworthy that we identified a significant overlap between regulated genes found in our study and the B. thetaiotaomicron colitis study (W. Zhu, M. G. Winter, L. Spiga, E. R. Hughes et al., Cell Host Microbe 27:376-388, 2020, http://dx.doi.org/10.1016/j.chom.2020.01.010). Many of those commonly regulated genes were also iron regulated in the oral bacterial genera. Overall, this work points to iron being the master regulator enabling bacterial persistence in the host and paves the way for a more generalized investigation of the molecular mechanisms of iron homeostasis in Bacteroidetes. IMPORTANCE Bacteroidetes are an important group of anaerobic bacteria abundant both in the oral and gut microbiomes. Although iron is a required nutrient for most living organisms, the molecular mechanisms of adaptation to the changing levels of iron are not well known in this group of bacteria. We defined the iron stimulon of Bacteroidetes by examination of the transcriptomic response of Porphyromonas gingivalis and Prevotella intermedia (both belong to the oral microbiome) and Bacteroidetes thetaiotaomicron (belongs to the gut microbiome). Our results indicate that many of the iron-regulated operons are shared among the three genera. Furthermore, using bioinformatics analysis, we identified a significant overlap between our in vitro studies and transcriptomic data derived from a colitis study, thus underscoring the biological significance of our work. Defining the iron-dependent stimulon of Bacteroidetes can help to identify the molecular mechanisms of iron-dependent regulation as well as better understand the persistence of the anaerobes in the human host.