Acarbose Impairs Gut Bacteroides Growth by Targeting Intracellular GH97 Enzymes.

Acarbose is a type-2 diabetes medicine that inhibits dietary starch breakdown into glucose by inhibiting host amylase and glucosidase enzymes. Numerous gut species in the Bacteroides genus enzymatically break down starch and change in relative abundance within the gut microbiome in acarbose-treated individuals. To mechanistically explain this observation, we used two model starch-degrading Bacteroides, Bacteroides ovatus (Bo) and Bacteroides thetaiotaomicron (Bt). Bt growth is severely impaired by acarbose whereas Bo growth is not. The Bacteroides use a starch utilization system (Sus) to grow on starch. We hypothesized that Bo and Bt Sus enzymes are differentially inhibited by acarbose. Instead, we discovered that although acarbose primarily targets the Sus periplasmic GH97 enzymes in both organisms, the drug affects starch processing at multiple other points. Acarbose competes for transport through the Sus beta-barrel proteins and binds to the Sus transcriptional regulators. Further, Bo expresses a non-Sus GH97 (BoGH97D) when grown in starch with acarbose. The Bt homolog, BtGH97H, is not expressed in the same conditions, nor can overexpression of BoGH97D complement the Bt growth inhibition in the presence of acarbose. This work informs us about unexpected complexities of Sus function and regulation in Bacteroides, including variation between related species. Further, this indicates that the gut microbiome may be a source of variable response to acarbose treatment for diabetes.

Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

When simplicity triumphs: niche specialization of gut bacteria exists even for simple fiber structures.

Structurally complex corn bran arabinoxylan (CAX) was used as a model glycan to investigate gut bacteria growth and competition on different AX-based fine structures. Nine hydrolyzate segments of the CAX polymer varying in chemical structure (sugars and linkages), CAX, five less complex non-corn arabinoxylans, and xylose and glucose were ranked from structurally complex to simple. The substrate panel promoted different overall growth and rates of growth of eight Bacteroides xylan-degrading strains. For example, Bacteroides cellulosilyticus DSM 14838 (Bacteroides cellulosilyticus) grew well on an array of complex and simple structures, while Bacteroides ovatus 3-1-23 grew well only on the simple structures. In a competition experiment, B. cellulosilyticus growth was favored over B. ovatus on the complex AX-based structure. On the other hand, on the simple structure, B. ovatus strongly outcompeted B. cellulosilyticus, which was eliminated from the competitive environment by Day 11. This adaptation to fine structure and resulting competition dynamics indicate that dietary fiber chemical structures, whether complex or simple, favor certain gut bacteria. Overall, this work supports a concept that fiber degraders diversify their competitive abilities to access substrates across the spectrum of heterogeneity of fine structural features of dietary fibers.

Opposing diet, microbiome, and metabolite mechanisms regulate inflammatory bowel disease in a genetically susceptible host.

Inflammatory bowel diseases (IBDs) are chronic conditions characterized by periods of spontaneous intestinal inflammation and are increasing in industrialized populations. Combined with host genetics, diet and gut bacteria are thought to contribute prominently to IBDs, but mechanisms are still emerging. In mice lacking the IBD-associated cytokine, interleukin-10, we show that a fiber-deprived gut microbiota promotes the deterioration of colonic mucus, leading to lethal colitis. Inflammation starts with the expansion of natural killer cells and altered immunoglobulin-A coating of some bacteria. Lethal colitis is then driven by Th1 immune responses to increased activities of mucin-degrading bacteria that cause inflammation first in regions with thinner mucus. A fiber-free exclusive enteral nutrition diet also induces mucus erosion but inhibits inflammation by simultaneously increasing an anti-inflammatory bacterial metabolite, isobutyrate. Our findings underscore the importance of focusing on microbial functions-not taxa-contributing to IBDs and that some diet-mediated functions can oppose those that promote disease.

Ruminococcus torques is a keystone degrader of intestinal mucin glycoprotein, releasing oligosaccharides used by Bacteroides thetaiotaomicron.

Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how Ruminococcus torques- a known mucin-degrader that remains poorly studied despite its implication in inflammatory bowel diseases (IBDs)- degrades mucin glycoproteins or their component O -linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that R. torques utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by R. torques revealed strong fucosidase, sialidase and ß1,4-galactosidase activities. There was a lack of detectable sulfatase and weak ß1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, Bacteroides thetaiotaomicron grows poorly on mucin glycoproteins, we demonstrate a clear ability of R. torques to liberate products from mucins, making them accessible to B. thetaiotaomicron . This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of R. torques to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which may contribute to its association with IBD. Importance: An important facet of maintaining healthy symbiosis between host and intestinal microbes is the mucus layer, the first defense protecting the epithelium from lumenal bacteria. Some gut bacteria degrade different components of intestinal mucins, but detailed mechanisms used by different species are still emerging. It is imperative to understand these mechanisms as they likely dictate interspecies interactions and may illuminate particular species associated with bacterial mucus destruction and subsequent disease susceptibility. Ruminococcus torques is positively associated with IBD in multiple studies. We identified mucin glycan-degrading enzymes in R. torques and found that it shares mucin degradation products with another gut bacterium implicated in IBD, Bacteroides thetaiotaomicron . Our findings underscore the importance of understanding the mucin degradation mechanisms of different gut bacteria and their consequences on interspecies interactions, which may identify keystone bacteria that disproportionately contribute to defects in mucus protection and could therefore be targets to prevent or treat IBD.

Fiber-deficient diet inhibits colitis through the regulation of the niche and metabolism of a gut pathobiont.

Exclusive enteral nutrition (EEN) with fiber-free diets is an effective steroid-sparing treatment to induce clinical remission in children with Crohn's disease (CD). However, the mechanism underlying the beneficial effects of EEN remains obscure. Using a model of microbiota-dependent colitis with the hallmarks of CD, we find that the administration of a fiber-free diet prevents the development of colitis and inhibits intestinal inflammation in colitic animals. Remarkably, fiber-free diet alters the intestinal localization of Mucispirillum schaedleri, a mucus-dwelling pathobiont, which is required for triggering disease. Mechanistically, the absence of dietary fiber reduces nutrient availability and impairs the dissimilatory nitrate reduction to ammonia (DNRA) metabolic pathway of Mucispirillum, leading to its exclusion from the mucus layer and disease remission. Thus, appropriate localization of the specific pathobiont in the mucus layer is critical for disease development, which is disrupted by fiber exclusion. These results suggest strategies to treat CD by targeting the intestinal niche and metabolism of disease-causing microbes.

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.

Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection.

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients, such as iron. Pathogens scavenge iron using siderophores, including enterobactin; however, this strategy is counteracted by host protein lipocalin-2, which sequesters iron-laden enterobactin. Although this iron competition occurs in the presence of gut bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron and sustains its resilience in the inflamed gut by utilizing siderophores produced by other bacteria, including Salmonella, via a secreted siderophore-binding lipoprotein XusB. Notably, XusB-bound enterobactin is less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. Because the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the host-pathogen interactions and nutritional immunity.

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection.

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients such as iron. Pathogens scavenge iron using siderophores, which is counteracted by the host using lipocalin-2, a protein that sequesters iron-laden siderophores, including enterobactin. Although the host and pathogens compete for iron in the presence of gut commensal bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron in the inflamed gut by utilizing siderophores produced by other bacteria including Salmonella, via a secreted siderophore-binding lipoprotein termed XusB. Notably, XusB-bound siderophores are less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella , allowing the pathogen to evade nutritional immunity. As the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the interactions between pathogen and host nutritional immunity.

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.

Multiple TonB Homologs are Important for Carbohydrate Utilization by Bacteroides thetaiotaomicron.

The human gut microbiota is able to degrade otherwise undigestible polysaccharides, largely through the activity of the Bacteroides. Uptake of polysaccharides into Bacteroides is controlled by TonB-dependent transporters (TBDT) whose transport is energized by an inner membrane complex composed of the proteins TonB, ExbB, and ExbD. Bacteroides thetaiotaomicron (B. theta) encodes 11 TonB homologs which are predicted to be able to contact TBDTs to facilitate transport. However, it is not clear which TonBs are important for polysaccharide uptake. Using strains in which each of the 11 predicted tonB genes are deleted, we show that TonB4 (BT2059) is important but not essential for proper growth on starch. In the absence of TonB4, we observed an increase in abundance of TonB6 (BT2762) in the membrane of B. theta, suggesting functional redundancy of these TonB proteins. Growth of the single deletion strains on pectin galactan, chondroitin sulfate, arabinan, and levan suggests a similar functional redundancy of the TonB proteins. A search for highly homologous proteins across other Bacteroides species and recent work in B. fragilis suggests that TonB4 is widely conserved and may play a common role in polysaccharide uptake. However, proteins similar to TonB6 are found only in B. theta and closely related species suggesting that the functional redundancy of TonB4 and TonB6 may be limited across the Bacteroides. This study extends our understanding of the protein network required for polysaccharide utilization in B. theta and highlights differences in TonB complexes across Bacteroides species.

Inulin diet uncovers complex diet-microbiota-immune cell interactions remodeling the gut epithelium.

BACKGROUND: The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure. METHODS: Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment. RESULTS: We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk. CONCLUSION: This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstract.

Unravelling specific diet and gut microbial contributions to inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic condition characterized by periods of spontaneous intestinal inflammation and is increasing in industrialized populations. Combined with host genetic predisposition, diet and gut bacteria are thought to be prominent features contributing to IBD, but little is known about the precise mechanisms involved. Here, we show that low dietary fiber promotes bacterial erosion of protective colonic mucus, leading to lethal colitis in mice lacking the IBD-associated cytokine, interleukin-10. Diet-induced inflammation is driven by mucin-degrading bacteria-mediated Th1 immune responses and is preceded by expansion of natural killer T cells and reduced immunoglobulin A coating of some bacteria. Surprisingly, an exclusive enteral nutrition diet, also lacking dietary fiber, reduced disease by increasing bacterial production of isobutyrate, which is dependent on the presence of a specific bacterial species, Eubacterium rectale. Our results illuminate a mechanistic framework using gnotobiotic mice to unravel the complex web of diet, host and microbial factors that influence IBD.

Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Fitness advantage of Bacteroides thetaiotaomicron capsular polysaccharide in the mouse gut depends on the resident microbiota.

Many microbiota-based therapeutics rely on our ability to introduce a microbe of choice into an already-colonized intestine. In this study, we used genetically barcoded Bacteroides thetaiotaomicron (B. theta) strains to quantify population bottlenecks experienced by a B. theta population during colonization of the mouse gut. As expected, this reveals an inverse relationship between microbiota complexity and the probability that an individual wildtype B. theta clone will colonize the gut. The polysaccharide capsule of B. theta is important for resistance against attacks from other bacteria, phage, and the host immune system, and correspondingly acapsular B. theta loses in competitive colonization against the wildtype strain. Surprisingly, the acapsular strain did not show a colonization defect in mice with a low-complexity microbiota, as we found that acapsular strains have an indistinguishable colonization probability to the wildtype strain on single-strain colonization. This discrepancy could be resolved by tracking in vivo growth dynamics of both strains: acapsular B.theta shows a longer lag phase in the gut lumen as well as a slightly slower net growth rate. Therefore, as long as there is no niche competitor for the acapsular strain, this has only a small influence on colonization probability. However, the presence of a strong niche competitor (i.e., wildtype B. theta, SPF microbiota) rapidly excludes the acapsular strain during competitive colonization. Correspondingly, the acapsular strain shows a similarly low colonization probability in the context of a co-colonization with the wildtype strain or a complete microbiota. In summary, neutral tagging and detailed analysis of bacterial growth kinetics can therefore quantify the mechanisms of colonization resistance in differently-colonized animals.

Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.