Investigation and Alteration of Organic Acid Synthesis Pathways in the Mammalian Gut Symbiont Bacteroides thetaiotaomicron.

Members of the gut-dwelling Bacteroides genus have remarkable abilities in degrading a diverse set of fiber polysaccharide structures, most of which are found in the mammalian diet. As part of their metabolism, they convert these fibers to organic acids that can in turn provide energy to their host. While many studies have identified and characterized the genes and corresponding proteins involved in polysaccharide degradation, relatively little is known about Bacteroides genes involved in downstream metabolic pathways. Bacteroides thetaiotaomicron is one of the most studied species from the genus and is representative of this group in producing multiple organic acids as part of its metabolism. We focused here on several organic acid synthesis pathways in B. thetaiotaomicron, including those involved in formate, lactate, propionate, and acetate production. We identified potential genes involved in each pathway and characterized these through gene deletions coupled to growth assays and organic acid quantification. In addition, we developed and employed a Golden Gate-compatible plasmid system to simplify alteration of native gene expression levels. Our work both validates and contradicts previous bioinformatic gene annotations, and we develop a model on which to base future efforts. A clearer understanding of Bacteroides metabolic pathways can inform and facilitate efforts to employ these bacteria for improved human health or other utilization strategies. IMPORTANCE Both humans and animals host a large community of bacteria and other microorganisms in their gastrointestinal tracts. This community breaks down dietary fiber and produces organic acids that are used as an energy source by the body and can also help the host resist infection by various pathogens. While the Bacteroides genus is one of the most common in the gut microbiota, it is only distantly related to bacteria with well-characterized metabolic pathways and it is therefore unclear whether research insights on organic acid production in those species can also be directly applied to the Bacteroides. By investigating multiple genetic pathways for organic acid production in Bacteroides thetaiotaomicron, we provide a basis for deeper understanding of these pathways. The work further enables greater understanding of Bacteroides-host relationships, as well as inter-species relationships in the microbiota, which are of importance for both human and animal gut health.

Bacteroides thetaiotaomicron-Infecting Bacteriophage Isolates Inform Sequence-Based Host Range Predictions.

Our emerging view of the gut microbiome largely focuses on bacteria, while less is known about other microbial components, such as bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the gut's "viral dark matter" and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.

Phase-variable capsular polysaccharides and lipoproteins modify bacteriophage susceptibility in Bacteroides thetaiotaomicron.

A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides-phage interactions, we isolated 71 Bacteroides thetaiotaomicron-infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts.

Polysaccharide Capsules Equip the Human Symbiont Bacteroides thetaiotaomicron to Modulate Immune Responses to a Dominant Antigen in the Intestine.

Bacteria express multiple diverse capsular polysaccharides (CPSs) for protection against environmental and host factors, including the host immune system. Using a mouse TCR transgenic CD4+ T cell, BθOM, that is specific for B. thetaiotaomicron and a complete set of single CPS-expressing B. thetaiotaomicron strains, we ask whether CPSs can modify the immune responses to specific bacterial Ags. Acapsular B. thetaiotaomicron, which lacks all B. thetaiotaomicron CPSs, stimulated BθOM T cells more strongly than wild-type B. thetaiotaomicron Despite similar levels of BθOM Ag expression, many single CPS-expressing B. thetaiotaomicron strains were antistimulatory and weakly activated BθOM T cells, but a few strains were prostimulatory and strongly activated BθOM T cells just as well or better than an acapsular strain. B. thetaiotaomicron strains that expressed an antistimulatory CPS blocked Ag delivery to the immune system, which could be rescued by Fc receptor-dependent Ab opsonization. All single CPS-expressing B. thetaiotaomicron strains stimulated the innate immune system to skew toward M1 macrophages and release inflammatory cytokines in an MyD88-dependent manner, with antistimulatory CPS activating the innate immune system in a weaker manner than prostimulatory CPS. The expression of antistimulatory versus prostimulatory CPSs on outer membrane vesicles also regulated immune responses. Moreover, antistimulatory and prostimulatory single CPS-expressing B. thetaiotaomicron strains regulated the activation of Ag-specific and polyclonal T cells as well as clearance of dominant Ag in vivo. These studies establish that the immune responses to specific bacterial Ags can be modulated by a diverse set of CPSs.

Bacteroides thetaiotaomicron.

This infographic on Bacteroides thetaiotaomicron (Bt) explores the ability of this microbe to digest a broad array of complex carbohydrates, alter its surface features, and its emerging role in gastrointestinal diseases. The infographic of Bacteroides thetaiotaomicron (Bt) illustrates two key facets of its symbiotic lifestyle in the human gut: a broad ability to digest dietary fiber polysaccharides and host glycans, and a dynamic cell-surface architecture that promotes both interactions with and evasion of the host immune system. The starch-utilization system (Sus) is a cell-surface and periplasmic system involved in starch cleavage and transport. Over 80 additional Sus-like systems utilize substrates ranging from host glycans to plant cell wall pectins. Bt has evolved intricate strategies to interact with other microbes or its host, including modification of its surface. Some nutrient utilization pathways select for or directly trigger changes in capsular polysaccharide (CPS) expression. Like other fermentative members of the gut microbiome, Bt produces host absorbable short-chain and organic acids, which can all be absorbed by the host as a source of energy.

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence.

When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome.IMPORTANCE The microorganisms that reside in the human colon fulfill their energy requirements mainly from diet- and host-derived complex carbohydrates. Members of this ecosystem possess poorly understood strategies to prioritize and compete for these nutrients. Based on direct carbohydrate measurements and corresponding transcriptional analyses, our findings showed that individual bacterial species exhibit different preferences for the same set of glycans and that this prioritization is maintained in a competitive environment, which may promote stable coexistence. Such understanding of gut bacterial glycan utilization will be essential to eliciting predictable changes in the gut microbiota to improve health through the diet.

A Subset of Polysaccharide Capsules in the Human Symbiont Bacteroides thetaiotaomicron Promote Increased Competitive Fitness in the Mouse Gut.

Capsular polysaccharides (CPSs) play multiple roles in protecting bacteria from host and environmental factors, and many commensal bacteria can produce multiple capsule types. To better understand the roles of different CPSs in competitive intestinal colonization, we individually expressed the eight different capsules of the human gut symbiont Bacteroides thetaiotaomicron. Certain CPSs were most advantageous in vivo, and increased anti-CPS immunoglobulin A correlated with increased fitness of a strain expressing one particular capsule, CPS5, suggesting that it promotes avoidance of adaptive immunity. A strain with the ability to switch between multiple capsules was more competitive than those expressing any single capsule except CPS5. After antibiotic perturbation, only the wild-type, capsule-switching strain remained in the gut, shifting to prominent expression of CPS5 only in mice with intact adaptive immunity. These data suggest that different capsules equip mutualistic gut bacteria with the ability to thrive in various niches, including those influenced by immune responses and antibiotic perturbations.

The Critical Roles of Polysaccharides in Gut Microbial Ecology and Physiology.

The human intestine harbors a dense microbial ecosystem (microbiota) that is different between individuals, dynamic over time, and critical for aspects of health and disease. Dietary polysaccharides directly shape the microbiota because of a gap in human digestive physiology, which is equipped to assimilate only proteins, lipids, simple sugars, and starch, leaving nonstarch polysaccharides as major nutrients reaching the microbiota. A mutualistic role of gut microbes is to digest dietary complex carbohydrates, liberating host-absorbable energy via fermentation products. Emerging data indicate that polysaccharides play extensive roles in host-gut microbiota symbiosis beyond dietary polysaccharide digestion, including microbial interactions with endogenous host glycans and the importance of microbial polysaccharides. In this review, we consider multiple mechanisms through which polysaccharides mediate aspects of host-microbe symbiosis in the gut, including some affecting health. As host and microbial metabolic pathways are intimately connected with diet, we highlight the potential to manipulate this system for health.

Lipopolysaccharide Biosynthesis Genes of Yersinia pseudotuberculosis Promote Resistance to Antimicrobial Chemokines.

Antimicrobial chemokines (AMCs) are a recently described family of host defense peptides that play an important role in protecting a wide variety of organisms from bacterial infection. Very little is known about the bacterial targets of AMCs or factors that influence bacterial susceptibility to AMCs. In an effort to understand how bacterial pathogens resist killing by AMCs, we screened Yersinia pseudotuberculosis transposon mutants for those with increased binding to the AMCs CCL28 and CCL25. Mutants exhibiting increased binding to AMCs were subjected to AMC killing assays, which revealed their increased sensitivity to chemokine-mediated cell death. The majority of the mutants exhibiting increased binding to AMCs contained transposon insertions in genes related to lipopolysaccharide biosynthesis. A particularly strong effect on susceptibility to AMC mediated killing was observed by disruption of the hldD/waaF/waaC operon, necessary for ADP-L-glycero-D-manno-heptose synthesis and a complete lipopolysaccharide core oligosaccharide. Periodate oxidation of surface carbohydrates also enhanced AMC binding, whereas enzymatic removal of surface proteins significantly reduced binding. These results suggest that the structure of Y. pseudotuberculosis LPS greatly affects the antimicrobial activity of AMCs by shielding a protein ligand on the bacterial cell surface.

Love Thy Neighbor: Sharing and Cooperativity in the Gut Microbiota.

To persist in the competitive gastrointestinal ecosystem, microbes often enforce selfish strategies that limit resource loss to neighboring bacteria. In contrast, a recent study in Nature by Rakoff-Nahoum et al. (2016) reveals that one commensal bacterium releases nutrients to benefit another species, which reciprocally provides growth-promoting factors to the producer.

Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles.

Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically susceptible mice (dnKO) that develop spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron (B. theta), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta, which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Authentic active learning activities demonstrating the use of serial dilutions and plate counts.

Serial dilution and plate counting is often taught in courses for both microbiology and allied health students. Lecture examples and examination questions addressing how the method is used can sometimes be contrived: artificial data sets may have little or no meaning other than to have students perform a calculation. Here we provide a set of activities employing data sets acquired from the primary literature. Our objective was to have the students think critically about a real scenario in which serial dilution and plate count was used. Each activity requires students to read a paragraph describing the study, predict the results, perform the appropriate calculations, and then evaluate the results in light of their predictions. To test the efficacy of these activities, a pretest quiz was given to approximately 100 students in an allied health/general microbiology course. After a lecture on how microbes are enumerated, students were given a different quiz. The class was then divided randomly into groups of three or four students and assigned one of the activities. A postactivity quiz was also administered. Approximately two weeks later, a serial dilution/plate count question was used on an examination and served as a final posttest. Standardized learning gains were calculated for the quiz administered after each learning activity. Even though learning gains were significantly higher after the lecture, there was also a significant improvement between the lecture and the activity. Using an exercise based on an authentic set of data significantly improved student learning gains, and is a useful practice for teaching microbiology.