Nathan T. Porter

Bacteria from the Bacteroidota phylum are well-known for their exceptional carbohydrate degradation abilities, which are closely associated with their use of polysaccharide utilization loci (PULs). The native exchange of PULs between different Bacteroidota species via horizontal gene transfer has been confirmed experimentally, and imitating such transfers in vitro can enable detailed studies of PULs from unstudied species in readily tractable hosts and the creation of novel strains. Here, we present a new set of tools and workflow to transfer large genetic loci, such as PULs, into genetically tractable Bacteroides strains. Our method uses a newly designed pICKUP plasmid and derivatives that allow site-specific genome integration and subsequent removal of the plasmid backbone. Thus, an unlimited number of loci may be transferred using the same resistance markers. We validated the pICKUP-based workflow by transfer of a previously characterized mixed-linkage β -glucan utilization locus (MLGUL) into the genome of Bacteroides thetaiotaomicron, which enabled metabolism of mixed-linkage β -glucan by this strain. Subsequent transfer of a PUL from an uncultivated bacterium from the bovine rumen confers the ability to metabolize certain cellooligosaccharides and allows further scrutiny of various genes in the PUL and their involvement in cellooligosaccharide metabolism. The pICKUP-based method presented here expands the toolkit for investigation of PULs and other large loci, including those from intractable or uncharacterized members of the Bacteroidota. A robust method to transfer whole PULs between species also forms the basis for creating custom-tailored PULs and resulting strains, with potential applications in improved gut health and biomass valorization processes. IMPORTANCE: The gut environment is a highly competitive niche, where dietary fiber is a major source of carbon, and polysaccharide utilization loci (PULs) encoded by Bacteroidota species are linked to their dominance in this environment. Our proposed method to study such PULs, especially from non-cultivated or unculturable strains, by transferring them into genetically tractable hosts, represents a valuable alternative to typical studies of isolated PUL-derived proteins. By transferring PULs into culturable hosts, we enable both a better understanding of the PULs’ biological roles and modification of the individual encoded genes. Our results show how a single PUL transfer can confer hemicellulose-degrading ability to Bacteroides thetaiotaomicron , which is lacking in the wild-type strain. Transfer of a PUL from a distantly related strain, speculated to confer cellulose-degrading capacity, showcases that this annotation may be incorrect and indicates that the utilization of foreign genetic material depends on species relatedness in the same phylum.

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.

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.

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. Key Points B. thetaiotaomicron CPSs control the activation of T cells and the clearance of Ag in vivo. Antistimulatory CPSs activate innate immunity weaker than prostimulatory CPSs. Antistimulatory CPSs block APC access, which can be rescued by opsonization. Visual Abstract

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.

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.

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.