Cell Host & Microbe

Bacteriophages are major ecological drivers of gut microbial ecology, yet whether bacterial mechanisms that determine phage susceptibility have consequences for the mammalian host remains poorly understood. Here, we identify dipeptidyl peptidase 11 (Dpp11a), the predominant active serine protease of the prevalent gut commensal Phocaeicola vulgatus, as an unexpected bacterial defence factor. Dpp11a protects against environmental proteases and confers resistance to bacteriophage infection. Metatranscriptomic analyses further reveal increased expression of both dpp11a and P. vulgatus-associated phage transcripts in ulcerative colitis stool samples, indicating that both components of this interaction are transcriptionally active in disease-associated human microbiomes. Using the microfluidic gut-on-a-chip co-culture model HuMiX, we show that the absence of Dpp11 is accompanied by altered epithelial tight-junction remodelling during phage-bacterial infection. Together, our findings reveal that the consequences of bacterial phage defence can extend beyond phage-bacterium interactions to the mammalian epithelium.

Adherent-invasive Escherichia coli (AIEC) is frequently isolated from the dysbiotic gut microbiome in inflammatory bowel disease and is implicated in disease progression, highlighting its potential as a therapeutic target. Here we show that iron acquisition is essential for AIEC colonization of the inflamed gut. Mechanistically, four catecholate siderophore receptors act redundantly to support AIEC growth under iron-limited conditions and during colitis. Immunization of colitis-prone Il10-/- mice against catecholate siderophores reduced intestinal AIEC colonization, particularly its association with the mucosa, while largely preserving overall gut microbiome composition. Longitudinal metagenomic profiling identified immunization-associated shifts in Akkermansiaceae and Lactobacillaceae, and experiments in gnotobiotic Il10-/- mice demonstrated that Limosilactobacillus reuteri, a member of the Lactobacillaceae, limited AIEC colonization, whereas Akkermansiaceae exacerbated disease. Our findings establish catecholate siderophore-mediated iron acquisition as a therapeutic target in AIEC and provide proof-of-principle that virulence-targeted immunization can selectively antagonize a disease-associated bacterium while preserving the gut microbiota

Antibiotic disruption of gut microbiota creates opportunities for toxigenic Clostridioides difficile. One strategy is to occupy this niche with a non-toxigenic or weakly virulent strain, but even strains that cause little disease may fail to compete during pathogen challenge. We screened 21 sequence type 1 (ST1) isolates for their ability to protect mice co-colonized with toxigenic VPI10463, separating disease potential from protection. Some strains, such as ST1-75, caused little disease and protected, whereas others, such as ST1-68, caused little disease but failed to protect. Metabolomics showed that ST1-75 depleted more proline than ST1-68, whereas another protective strain, ST1-6, had a different metabolic profile; both protective strains nevertheless responded to many substrates. Substrate-utilization profiling revealed that protection across the 21 isolates correlated with amino-acid overlap with VPI10463, but not overall nutrient overlap or shared genome-encoded protein families. Relative amino-acid access explained variation beyond mono-colonization disease potential, and a consumer-resource model showed how nutrient overlap together with additional resource access can support persistence during competition. Additional experiments support that ST1-mediated protection occurs even in the absence of mature B and T cells. These findings distinguish low disease potential from protection, link protection to strain competition, and identify relative resource access as an informative phenotype for prioritizing strains for biotherapeutic testing.

Immunoglobulin A (IgA) is the most abundant antibody within the mammalian gut, where it binds to both pathogenic and commensal microbes. Despite its prevalence, the impact of IgA upon the biology and behavior of individual microbes remains poorly understood. Here we present Paired Immunoglobulin Sequencing (PIg-seq), a high-throughput method to identify IgA-associated changes in bacterial gene expression. Using PIg-seq we uncover widespread, and in many cases previously unrecognized, transcriptional responses associated with IgA-binding within the mouse gut. While many of these associations are conserved across diverse bacterial lineages, others vary dramatically even between closely related strains, revealing striking strain-level heterogeneity. Most notably, IgA binding is associated with altered expression of bacterial conjugation genes, and multiple prophages, suggesting an exciting link between adaptive immunity and horizontal gene transfer. Together, our findings establish IgA as a key regulator of microbial behavior, with potential consequences for both the ecology and the evolution of the gut microbiome.

Bacteria encode diverse antiviral defense systems, yet the evolutionary reservoirs that generate this immune innovation remain poorly defined. Although genomic context has been instrumental in discovering defense systems, genomic islands (GIs), variable chromosomal regions enriched in defense genes and proteins of unknown function, have not been systematically explored as sources of new bacterial immunity. Here, using comparative genomics of Streptococcus thermophilus, we identified 30 defense-associated GIs displaying extensive accessory gene diversity. Investigation of candidate loci from three GIs uncovered 15 previously uncharacterized antiviral systems, including 10 with novel architectures or features, revealing the potential within even a small subset of GIs. Several systems were not located near known defense genes, highlighting that GI-based discovery complements the traditional guilt-by-association approach, revealing systems that would otherwise remain undetected. Together, our findings establish GIs as reservoirs of antiviral innovation and provide a framework for uncovering the diversity of bacterial immune systems.

Gut microbes convert cholesterol into coprostanol, a poorly absorbed sterol that is largely excreted in feces and is linked to lower serum cholesterol levels. However, the enzymes responsible for this process remain poorly characterized. The second step of the pathway, cholestenone reduction into coprostanone, has long been established, yet the enzyme(s) responsible were unknown. Here, we use comparative genomics to identify SpiE, a cholestenone (4-cholesten-3-one) reductase that is part of the steroid processing (spi) pathway with SpiR. We confirm that SpiE binds to and reduces both cholestenone and progesterone into 5{beta}-reduced forms. SpiR and SpiE together convert cholesterol into coprostanol, indicating that both enzymes are sufficient for coprostanol formation. SpiE belongs to the short-chain dehydrogenase (SDR) family and represents an evolutionarily distinct lineage of SDR ene-reductases. Metagenomic analyses reveal that spiE relative abundance is strongly correlated with stool coprostanol (Spearman's {rho}=0.81, p=5.99e-65). In longitudinal patient cohorts, antibiotic treatment coincided with loss of SpiE and coprostanol production. Post antibiotics, the emergence of distinct SpiE-encoding genera rescued coprostanol production in some individuals. Our findings provide the biochemical basis of the cholesterol to coprostanol pathway and lay the groundwork for future studies linking it to host physiology.

The intestinal mucus layer forms the primary physical barrier separating the host epithelium from the gut microbiota. Antibiotic-induced dysbiosis compromises this barrier, yet the microbial metabolites and host mechanisms responsible for its rapid restoration remain poorly understood. Here, we identify pantothenic acid (vitamin B5) as a microbial metabolite that rapidly restores colonic mucus integrity following antibiotic administration. Integrated 16S rRNA gene sequencing and metabolomic profiling of murine feces revealed a positive association between Muribaculaceae abundance, fecal pantothenic acid level, and colonic mucus thickness. Among four isolated Muribaculaceae species, only Sangeribacter muris produced pantothenic acid. An exogenous pantothenic acid supplement quickly restored the thin mucus layer. This rapid response occurred without detectable transcriptional activation. This response did not recur in isolated colonic epithelium or colonic organoids, indicating that pantothenic acid acts through a non-epithelial, non-autonomous mechanism. Together, these findings identify a previously unrecognized microbe-metabolite-host signaling pathway that rapidly regulates colonic mucus barrier homeostasis and reveal a local barrier-protective function of microbiota-derived pantothenic acid.

Exchange of genetic loci encoding extracellular capsular polysaccharides in bacteria can change capsular antigens or serotype, which, in turn, can lead to evasion of vaccines targeting the capsule. However, the evolutionary dynamics and drivers of bacterial capsule switching remain poorly understood. Here, we investigate the evolutionary dynamics and correlates of capsule switching in bacteria, using the human-restricted bacterial pathogen Streptococcus pneumoniae as an example, using 10,558 genetically and geographically diverse whole-genome sequences. Our phylogenomic and ancestral state reconstruction analysis revealed that the propensity for capsule switching varies by capsule type and genetic background. We found capsule switches only between ~4% of all possible capsule-type pairs, varying by specific pairs and switching direction. We also found associations between capsule switching patterns and genetic and ecological factors, including nasopharyngeal carriage duration, capsule biosynthesis locus size, and capsule thickness. Furthermore, the genetic similarity of the replacing and replaced capsule loci was primarily associated with the frequency of switching between specific capsule type pairs. Although a few specific non-capsule genomic loci were associated with the propensity for capsule switching across distinct lineages, the collective effect of the genetic background appears to influence capsule switching more strongly. These findings shed light on how bacterial capsule types and lineages undergo capsule switching influenced by genetic and ecological factors, informing our understanding of the emergence of novel capsule-genotype combinations that may impact the design of capsule-targeting glycoconjugate vaccines.

Gut microbes metabolize the ellagitannin castalagin to ellagic acid and urolithins, a process associated with health effects for the host. Despite the use of the castalagin-rich camu camu extracts in clinical trials, the genes and taxa responsible for castalagin hydrolysis remain elusive. We used multi-omics to discover a castalagin associated hydrolase (cah) operon sparsely distributed in Lachnospiraceae. Analysis of fecal samples from a camu camu supplementation clinical trial demonstrates functional redundancy with prevalence of cah and other ellagic acid metabolism genes in different taxa explaining the prevalence of metabolites. Employing genome mining strategies, we discovered and validated ellagitannin metabolism genes in uncharacterized and uncultured species. We further identified flavogallonic acid as a prevalent metabolite of castalagin in human fecal samples. Collectively, this work provides a genetic basis for castalagin metabolism to urolithins and flavogallonic acid end-metabolites and sets the stage to study the role of metabolism in clinical trials.

There is a critical window in early life for immune development that prepares a young mammal for postnatal exposure to environmental, dietary and microbial antigens. Both the maternal microbiota and endogenous colonization have emerged as pivotal drivers in this process. Using gnotobiotic mouse models and reversible colonization to model the effects of the maternal microbiota during pregnancy compared with early life colonization on the small intestinal epithelium we show that both microbial metabolites originating from the maternal microbiota and the postnatal endogenous microbiota drive intestinal epithelial maturation by modulating epigenetic signatures. Microbiota-derived signals from both the pregnant mother and from endogenous postnatal colonisation drove the BLIMP1-guided epithelial neonatal-to-adult transition at weaning through epigenetic mechanisms and direct transcriptional effects. These microbial signals also shaped the developmental trajectory of barrier-forming tight junctional claudins in the neonatal intestine, increased aerobic respiration, and increased the gene expression of xenosensors. Thus, both maternal and endogenous microbiota derived drivers of the offspring's epigenetic and transcriptomic intestinal epithelial landscape fine-tuned the timing of epithelial maturation and augmented functional plasticity at weaning.

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems. Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa. Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

Prophages are increasingly recognized as drivers of bacterial adaptation, yet how domesticated prophage genes shape microbial symbiosis remains poorly understood. Here, we identify GDPDXhp1, a glycerophosphodiester phosphodiesterase encoded by prophage Xhp1 of Schaalia odontolytica XH001, as a metabolic regulator linking bacterial physiology, episymbiosis and phage fitness. Association with the epibiotic bacterium Nanosynbacter lyticus TM7x strongly induced gdpdXhp1 expression and triggered GDPDXhp1-dependent lipid droplet accumulation in XH001. GDPDXhp1 orchestrated glycerophospholipid remodeling, membrane biophysical properties, respiration and cell-surface glycan composition in XH001, thereby promoting TM7x episymbiosis. Remarkably, GDPDXhp1 was also required for productive Xhp1 infection by remodeling susceptible bacterial host envelope structures to facilitate phage-host interactions. Together, our findings reveal a domesticated prophage enzyme that contributes to bacterial metabolism, interspecies symbiosis and viral reproduction, highlighting prophage as an overlooked source of metabolic innovation in microbial symbiosis.

Horizontal gene transfer (HGT) drives microbial evolution, but what determines when genes move between species within complex communities remains unclear. We developed a junction-resolved metagenomic framework using short-read sequencing to distinguish mobile genetic element activation from de novo transfer and track both longitudinally. In a defined human gut community colonizing mice, mobile element activation was widespread but successful transfer was rare. A prophage carried by invading Phocaeicola vulgatus transferred reproducibly to resident species when the community resisted invasion, but not when dietary perturbation allowed the invader to expand. Transfer required prophage activation and persistence of a permissive recipient and coincided with diversification of a phage receptor-binding protein. Dietary polysaccharides reproduced prophage-associated fitness costs in vitro, and rhamnogalacturonan I induced cell lysis consistent with productive prophage activation. The framework also detected interspecies transfer retrospectively in longitudinal human metagenomes. Thus, diet and community ecology can redirect gene flow through the gut microbiome.

Clostridioides difficile infection (CDI) is characterized by toxin-mediated epithelial injury, intestinal inflammation, and a high frequency of disease recurrence. Although complement components are produced locally within the gastrointestinal tract, their contribution to C. difficile pathogenesis remains poorly understood. Here, we investigated the interaction of the complement proteins C1q and C3 with C. difficile spores and their contribution to spore-host interactions and disease outcome. We found that C1q and C3 are accessible within the healthy ileal mucosa and that TcdB intoxication differentially remodels their spatial availability, decreasing accessible C1q while increasing accessible C3 at higher toxin concentrations. C. difficile spores associated with both complement components in vivo and directly interacted with purified human C1q and C3 in vitro, with immunogold electron microscopy localizing these interactions to the outer exosporium. Although the collagen-like BclA proteins modulated complement binding under some conditions, they were not required for C1q or C3 association. Far-Western analysis coupled to mass spectrometry identified the exosporium proteins CotE and CdeM as candidate complement-interacting proteins, and purified CdeM inclusion bodies directly associated with both C1q and C3. Exposure of spores to human serum promoted their interaction with intestinal epithelial cells, whereas depletion of either C1q or C3 markedly reduced spore internalization and reconstitution with the corresponding purified protein partially restored entry. C3-derived species, including C3dg, were selectively associated with spores but not vegetative cells, without reducing spore viability. Finally, C1q deficiency did not alter the initial onset of CDI but accelerated recovery and markedly attenuated recurrent disease following vancomycin treatment. Together, these findings identify intestinal complement as a previously unrecognized component of the C. difficile spore-host interface and reveal C1q and C3 as modulators of spore epithelial entry, persistence, and recurrent disease.

As fungal pathogens pose mounting global challenges, decoding their complex adaptive traits remains a paramount academic priority. While previous studies have provided foundational insights into protein-coding variations and orphan genes, fully understanding such traits requires exploring upstream non-coding regulatory networks. Here, we present a high-resolution, rRNA-depleted temporal transcriptomic framework across reciprocal dimorphic transitions to decode how non-coding network rewiring involving in fungal adaption. Integrating temporal profiling with comparative genomics reveals that early ATP-related metabolic reprogramming and melanin biosynthesis direct morphogenesis via species-specific motifs. Further temporal network modeling uncovers a functional stratification where long non-coding RNAs orchestrate early stress responses, whereas circular RNAs drive morphological conversion. Functional validation confirms that hub non-coding transcripts mediate stress adaptation, with cross-species perturbation in the closest non-pathogen species altering phenotypic outcomes. Together, these findings demonstrate how system-level non-coding dynamics underlie the evolutionary innovation of thermal dimorphism, establishing a robust paradigm for understanding specialized fungal pathogenicity and evolution adaption.

The microbial ecosystem in the human gut is fundamentally shaped by the resources it receives. Yet, while quantitative information is available for many individual resource fluxes, a comprehensive description of the overall resource landscape is lacking. Here, I systematically integrate physiological measurements and food composition data to map diet and digestion to the fluxes of more than 30 major resources entering the human large intestine, including carbohydrates, proteins, mucus, bile acids, ions, and electron acceptors. Applying this reconstruction across dietary variation in the U.S. population and analyzing the resulting influxes of biomass-forming elements, I find that microbial growth is carbon-limited for most individuals, whereas phosphorus is limiting for a subset; nitrogen, by contrast, is not limiting once host-derived urea recycling is accounted for. Resource supply also determines whether microbial auxotrophies impose physiological constraints. While amino acid dependencies can often be satisfied by protein-derived influx, characterized dietary cobamide influx is insufficient to account for the fecal cobamide pool, pointing to a much greater contribution from microbial production. Together, these results provide a quantitative resource landscape for the human gut, defining physiological constraints on microbial growth and providing a basis for predictive models of microbiome function and its impact on the host.

The generation of appropriate responses to the intestinal microbiota is an essential aspect of immune homeostasis. Studies of microbes that elicit stereotyped, antigen-specific CD4 T cell responses support the view that immune recognition of the microbiota is specific, relatively limited, and largely skewed toward Tregs, but recent profiling efforts have suggested that recognition of the microbiota may be broader and less regulatory. Such studies have mostly tracked responses to single antigens or used mice that acquire their microbiota after weaning, which has made it difficult to generalize the rules that govern microbiota-mediated T cell responses. Here we comprehensively profile, for the first time, the T cell response to the microbiota at homeostasis, and develop a panel of tools to track multiple, endogenous microbiota-reactive CD4 T cell populations in vivo. Our profiling reveals a broad microbiota-reactive T cell response that is Treg-biased but that also contains non-Treg responses to specific microbes. We find that certain microbiota-reactive T cells require priming during early-life to efficiently establish their Treg identity, while others do not. Our findings illustrate an unappreciated antigen-specific complexity in programming of microbiota-reactive T cells and underscore the importance of studying responses to the microbiota under homeostatic conditions.

Diet exerts selection on bacterial species within the mammalian gut microbiota1,2,3,4,5, but the extent, modes, genomic bases, and repeatability of gut bacterial adaptive evolution in response to dietary variation within hosts are poorly understood. Here, we show that within-host dietary transitions drive ubiquitous and highly reproducible adaptive evolution of gut bacterial populations. We deeply profiled allele-frequency dynamics in gut microbiota of an experimental cohort of litter-matched wild-derived isogenic mice either transitioned from low-fat/high-carbohydrate diets to high-fat/low-carbohydrate diets or maintained on their ancestral low-fat/high-carbohydrate diet. Dietary transitions induced significantly parallel allele-frequency changes across replicates in 61/62 core gut bacterial species within five months, whereas 0/62 species displayed significant across-replicate parallelism in the control group, indicating deterministic adaptive evolutionary responses across the gut microbiota. Adaptation proceeded through multiple modes, including strain sorting and gene-wise responses. The latter were enriched in a subset of gene families and functions across bacterial species, including invertases. Culturing revealed an invertible DNA switch in Phocaeicola sartorii that, in all replicates transitioned to high-fat/low-carbohydrate diets, localized a promoter sequence upstream of cell-surface glycan foraging genes. These findings elucidate widespread gut bacterial adaptive evolution mediated by within-host dietary variation.

The oral cavity harbors one of the body's most diverse microbial communities, which continuously seeds the bloodstream and gastrointestinal tract, particularly in periodontitis, a disease in which both barrier permeability and microbial burden increase. Despite this constant systemic exposure, overt bacteremia from oral organisms is rare, suggesting an active surveillance system that contains this reservoir. Here, we apply IgG-sequencing to comprehensively map mucosal and systemic antibody recognition of the oral microbiome across paired oral fluid and serum from healthy individuals and patients with periodontitis. We find that IgG-binding is only weakly predicted by microbial abundance, suggesting taxon-specific and largely conserved IgG-binding of commensals, many of them streptococci which dominate antibody recognition irrespective of disease state. Periodontitis broadens the repertoire of IgG-bound taxa to include particular disease-associated pathobionts while preserving robust targeting of health-associated symbionts. Comparison of salivary and serum antibody profiles further uncovers a subset of commensal streptococci, including species implicated in infective endocarditis, that acquire disproportionate systemic IgG binding in periodontitis. Together, these findings establish a taxon-level atlas of humoral immunosurveillance at a major mucosal-microbial interface and reveal how periodontal disease reshapes systemic antibody recognition of both pathobionts and commensals.