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

The human microbiome exerts broad influence in health and disease with associative studies implicating the microbiome in influencing immunity, cancer outcomes, and neurodegeneration. However, the molecular mediators of microbe-host communication remain poorly defined. Bacterial microproteins from the microbiome represent a largely uncharacterized class of potential regulators of host immunity. Here, we utilize functional genomics to interrogate 3,552 microproteins in order to identify novel microbial-immune interactions. We constructed a microproteome library from microbial metagenomic datasets, expressed it in macrophages and assayed for immunomodulatory activity. We identify several bacterial microproteins that drive macrophage M1 polarization. Among the strongest hits are a cluster of structurally related microproteins from Leptotrichia species, which are oral Gram-negative commensals associated with differential cancer outcomes. Genomic analysis reveals that Leptotrichia species encode these putative immunomodulatory microproteins in tandem arrays of up to 44 copies. These genes encode microproteins with varying sequences but conserved predicted structures. In an orthogonal approach, we demonstrate that bacterial expression of Leptotrichia microproteins influences macrophage cell state and function. As a whole, our findings identify novel microbial microproteins with immunomodulatory activity and provide a framework for future discovery of host-microbe interactions that influence human health.

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.

Fungal cerato-platanins (CPs) are small ([~]12 kDa) secreted proteins broadly conserved across filamentous fungi and implicated in a striking diversity of biological processes, ranging from fungal development to interactions with plant hosts. However, a core molecular activity unifying these diverse functions has remained elusive. Fungal pathogens secrete effector proteins, including CPs, to manipulate host physiology and promote colonization. Increasing evidence demonstrates that particular fungal effectors possess antimicrobial activity that enables pathogens to reshape host-associated microbiota during infection, and that antimicrobial activity is strongly enriched among evolutionarily conserved secreted proteins. These findings suggests that intermicrobial competition represents an ancient and fundamental fungal trait, and that several effectors that manipulate plant hosts evolved from ancient antimicrobials. Here, we identify antimicrobial activity as the conserved core function of the CP family, from which diverse roles in environmental adaptation and host interaction evolved. Structural analysis of the Verticillium dahliae secretome identified a structural cluster containing the CP protein CP1 and the previously characterized antimicrobial effector Ave1, providing the first structural link between CPs and antimicrobial activity. Accordingly, functional assays demonstrate that V. dahliae CPs exhibit selective antimicrobial activity in vitro. Expanding this analysis to a wide diversity of phylogenetic lineages and ecological lifestyles revealed that antimicrobial features are broadly conserved across the CP family. Together, our findings identify antimicrobial activity as an ancient and conserved molecular function that unifies the CP family and support a model in which host-manipulating effector functions evolved from ancestral proteins that mediate inter-microbial competition.

Hosts must control symbiont abundance to ensure stable associations with their beneficial microbes, while also minimizing potentially harmful unrestricted microbial proliferation. Yet, the microbial stimuli and involved host responses that shape this colonization control, are usually unknown, in spite of their central role in shaping host-microbe and, more broadly, host-microbiota interactions. Here, using the natural association between Caenorhabditis elegans and its symbiont Pseudomonas lurida MYb11, we combine an unbiased bacterial genetic screen with a host response reporter to identify the biosurfactant massetolide as a key regulator of host-microbe interaction. Our subsequent host functional genetic analyses revealed that massetolide triggers host TGF-beta and serotonergic signaling to promote intestinal peristalsis, limiting colonization. Indeed, massetolide deficiency enhances colonization, while its presence specifically facilitates serotonin-dependent expulsion. Together, our findings reveal a symbiont-derived biosurfactant that links host neuroimmune control of intestinal peristalsis with bacterial susceptibility to expulsion, coupling host and microbial physiology to regulate symbiont abundance.

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.

Metformin improves metabolic health and remodels the intestinal microbiota, yet how these microbial changes contribute to therapeutic benefits remains incompletely understood. We combined human metagenomics, quantitative analyses of bacterial behavior and ex vivo mucus models to investigate how metformin modulates host-microbiota interactions. Human metagenomics revealed metformin-associated enrichment of chemotaxis- and motility-related genes. At millimolar concentrations, metformin acted as a bacterial chemorepellent, an effect abolished by deletion of the chemotaxis regulator CheY and dependent on the chemoreceptor Tsr. Direct metformin exposure or fecal water from metformin-treated individuals reduced bacterial penetration into mucin, an effect reproduced in intestinal mucus from pigs fed a moderate-fat diet. Metformin treatment was further associated with reduced fecal flagellin activity and increased anti-flagellin IgA. Together, these findings identify bacterial chemotaxis as a target of metformin and reveal a mechanism by which metformin may limit bacterial access to the intestinal mucus barrier.

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.

CD4+ T cells recognize antigens from microbiota, diet, and pathogens via T cell receptors (TCRs) and orchestrate immunity by differentiating into tolerogenic regulatory (Treg) or pro-inflammatory effector (Teff) lineages (e.g. TH1 or TH17) (1). Dysregulation of these responses underlies numerous gastrointestinal inflammatory and infectious diseases (2-6). The prevailing paradigm suggests that individual microbes and dietary antigens drive distinct cell fates (e.g., segmented filamentous bacteria [SFB] induce TH17 cells (7) whereas Helicobacter hepaticus (8) and diet (9) induce Tregs). However, the generality of this model is uncertain: several key organisms are atypical, and foundational studies often omitted a complex microbiome or a diverse polyclonal TCR repertoire. Here we develop a high-throughput pipeline to screen hundreds of TCRs from mice colonized from birth with a 116-strain human microbiota (hCom2v), demonstrating that TCRs recognizing microbiota or dietary antigens are overwhelmingly enriched in the induced Treg (iTreg) lineage. Endogenous CD4+ T cells specific for these antigens adopt a uniform iTreg phenotype in vivo, both in hCom2v-colonized and conventional mice. This baseline tolerance is robust to acute inflammation but breaks down following a 'two-hit' combination of inflammation and genetic susceptibility, allowing Teff to emerge against otherwise Treg-restricted antigens. These data support a revised paradigm in which antigen-specific Treg induction is the default response to foreign antigens in the healthy gut, and effector responses are an exception reflecting a perceived threat. Reframing gastrointestinal immunity as a tolerance-first system provides a framework for understanding inflammatory disease pathogenesis and suggests that therapeutic strategies should aim to restore a Treg-predominant baseline.

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.

Antibiotic-induced disruption of plant-associated microbiomes has the potential to alter host health beyond the directly exposed generation, yet whether the effects of dysbiosis are transmitted through the seed microbiome remains unknown. Here, we investigated the intergenerational impacts of streptomycin-induced dysbiosis in tomato (Solanum lycopersicum), demonstrated that seed microbiome transfer (SMT) restores progeny microbiome function and disease resistance, and characterized the underlying physiological and genetic mechanisms. Parental streptomycin exposure altered the composition of progeny rhizosphere bacterial communities, reduced expression of defense-associated genes, and increased susceptibility to Xanthomonas perforans. Suppression of immune gene expression was strongly associated with increased disease severity, indicating that parental dysbiosis impaired progeny plants ability to mount effective immune responses. Transfer of the seed microbiome from healthy plant donors partially restored rhizosphere community composition, reduced disease severity and recovered defense gene expression of three genes. Together, our findings demonstrated that antibiotic exposure microbiome disturbance generates intergenerational legacy effects that influence plant immunity and disease susceptibility and seed microbiome transfer can counteract this dysbiosis across generations.

Bacteroidales secreted antimicrobial proteins (BSAPs) are diffusible MACPF-domain toxins that mediate intra-species antagonism in the gut microbiota. Here we define the mechanism of action of BSAP-1 from Bacteroides fragilis, showing how target specificity encoded within the N- and C-terminal domains is coordinated with pore-forming activity of the MACPF. We show that specificity of the toxin for its receptor is mediated by an extended interface comprised of the BSAP-1 C-terminal domain and residues on the receptor that differ from the orthologous protein of BSAP-1 producing strains. On the surface of susceptible cells, BSAP-1 undergoes proteolytic cleavage of an N-terminal regulatory domain, triggering its assembly into oligomeric pores. Cryo-electron microscopy of membrane-inserted BSAP-1 reveals a 13-subunit transmembrane {beta}-barrel pore formed through canonical MACPF rearrangements. Comparative modelling supports a conserved oligomerization mechanism across the BSAP family despite diversification of receptor-binding domains that target either proteins or glycan receptors. Together, these findings establish BSAP-1 as a receptor-targeted, protease-activated antibacterial MACPF toxin and provide a framework for understanding how gut Bacteroidales spatially restrict toxin activation to shape strain-level competition.

Fecal microbiota transplantation cures approximately 90% of recurrent Clostridioides difficile infection, yet it remains unknown whether all healthy donor microbiota confer equivalent protection. We colonized germ-free C57BL/6 mice with stool microbiota from 30 healthy human donors and challenged them with C. difficile in the absence of antibiotic pretreatment. Donor microbiota conferred a spectrum of colonization resistance phenotypes: Resistant (no detectable colonization or toxin), Carrier (asymptomatic colonization with detectable toxin), Symptomatic (non-lethal diarrheal illness), and Susceptible (lethal infection). Of these, 8 conferred Resistant phenotypes, 12 Carrier, 6 mixed Resistant-Carrier outcomes, and 4 Symptomatic or Susceptible phenotypes. While 16S rRNA gene sequencing of donor stool did not distinguish phenotypes across any diversity or compositional metric tested, humanized mouse microbiomes exhibited clear phenotype-dependent differences after engraftment. Richness (observed amplicon sequence variants, Chao1) and diversity (Shannon and Faith's phylogenetic diversity) declined progressively from Resistant to Susceptible phenotypes, although substantial overlap was observed between groups. Differential abundance analysis identified taxa depleted across non-resistant phenotypes, including Lachnospiraceae taxa such as Hungatella and Sellimonas, and Bacteroides intestinalis. Shotgun metagenomics confirmed these associations and revealed coordinated depletion of biosynthetic and carbohydrate metabolism pathways in non-resistant phenotypes, consistent with broad loss of community metabolic capacity rather than loss of a single dominant function. These findings demonstrate colonization resistance is a graded, microbiota-associated ecological property, evident after host engraftment rather than being a binary trait encoded in donor stool. This has implications for donor screening in fecal microbiota transplantation and the rational design of microbiome-based therapeutics.

Cultured phage collections provide essential access to viral biology, but they capture only a small and biased fraction of natural phage diversity. Key barriers to diverse phage isolation are likely to include the prophages and defence systems present in the genomes of isolation hosts. Here, we show that removal of these host genetic barriers expands recoverable phage diversity in Pseudomonas aeruginosa. We constructed a series of progressively more permissive hosts derived from MPAO1 by sequentially deleting intracellular defence systems and resident prophages, culminating in viPAO1 (virus-isolation PAO1). Sequential removal of these barriers increased phage susceptibility, with Gabija and type I restriction-modification acting as major barriers to phage infection. In comparative isolation experiments, removal of intracellular barriers produced the largest increase in phage recovery, while additional receptor modification altered the composition of recovered phage subsets. viPAO1 also substantially increased recovery of phage contigs from environmental samples. Scaling this approach across 1090 clinical isolates enabled recovery of a curated collection of 69 individually purified temperate P. aeruginosa phages spanning 24 predicted genera, including 15 putative novel genera. Together, these findings demonstrate that removal of host genetic barriers to infection enhances recoverable phage diversity and establish permissive host engineering as a practical strategy for uncovering under-sampled regions of cultured phage diversity.

Streptomyces sp. AgN23 is an epiphytic rhizobacterium that establishes in the Arabidopsis rhizosphere by activating plant immune responses. This activity depends on the secretion of polyketide galbonolides, which inhibit host inositol phosphoceramide synthase (IPCS) and thereby perturb sphingolipid homeostasis. However, the downstream signalling events linking IPCS inhibition to AgN23 enrichment in the rhizosphere remain unclear. Here, we show that AgN23 activates ethylene- and salicylic acid-dependent immune signalling, leading to coordinated stimulation of phenylalanine- and tryptophan-derived secondary metabolism. Using Arabidopsis mutants defective in these pathways, we show that these metabolites mitigate AgN23-induced root growth inhibition. We further show that the npr1 mutant is strongly compromised in AgN23-triggered secondary metabolic responses, resulting in reduced rhizosphere colonization by AgN23. By comparing rhizosphere microbiota from wild-type and npr1 plants, we distinguished direct AgN23 effects linked to intermicrobial competition from indirect effects mediated by host metabolic activation. In particular, AgN23 colonization occurred at the expense of several Streptomycetaceae ASVs and coincided with changes in bacterial and fungal taxa belonging to Flavobacteriaceae and Mucoromycota. Together, these findings define a mechanistic framework in which Streptomyces AgN23 interacts with NPR1-dependent signalling to reprogram root metabolism and rhizosphere community structure, notably through the production of specialized metabolites such as galbonolides.

Microbial colonization is essential for intestinal maturation, yet how spatial organization of the microbiome shapes host tissue function remains unresolved. Here, we applied Stereo-seq V2 spatial platform to simultaneously profile the host transcriptome at single- cell resolution and microbial meta-transcriptome at 5 x 5 m resolution across the proximal, middle, and distal colon of germ-free (GF) mice and mice reconstituted by fecal microbiota transplantation (FMT), integrated with time-course fecal metagenomics. We observed that, four weeks after FMT, microbial colonization established a mature colonic architecture, increased goblet cell number and mucus layer thickness, diversified epithelial lineages, and expanded stem/transit-amplifying, myeloid, and T-cell populations. Metagenomic profiling showed succession from early colonizers to a metabolically mature, short-chain fatty acid (SCFA)-producing community that stabilized by four weeks. Distance-resolved spatial analysis resolved two reproducible strata of colonized microbiota along the radial host-lumen axis, separated at approximately 150 m. The epithelium-proximal stratum was enriched for mucus-associated taxa such as Bacteroides thetaiotaomicron, whereas the luminal stratum harbored fiber-associated taxa such as Ruminococcus champanellensis. This radial organization was underpinned by co-occurrence networks of spatial co-localization and co-exclusion. Finally, we identified a butyrate-producing guild that preferentially colonized the epithelium-proximal stratum, localized closer to epithelial and stromal cells, and showed active butyrate- responsive transcriptional activity. Colonization therefore establishes a spatially integrated host-microbiome interface with quantifiable, stratified microbial niches in which location, and not composition alone, coordinates epithelial and immune maturation.