Cell Host & Microbe

The mechanisms by which maturation of the infant gut microbiota promotes resistance to pathogen colonization remain poorly understood. Infant botulism, a potentially fatal disease caused by intestinal colonization by Clostridium botulinum, provides a striking example of age-dependent susceptibility to infection1. Although the gut microbiota has long been implicated in protection against C. botulinum2-6, the responsible bacterial species and underlying mechanisms have yet to be elucidated. Here, we show that acquisition of Stickland-metabolizing Clostridia during infant gut microbiota maturation confers resistance to C. botulinum colonization through competition for shared amino-acid-dependent nutritional niches. In human fecal microbiota-transplanted mice, longitudinally collected infant microbiotas exhibited a clear transition from susceptibility to resistance. Intestinal metabolomic analysis identified 5-aminovalerate as a hallmark metabolite of the resistant microbiota, implicating Stickland metabolism, an amino acid metabolic pathway also utilized by C. botulinum. Guided by this finding, metagenomic analysis revealed enrichment of Stickland-metabolizing Clostridia in resistant microbiotas, including Clostridioides difficile, a bacterium frequently carried by healthy infants. Intestinal metabolic signatures of C. botulinum and infant-derived Stickland-metabolizing Clostridia suggested competition for shared amino-acid-dependent nutritional niches. Consistent with this model, C. difficile suppressed C. botulinum expansion through nutrient competition. Together, these findings identify nutritional niche competition as a mechanism by which microbiota maturation promotes resistance to C. botulinum colonization. This work demonstrates how acquisition of specific microbial metabolic functions during early life can shift the gut microbiota from a susceptible to a resistant state.

Cholera causes severe diarrheal illness in young children, but the mechanisms underlying age-dependent susceptibility remain unclear. Experimental cholera in neonatal mice recapitulates age-dependent susceptibility: suckling mice are susceptible to Vibrio cholerae colonization and cholera toxin (CT)-dependent disease, whereas adult mice are not readily colonized and do not develop cholera-like disease. Here, we define a developmental window in which susceptibility declines sharply over the first two postnatal weeks. Maternal antibiotic exposure disrupted vertical transmission of maternal microbiota to offspring and altered distal small intestinal microbiota assembly, extending the window of susceptibility to CT-dependent V. cholerae colonization and disease. Pups born to antibiotic-treated dams exhibited reduced Lactobacillaceae and increased Enterobacteriaceae, and reintroduction of an endogenous Lactobacillus isolate restored offspring lactobacilli levels and reestablished resistance to experimental cholera at two weeks of age. Consistent with a direct protective role, increasing lactobacilli in susceptible neonatal mice reduced experimental cholera burden, and cultures of the endogenous Lactobacillus isolate as well as spent culture media acidified the in vitro growth environment and rapidly eliminated recoverable V. cholerae. Together, these findings identify vertical transmission of maternal microbiota to offspring and lactobacilli-associated antagonism as determinants of early-life resistance to cholera.

Intra-species genomic variation results from diversity-generating processes and supplies the raw material for subsequent natural selection. Environmental stress can be regarded as the ultimate accelerator of these processes, especially for microorganisms, which can alter their DNA if presented with nutrient limitation, toxins, or pathogen attack. Chronic intestinal inflammation, as in inflammatory bowel diseases (IBD), may be regarded as prolonged environmental stress for gut commensal bacteria, bringing a large number of enteric species down to undetectable levels. However, it remains unclear how the microbes that survive the IBD gut environment actually respond to IBD stress, and whether their stress response may leave a transient or permanent signature in their genomes. To investigate whether IBD stress induces and selects for certain genetic diversity, we performed metagenomic analyses on gut species in IBD patients and Controls. We focused on strain diversity within a single individual, which might be the result of more recent diversification processes under stress. We found measurable differences at the genome level between IBD and Controls, yet this was species-dependent. We then investigated gene-level diversity and found that certain functions were more likely to be enriched with either neutral divergence, functional divergence, or both. Functions that were enriched in IBD with both kinds of diversity were associated with motility and iron-scavenging, among others. These results may point towards functions that are under selection in the context of IBD stress, and could inform future mechanistic work, exploring previously unknown routes of bacterial diversification and adaptation to stress in the gut microbiome.

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

Perturbation to the early-life microbiota has long-term detrimental effects on health and development, leading to increased risk for metabolic dysfunction and childhood obesity. Despite the central role of the small intestine (SI) in energy balance, the impact of SI microbiota establishment on the regulation of host metabolism and early-life adiposity remains unclear. Here, we report that disruption of a critical SI microbiota-intestinal epithelial cell circuit, specifically during a critical early-life period, drives long-lasting obesity. We demonstrate that the SI microbiota expands in abundance and diversity significantly between 2 and 3 weeks of life, and that segmented filamentous bacteria (SFB) and Lactobacillus intestinalis establish residence. Disruption of the early-life SI microbiota with antibiotics leads to enhanced lipid uptake and adiposity, driven by increased peroxisome proliferator-activated receptor alpha (PPAR) expression and activity in SI epithelial cells (IECs). We demonstrate that SFB and L. intestinalis are key regulators of PPAR in SI IECs by increasing intestinal IL-22 levels specifically during weaning, which is necessary for inhibition of antibiotic-induced adiposity in a PPAR-dependent manner. Together, this work provides mechanistic insights into beneficial microbiota-induced epithelial-immune crosstalk in the SI that is specific to early life, a critical protective mechanism against excessive adiposity in infancy, and offers insight into how antibiotics during infancy may increase the risk of childhood obesity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/731695v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1b32f07org.highwire.dtl.DTLVardef@d4a5a6org.highwire.dtl.DTLVardef@c76d1borg.highwire.dtl.DTLVardef@cc479d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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 exposure disrupts enteric pathogen colonization resistance, yet how antibiotics reshape pathogen population dynamics, infection bottlenecks, and strain-level heterogeneity in the gut remains poorly understood. Here, we combine high-resolution pathogen barcoding, transcriptomic, and metabolomic analyses to quantify how short-term vancomycin perturbation alters infection ecology in vivo. We use Citrobacter rodentium as a model for human infection by pathogenic Escherichia coli-an antimicrobial resistance priority group-to demonstrate that just two days of vancomycin pre-treatment profoundly reshapes infection trajectories, driving rapid, global gut colonization, a dramatic increase in pathogen founding population size, and preservation of strain diversity across intestinal sites. Notably, vancomycin eliminated the hallmark heterogeneity of C. rodentium infection, resulting in fully reproducible colonization across hosts. Population-level analysis revealed that antibiotic treatment relaxes competitive constraints both with the resident microbiota and among clonal pathogen lineages, allowing early-established founders to persist and expand. Despite accelerated pathogen engraftment and tissue pathology, transcriptomic analysis revealed reduced virulence gene expression. Instead, antibiotic-induced metabolic restructuring of the gut created permissive conditions for pathogen expansion. Interactions with a vancomycin-altered microbiota, dominated by Akkermansia and Bacteroides, further promoted nutrient cross-feeding and influenced epithelial attachment. Together, we illustrate how short-term antibiotic exposure reshapes enteric infection by removing ecological bottlenecks that normally constrain strain diversity and infection outcomes. These findings have implications for antibiotic use, antimicrobial resistance transmission, and therapeutic strategies that rely on competition-driven dynamics, such as strain replacement.

Bacteriophage therapy is needed to treat antibiotic resistant infections; however, when a clinical isolate resists a given phage, it is often unclear why. It is therefore currently unknown how to rationally fortify phage therapies to circumvent a priori resistance. Using a family of broad host range therapeutic Pseudomonas aeruginosa phages (Pbunaviruses), we show that cell surface receptor masking and intracellular defenses are both common barriers in distinct clinical isolates. In some cases these barriers can be bypassed by intrafamily phage engineering. Using unbiased genome-wide CRISPRi screens, we reveal that the broadly conserved L-Rhamnose in the core polysaccharide is the receptor for Pbunavirus family. This molecule is often masked by diverse O-antigen structures. In other isolates with the L-Rha receptor accessible, internal defense mechanisms commonly prevent Pbunavirus DNA replication. A single anti-defense locus often encoding 8-11 different genes within the Pbunavirus family is required for optimal host range, providing anti-defense genes that enable replication of both Pbunavirus phages and phages of other families. Our work demonstrates the importance of both internal and surface defense mechanisms in clinical isolates causally antagonizing a commonly used phage therapeutic and presents phage engineering strategies to circumvent a priori resistance.

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.

Pneumonia is a leading cause of death globally and disproportionately affects children in lower- and middle-income countries. To explore microbial and immune correlates of disease and death, we performed metagenomic sequencing of upper respiratory tract (URT) microbiome in 309 children in Mali with pneumonia and 150 age- and season- and site-matched controls. We show that the URT microbiome matures throughout early life and is influenced by breastfeeding. URT microbiome maturation was disrupted during pneumonia resulting in loss of commensal species and expansion of pathobionts, which was linked to disease severity and death. Analysis of serum antibody levels revealed that low levels of passively acquired antibody from mothers, deficient antibody responses to RSV, and persistent autoantibody to cytokines were associated with pneumonia mortality in an age-dependent manner. These findings underscore the complex nature of pneumonia and identify microbial and immune factors for risk stratification and therapeutic interventions in pediatric pneumonia.

HLA-DQ2/8 haplotypes are established genetic risk factors for autoimmune diseases and are known to influence gut microbiota assembly in early life. However, their impact on the adult microbiome and functional consequences for host physiology remain unclear. Here, we performed a genotype-stratified multi-omics analysis of 60 healthy adults, including 28 HLA-DQ2/8 carriers and 32 non-carriers. We found that host HLA-DQ2/8 genotype was significantly associated with gut microbiome composition, with an effect size exceeding that of sex and BMI. HLA-DQ2/8 carriers exhibited higher gut microbial alpha-diversity, lower virulence factor abundance, and a distinct species profile enriched in butyrate-producing taxa. We identified pervasive intra-species phylogenetic and functional divergence linked to the DQ2/8 genotype. This diversification reflects predicted HLA-restricted microbial peptide-binding specificities, suggesting a possible role for antigen presentation-mediated immune selection, a mechanism further supported by AlphaFold3 structural modeling. We found an enrichment of microbial pathways for pantothenate and coenzyme A (CoA) biosynthesis in carriers, primarily driven by functionally divergent Blautia obeum strains. This functional shift paralleled lower levels of serum pantothenate and HDL-cholesterol in the host. Our findings suggest a potential genotype-microbiome-host axis where antigen presentation-mediated immune selection may modulate microbial adaptation, with possible implications for the host availability of essential cofactor precursors and lipid metabolism.

Molecular hydrogen (H2) and hydrogen sulfide (H2S) are central gut metabolites that shape microbial metabolism and affect host health. In Crohns disease (CD), the shift in microbiota composition ( dysbiosis) is associated with intestinal accumulation of these gases, but the responsible microbes remain poorly resolved. Here, we analysed 4,644 bacterial and archaeal species-level genomes from the Unified Human Gastrointestinal Genome Collection to identify H2-cycling microbes, assessed their prevalence in ca. 1,700 stool metagenomes from healthy and diseased individuals, and validated their activity using culture-based incubations of stool isolates and biopsy samples. Approximately half of all species encoded H2-producing abilities, with acetate- and propionate-forming fermenters such as Phocaeicola and Bacteroides dominating healthy cohorts, whereas comparatively few taxa, including Escherichia and Megamonas, encoded H2 consuming abilities. In CD, H2 producers became more abundant but less diverse, favouring species with multiple H2-evolving hydrogenases and more fermentation routes, especially Clostridium and Enterocloster species. Consistently, isolates enriched in CD produced H2 faster and at higher concentrations than health-associated isolates. Increased H2S-producing capacity in CD was driven mainly by these H2-producing fermenters carrying anaerobic sulfite reductases (Asr), rather than sulfate-reducing bacteria, and was supported by elevated H2S production in Asr-positive isolates, likely providing an additional electron sink. These findings provide a species-resolved view of gut gas metabolism and implicate metabolically flexible fermenters in excessive gas and sulfide production in gut disorders.

Many commensal bacteria that peacefully reside in the human microbiome are also able to cause acute opportunistic infections. Emerging evidence suggests that within-host evolution contributes to infection, but the genetic mechanisms facilitating the progression of opportunistic pathogens from carriage to acute infection remain unknown. Here, we prospectively collected native samples from four microbiome niches of 13 critically ill patients to assess the evolutionary dynamics leading up to infection. Among three patients we have observed eleven healthcare-associated infections (HAI) caused by nine pathogen species. Leveraging a culture-based approach, we demonstrate that the microbiome is frequently (73%) colonized by the pathogen lineage already before or at the time of diagnosis. Moreover, we identify a short-lived, non-synonymous mutation (F126L) within the fimbriae regulator gene fimZ of Enterobacter hormaechei, first detectable within the gut and subsequently associated with HAI before becoming replaced by body-wide sweeps of independent treatment-associated mutations. Despite fimZ [F126L] being globally undetected, we can show in vitro and in vivo that the F126L mutation leads to elevated biofilm formation, cell adhesion and virulence, suggesting a role during HAI. Our work highlights the power of prospective, population-wide investigation of pathogens to elucidate rapid evolution linked to disease.

The IL-23/Th17 axis is a central driver of intestinal and spondyloarthritic inflammation, yet upstream regulatory mechanisms linking microbial signals to IL-23 production remain incompletely defined. NF-{kappa}B signalling, particularly via the c-Rel subunit, is a critical transcriptional regulator of IL23A (p19), positioning c-Rel as a nodal checkpoint in mucosal inflammation. Here, we demonstrate that cell-free supernatant derived from Enterococcus faecalis AHG0090 (AHG0090-CS) suppresses c-Rel-dependent IL-23 signalling and attenuates inflammatory pathology across murine models of gut and joint disease. In the ZAP-70 mutant SKG model of spondyloarthritis and ileitis, AHG0090-CS significantly reduced weight loss, joint scores, and histological gut inflammation following curdlan challenge. In the Winnie model of spontaneous colitis, treatment similarly diminished inflammatory cytokine production. Mechanistically, AHG0090-CS reduced IL-23p19 mRNA and protein expression in intestinal tissue and lamina propria myeloid cells, accompanied by decreased nuclear c-Rel intensity. Suppression extended to downstream IL-23-associated cytokines including IL-17A, GM- CSF, MCP-1 and IL-6. In human peripheral blood mononuclear cells and macrophages, AHG0090-CS attenuated LPS-induced IL-23 and pro-inflammatory cytokine production, supporting translational relevance. Collectively, these findings identify microbial modulation of c-Rel-dependent IL-23 signalling as a tractable mechanism to restrain gut-joint inflammation and highlight targeting upstream NF-{kappa}B pathways as a therapeutic strategy in IL-23-driven immune-mediated disease.

The lung mucosal barrier thwarts many inhaled pathogens, including Coccidioides, the causative agent of Valley Fever. Because the earliest stages of pathogen recognition in the lung remain obscure, we investigated the initial events of barrier immunity in a murine model involving inhalation of Coccidioides sp. arthroconidia. Neutrophils accumulated rapidly, within 24 hours, near fungal spores in the bronchiolar airways. This response was driven by pattern recognition via the lectin complement pathway. Bronchiolar club cells propagated C3a signals and amplified the response via convergent C3aR and P2X7 signaling. We identified several MBL2 and P2RX7 polymorphisms that correlated with progressive disease in humans. Our assays revealed that these mutations caused functional impairments in C3a generation and P2X7 responsiveness. Our findings establish how complement signaling and epithelial sensing coordinate early immune responses to fungal infection, offering insights into essential host defense mechanisms and risk factors for disease progression in coccidioidomycosis.

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.

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.

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.