Cell Host & Microbe

Bacteria inhabiting the mammalian gut coexist in dense communities where contact-dependent antagonism mechanisms are widespread. The type VI secretion system (T6SS) is an interbacterial toxin delivery pathway prevalent among gut Bacteroidales, yet its function in naturally evolved microbiomes remains poorly defined. Here, we examine the physiological role of the T6SS in Bacteroides within a gut community derived from wild mice (the WildR microbiome). Using newly developed genetic tools and a strategy for functional replacement of strains within the WildR community, we demonstrate that the WildR isolate B. acidifaciens employs a T6SS to antagonize co-resident Bacteroidales. We also show that loss of T6SS function compromises the long-term maintenance of B. acidifaciens in the community but not its initial colonization, establishing the system as a determinant of strain persistence. The T6SS we identified resides on an integrative and conjugative element (ICE). ICE-seq, a targeted sequencing approach, reveals that the T6SS-ICE is distributed among select Bacteroidales and Muribaculaceae species in the WildR microbiome, between which it appears to be recently exchanged. We also show that transfer of the T6SS-ICE to WildR isolate Phocaeicola vulgatus confers transient colonization benefits in mice, but an eventual fitness cost. Our findings demonstrate that the T6SS can stabilize the presence of specific strains within a complex, co-evolved gut microbiome, yet its value is context dependent and constrained by the ecological and physiological landscape of the host community.

Inflammation boosts the availability of electron acceptors in the intestinal lumen creating a favorable niche for pathogenic Enterobacteriaceae. However, the mechanisms linking intestinal inflammation-mediated changes in luminal metabolites and pathogen expansion remain unclear. Here, we show that mucosal inflammation induced by Salmonella enterica serovar Typhimurium (S. Tm) infection and chemical colitis results in increased intestinal levels of the amino acid aspartate. The S. Tm and E. coli genomes encode an aspartate ammonia-lyase (aspA) which converts aspartate into fumarate, an alternative electron acceptor. S. Tm and pathogenic E. coli used aspA-dependent fumarate respiration for growth in the murine gut only during inflammation. Such growth advantage was abolished in the gut of germ-free mice. However, mono-association of gnotobiotic mice with members of the classes Bacteroidia and Clostridia restored the benefit of aspartate utilization to the pathogens. Our findings demonstrate the role of microbiota-derived amino acids in driving respiration-dependent Enterobacteriaceae expansion during colitis.

Candida albicans is a ubiquitous fungus in the human gut microbiome as well as a prevalent cause of opportunistic mucosal and systemic disease. There is currently little understanding, however, as to how crosstalk between C. albicans and the host regulates colonization of this key niche. Here, we performed expression profiling on ileal and colonic tissues in germ-free mice colonized with C. albicans to define the global response to this fungus. We reveal that Duox2 and Duoxa2, encoding dual NADPH oxidase activity, are upregulated in both the ileum and colon, and that induction requires the C. albicans yeast-hyphal transition and the hyphal-specific toxin candidalysin. Hosts lacking the IL-17 receptor failed to upregulate Duox2/Duoxa2 in response to C. albicans, while addition of IL-17A to colonoids induced these genes together with the concomitant production of hydrogen peroxide. To directly define the role of Duox2/Duoxa2 in fungal colonization, antibiotic-treated mice lacking intestinal DUOX2 activity were evaluated for C. albicans colonization and host responses. Surprisingly, loss of DUOX2 function reduced fungal colonization at extended time points (>17 days colonization) and increased the proportion of hyphal cells in the gut. IL-17A levels were also elevated in C. albicans-colonized mice lacking functional DUOX2 highlighting cross-regulation between this cytokine and DUOX2. Together, these experiments reveal novel links between fungal cells, candidalysin toxin and the host IL-17-DUOX2 axis, and that a complex interplay between these factors regulates C. albicans filamentation and colonization in the gut.

Enteric pathogens engage in complex interactions with the host and the resident microbiota to establish gut colonization. Although mechanistic interactions between enteric pathogens and bacterial commensals have been extensively studied, whether and how commensal fungi affect pathogenesis of enteric infections remains largely unknown. Here we show that colonization with the common human gut commensal fungus Candida albicans worsened infections with the enteric pathogen Salmonella enterica serovar Typhimurium. Presence of C. albicans in the mouse gut increased Salmonella cecum colonization and systemic dissemination. We investigated the underlying mechanism and found that Salmonella binds to C. albicans via Type 1 fimbriae and uses its Type 3 Secretion System (T3SS) to deliver effector proteins into C. albicans. A specific effector, SopB, was sufficient to manipulate C. albicans metabolism, triggering increased arginine biosynthesis in C. albicans and the release of millimolar amounts of arginine into the extracellular environment. The released arginine, in turn, induced T3SS expression in Salmonella, increasing its invasion of epithelial cells. C. albicans deficient in arginine production was unable to increase Salmonella virulence in vitro or in vivo. In addition to modulating pathogen invasion, arginine also directly influenced the host response to infection. Arginine-producing C. albicans dampened the inflammatory response during Salmonella infection, whereas C. albicans deficient in arginine production did not. Arginine supplementation in the absence of C. albicans increased the systemic spread of Salmonella and decreased the inflammatory response, phenocopying the presence of C. albicans. In summary, we identified C. albicans colonization as a susceptibility factor for disseminated Salmonella infection, and arginine as a central metabolite in the cross-kingdom interaction between fungi, bacteria, and host.

How bacterial strains within a complex human microbiota collectively shape intestinal T cell homeostasis is not well-understood. Methods that quickly identify effector strains or species that drive specific mucosal T cell phenotypes are needed to define general principles for how the microbiota modulates host immunity. We colonize germ-free mice with defined communities of cultured strains and profile antigen-specific responses directed towards individual strains ex vivo. We find that lamina propria T cells are specific to bacterial strains at the species level and can discriminate between strains of the same species. Ex vivo restimulations consistently identify the strains within complex communities that induce Th17 responses in vivo providing the potential to shape baseline immune tone via community composition. Using an adoptive transfer model of colitis, we find that lamina propria T cells respond to different bacterial strains in conditions of inflammation versus homeostasis. Collectively, our approach represents a novel method for efficiently predicting the relative impact of individual bacterial strains within a complex community and for parsing microbiota-dependent phenotypes into component fractions. SIGNIFICANCEDetermining the mechanisms by which the gut microbiome modulates the host immune system has translational potential for treating or preventing immune mediated disease. A key challenge is identifying the immunogenic bacterial strains in the setting of a complex microbiota. We use a combination of anaerobic culturing, in vitro T cell assays, gnotobiotic mouse models, and ex vivo T cell restimulations to explore the influence of species and strain diversity on the specificity of mucosal T cells. Our approach efficiently predicts the relative impact of individual bacterial strains within a complex community and can be used to parse microbiota-dependent phenotypes into component fractions.

The carbohydrates that fuel gut colonization by S. Typhimurium are not fully known. To investigate this, we designed a quality-controlled mutant pool to probe the metabolic capabilities of this enteric pathogen. Using WISH-barcoding, we tested 35 metabolic mutants across five different mouse models, allowing us to differentiate between context-dependent and context-independent nutrient sources. Results showed that S. Typhimurium uses D-glucose, D-mannose, D-fructose, and D-galactose as context-independent carbohydrates across all models. The utilization of N-acetylglucosamine and hexuronates, on the other hand, was context-dependent. Furthermore, we showed that D-fructose is important in strain-to-strain competition between Salmonella serovars. Complementary experiments confirmed that D-glucose, D-fructose, and D-galactose are excellent niches for S. Typhimurium to exploit during colonization. Quantitative measurements revealed sufficient amounts of D-glucose and D-galactose in the murine cecum to drive S. Typhimurium colonization. Understanding these key substrates and their context-dependent use by enteric pathogens will inform the future design of probiotics and therapeutics to prevent diarrheal infections such as non-typhoidal salmonellosis.

In addition to individual genetics, environmental factors (e.g., dietary changes) may influence host susceptibility to gastrointestinal infection through unknown mechanisms. Herein, we developed a model in which CBA/J mice, a genetically resistant strain that tolerates intestinal colonization by the enteric pathogen Salmonella Typhimurium (S. Tm), rapidly succumb to infection after exposure to a diet rich in L-amino acids (AA). In mice, S. Tm-gastroenteritis is restricted to the large intestine (cecum), limiting their use to understand S. Tm small intestine (ileum) colonization, a feature of human Salmonellosis. Surprisingly, CBA mice fed AA diet developed ileitis with enhanced S. Tm ileal colonization. Using germ-free mice and ileal-fecal slurry transplant, we found diet-mediated S. Tm ileal expansion to be microbiota-dependent. Mechanistically, S. Tm relied on Fructosyl-asparagine utilization to expand in the ileum during infection. We demonstrate how AA diet overrides host genetics by altering the gut microbiotas ability to prevent S. Tm ileal colonization.

DNA viruses and retroviruses need to consume large quantities of deoxynucleotides (dNTPs) when replicating within infected cells. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral life cycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. In this study we report that bacteria employ a similar strategy to defend against phage infection. We found a family of defensive dCTP deaminase proteins that, in response to phage infection, convert dCTP into deoxy-uracil nucleotides. A second family of phage resistance genes encode dGTPase enzymes, which degrade dGTP into phosphate-free deoxy-guanosine (dG) and are distant homologs of the human SAMHD1. Our results show that the defensive proteins completely eliminate the specific deoxynucleotide (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Both defensive genes are found in a diverse set of bacterial species and are specifically enriched in Vibrio genomes. Our study demonstrates that manipulation of the deoxynucleotide pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

Bacteriophages (phages) are abundant members of the gut microbiota and regulators of bacterial communities. During homeostasis, gut phage communities are longitudinally stable and lysogenic replication is dominant. In chronic gut inflammatory disorders, such as inflammatory bowel diseases (IBDs), there are alterations in phage diversity, which may result from changes in phage replication cycle dynamics. Here, we used a combination of in vitro, simplified community, and whole-community bioinformatics approaches to investigate whether prophage induction contributes to these alterations. We identified several compounds associated with gut inflammation that induced prophages in commensal gut bacterial isolates. Analysing data from two mouse models of colitis, we observed that shifts in the composition of temperate phages occur over the course of inflammation, supporting a switch from lysogenic to lytic replication. Collectively, our observations support the idea that prophage induction contributes to alterations in the phageome associated with inflammation.

In order to investigate SARS-CoV-2 mutations and their impact on immune evasion and infectivity, we developed a Deep Mutational Scanning (DMS) platform utilizing an inverted infection assay to measure spike expression, ACE2 affinity, and viral infectivity in human cells. Surprisingly, our analysis reveals that spike protein expression, rather than ACE2 affinity, is the primary factor affecting viral infectivity and correlated with SARS-CoV-2 evolution. Notably, within the N-terminal domain (NTD), spike expression and infectivity-enhancing mutations are concentrated in flexible loops. We also observed that Omicron variants BA.1 and BA.2 exhibit immune evasion through receptor binding domain (RBD) mutations, although these mutations reduce structural stability. Interestingly, the NTD has evolved to increase stability, compensating for the RBD instability and resulting in heightened overall infectivity. Our findings, available in SpikeScanDB, emphasize the importance of spike expression levels and compensatory mutations in both the NTD and RBD domains for shaping Omicron variant infectivity.

Bacteria encode diverse mechanisms for mediating interbacterial antagonism through the exchange of toxic effector proteins. Although the structure, function, and regulation of these pathways has been well established for many organisms, an understanding of their ecological and evolutionary dynamics lags behind. Type VI secretion systems (T6SS) deliver effectors between competing Gram-negative bacteria, including among mammalian gut Bacteroidales, resulting in the evolution of elaborate defense mechanisms that protect against T6SS attack. One such mechanism is the recombinase-associated acquired interbacterial defence (rAID) system, which harbors arrays of orphan immunity genes that diverge in sequence from T6SS-associated cognate immunity genes. It is not known if such sequence divergence impacts rAID orphan immunity function, or how rAID distribution across microbiomes relates to the T6SS. Here, we show that divergent rAID orphan immunity factors that possess SUKH domains allow bacteria to survive intoxication by cognate effectors. Such protection is due to high affinity protein-protein interactions between orphan immunity and effector that are comparable to that of cognate effector-immunity. Unlike other examples of T6SS effector-immunity interactions, we find that the binding interface is comprised of electrostatic interactions with a high degree of redundancy underlying its protective capacity. Finally, we quantify orphan immunity and effector gene abundance and dynamics across human gut metagenomes, revealing patterns of co-occurrence indicative of positive selection. Population genetic analyses of longitudinal data suggests that orphan immunity genes accumulate non-synonymous mutations that lie at the predicted effector-immunity interface. Together, our findings establish rAID orphan immunity genes as important bacterial fitness determinants in the human gut.

The functional potential of the gut microbiota remains largely uncharacterized. Efforts to understand how the immune system responds to commensal organisms have been hindered by the large number of strains that comprise the human gut microbiota. We develop a screening platform to measure innate immune responses towards 277 bacterial strains isolated from the human gut microbiota. We find that innate immune responses to gut derived bacteria are as strong as responses towards pathogenic bacteria, and vary from phylum to strain. Myeloid cells differentially rely upon TLR2 or TLR4 to sense particular taxa, an observation that predicts in vivo function. These innate immune responses can be modeled using combinations of up to 8 TLR agonists. Furthermore, the immunogenicity of strains is stable over time and following transplantation into new humans. Collectively, we demonstrate a powerful high-throughput approach to determine how commensal microorganisms shape innate immune phenotypes.

Adherent-invasive Escherichia coli (AIEC) exhibit proinflammatory properties and have been implicated in the pathogenesis of Crohns disease (CD), a form of inflammatory bowel disease (IBD). Antibiotic use in CD lacks specificity and may worsen microbiome disruption, prompting interest in bacteriophages (phages) for targeted microbiome editing. Here, we identified HER259, a phage active against the clinical AIEC strain NRG857c. Using gnotobiotic models of AIEC-driven colitis, we show that HER259 attenuates AIEC virulence, including suppression of the FimH adhesin through inversion of the fimS promoter to its off orientation. Withdrawal of HER259 treatment leads to reversion of the fimS promoter and reactivated colitis in mice. HER259 phage also enhances the therapeutic effect of sub-therapeutic budesonide, independent of microbial drug metabolism. These findings support targeted phage therapy as an adjunct treatment approach in IBD, demonstrating modulation of bacterial virulence and improved response to conventional treatments which may reduce drug-related side effects. One Sentence SummaryBacteriophage HER259 improves colitis severity mediated by Crohns disease Escherichia coli NRG857c, and increases efficacy of budesonide.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

Commensal skin bacteria elicit potent, antigen-specific immune responses in the skin without barrier breach or visible inflammation. While microbial modulation of immune homeostasis has profound consequences for epithelial health and inflammatory skin diseases, the mechanisms of microbe-immune crosstalk in the skin are largely unknown. A key barrier to mechanistic work has been genetic intractability of one of the most prevalent skin colonists, Staphylococcus epidermidis (S. epidermidis). Here, we develop a novel method to create a library of mutants with defined cell envelope alterations in primary human S. epidermidis isolates. By colonizing mice with these mutants, we uncover bacterial molecules involved in the induction of defined immune signatures. Notably, we show that under conditions of physiologic colonization, S. epidermidis cell envelope glycolipids are sensed by C-type lectin receptors, likely in non-myeloid cells, in conjunction with Toll-like receptors. This combinatorial signaling determines the quality of T cell responses and results in the potential for greater specificity toward commensal microbiota than previously appreciated. Additionally, the microbial molecules required for the colonization-induced immune response are dispensable for T cells responses in a model of S. epidermidis infection, but differentially modulate innate inflammatory responses. Thus, the same microbe uses distinct sets of molecules to signal to the immune system commensal versus pathogenic behavior, and differential sensing of these microbial signals depends on host context.

The gut microbiota plays a critical role in constraining Candida albicans (Ca) colonization of the gastrointestinal (GI) tract, a key precursor to disseminated fungal infection in immunocompromised hosts. Depletion of commensal microbiota increases Ca burden and promotes dissemination, yet the mechanisms of microbiota-mediated Ca colonization resistance remain poorly defined. Here, we show that gut microbiota-derived short-chain fatty acids (SCFAs) directly inhibit Ca growth by impairing hexose uptake, disrupting central carbon metabolism, and inducing intracellular acidification. In vivo, SCFAs enhance Ca colonization resistance only in the presence of an intact gut microbiome, which is required to drive SCFA-induced taxonomic shifts that further augment resistance. Commensal microbiota lacking SCFA production exhibit diminished capacity to restrict Ca colonization, while prebiotic therapy that increases luminal SCFA levels enhances Ca clearance. These findings define a critical microbiota-metabolite mechanism underlying Ca colonization resistance and suggest strategies to modulate GI fungal burden and prevent invasive disease.

Bacteria and archaea typically have multiple defence systems that protect them against viral predation. Recently, many new defence systems have been discovered, yet the full scope of the prokaryotic pan-immune system remains to be determined. In this study, we observed that many multi-gene defence systems have additional genes nested or embedded within them. Based on this observation, we present a new approach to predict new defence systems, where defence function of uncharacterised genes is inferred based on their genetic embedding in known defence systems. Applying this guilt-by-embedding method, we identified and confirmed anti-phage function for seven defence systems and predicted 145 additional candidates. Our findings expand the known immune repertoire of prokaryotes, provide a wealth of new systems for future functional studies, and demonstrate a simple, efficient approach to identify new antiviral defences.

Fecal microbiota transplantation (FMT) has shown immunotherapeutic promise across multiple malignancies, yet its clinical efficacy in non-small-cell lung cancer (NSCLC) remains unexplored. Here, we report results from a single-arm trial demonstrating that FMT significantly enhances anti-PD-1 efficacy and prolongs progression-free survival in patients with advanced PD-L1-negative NSCLC. To understand the mechanistic basis of variable FMT outcomes, we developed a high-resolution strain-tracking framework and analyzed over 2,000 samples from FMT and longitudinal studies across NSCLC, melanoma, inflammatory bowel syndrome, recurrent Clostridioides difficile infection, type 2 diabetes, and healthy individuals. Our analysis reveals that genetically distinct strains within the same bacterial species exert opposing therapeutic effects, explaining contradictory findings in previous reports. We discovered universal ecological principles governing strain persistence and engraftment that transcend disease contexts: engraftment success correlates with species-intrinsic fitness traits encoded in core metabolic and immune evasion pathways. Phylogenetic analysis revealed that key species segregate into functionally distinct clades with divergent clinical associations. Longitudinal tracking demonstrated that successful colonization by beneficial strain variants strongly associates with positive clinical outcomes. By integrating colonization dynamics with functional genomics, we identified 39 priority species exhibiting robust engraftment potential and strain-specific therapeutic effects as candidates for precision microbiome therapeutics. These findings establish a strain-function-efficacy paradigm that resolves inconsistent clinical outcomes in microbiome interventions and provides a framework for next-generation therapeutic development.

The microbiota can promote host health by inhibiting pathogen colonization, yet how host-resident fungi, or the mycobiota, contribute to this process remains unclear. The human skin mycobiota is uniquely stable compared to other body sites and dominated by yeasts of the genus Malassezia. We observe that colonization of human skin by Malassezia sympodialis significantly reduces subsequent colonization by the prominent bacterial pathogen Staphylococcus aureus. M. sympodialis secreted products possess potent bactericidal activity against S. aureus and are sufficient to impair S. aureus skin colonization. This bactericidal activity requires an acidic environment and is exacerbated by free fatty acids, demonstrating a unique synergy with host-derived epidermal defenses. Leveraging experimental evolution to pinpoint mechanisms of S. aureus adaptation in response to the skin mycobiota, we identified multiple mutations in the stringent response regulator Rel that promote survival against M. sympodialis. Similar Rel alleles have been reported in S. aureus clinical isolates, and natural Rel variants are sufficient for tolerance to M. sympodialis antagonism. Partial stringent response activation underlies tolerance to clinical antibiotics, with both laboratory-evolved and natural Rel variants conferring multidrug tolerance. These findings demonstrate the ability of the mycobiota to mediate pathogen colonization resistance, identify new mechanisms of bacterial adaptation in response to fungal antagonism, and reveal the potential for microbiota-driven evolution to shape pathogen antibiotic susceptibility. Highlights- M. sympodialis reduces colonization of human skin by S. aureus - Bactericidal activity of M. sympodialis is exacerbated by features of the skin niche - S. aureus Rel variants are sufficient for tolerance to Malassezia antagonism - Evolved tolerance to yeast antagonism coincides with S. aureus multidrug tolerance

Gastrointestinal (GI) colonization by Klebsiella pneumoniae is a risk factor for subsequent infection as well as transmission to other patients. Additionally, colonization is achieved by many strain types that exhibit high diversity in genetic content. Thus, we aimed to study strain-specific requirements for K. pneumoniae GI colonization by applying transposon insertion sequencing to three classical clinical strains: a carbapenem-resistant strain, an extended-spectrum beta-lactamase producing strain, and a non-epidemic antibiotic-susceptible strain. The transposon insertion libraries were screened in a murine model of GI colonization. At three days post-inoculation, 27 genes were required by all three strains for colonization. Isogenic deletion mutants for three genes/operons (acrA, carAB, tatABCD) confirmed colonization defects in each of the three strains. Additionally, deletion of acrA reduced bile tolerance in vitro, while complementation restored both bile tolerance in vitro and colonization ability in vivo. Transposon insertion sequencing suggested that some genes were more important for colonization of one strain than the others. For example, deletion of the sucrose porin-encoding gene scrY resulted in a colonization defect in the carbapenemase-producing strain but not in the extended-spectrum beta-lactamase producer or the antibiotic-susceptible strain. These findings demonstrate that classical K. pneumoniae strains use both shared and strain-specific strategies to colonize the mouse GI tract. IMPORTANCEKlebsiella pneumoniae is a common cause of difficult-to-treat infections due to its propensity to express resistance to many antibiotics. For example, carbapenem-resistant K. pneumoniae (CR-Kp) has been named an urgent threat by the United States Centers for Disease Control and Prevention. Gastrointestinal colonization of patients with K. pneumoniae has been linked to subsequent infection, making it a key process to control in prevention of multidrug-resistant infections. However, the bacterial factors which contribute to K. pneumoniae colonization are not well understood. Additionally, individual strains exhibit large amounts of genetic diversity, begging the question of whether some colonization factors are strain-dependent. This study identifies the enteric colonization factors of 3 classical strains using transposon mutant screens to define a core colonization program for K. pneumoniae as well as detecting strain-to-strain differences in colonization strategies.