The gut microbiome profoundly influences host health, with disrupted microbial communities implicated in inflammatory, metabolic, and neurological diseases. The aryl hydrocarbon receptor (AHR) shapes intestinal immunity and microbial composition, yet the mechanisms by which AHR mediates selective microbiome assembly remain poorly understood. Whether distant organ systems such as the nervous system actively participate in microbial community regulation is unexplored. Here we show that Caenorhabditis elegans neuronal AHR-1 orchestrates selective gut microbiome assembly through a neuroendocrine cascade that calibrates intestinal redox tone. Using a defined natural microbiome community, we demonstrate that ahr-1 loss triggers specific microbiome changes, as Enterobacterales and Pseudomonadales bloom up to 24-fold while Sphingobacteriales decline. Adult-specific AHR-1 depletion recapitulates this dysbiosis, separating the role of AHR-1 in neurodevelopment from its regulation of the microbiome. Neuronal AHR-1 regulates expression of the neuropeptide FLP-8 in oxygen-sensing URX neurons, which in turn signals to the intestine to modulate intestinal redox tone. Elevated intestinal redox tone in ahr-1 mutants favors growth of oxidative stress-tolerant Enterobacterales and Pseudomonadales, which also exhibit functional and genomic enrichment for stress resistance and AHR ligand biosynthesis pathways. This establishes a precise feedback circuit whereby AHR-1 senses bacterial metabolic output to regulate colonization by ligand-producing taxa. AHR-1 signaling regulates redox tone through downstream regulation of glutathione S-transferase GST-4 and alpha-arrestin ARRD-11. Intestinal gst-4 knockdowns had the greatest impact on restoring redox tone and microbiome compositional control in the gut. These findings reveal an evolutionarily ancient molecular circuit where AHR serves as a precision metabolic sensor that coordinates whole organismal physiology to maintain host-microbiome homeostasis.

Unresolved tissue damage is a common feature of Inflammatory Bowel Disease (IBD) that facilitates disease progression. Here, we showed that high animal fat diets (HFD), an environmental risk factor associated with IBD pathogenesis, suppress intestinal macrophage production of critical tissue repair responses after damage. This includes reduced IL-23 production, which drives downstream production of IL-22, which is needed for barrier repair. Indicating that dietary lipids interfere with responses to microbial molecules needed to induce barrier protective functions, we found oleic acid could directly suppress macrophage Il23a induction after lipopolysaccharide (LPS) treatment. Deleting the lipid transporter CD36 on macrophages restored the Il23a and Il22 response, reducing intestinal damage in HFD-fed DSS-treated mice. We found that CD36-mediated intracellular lipid accumulation, mainly oleic acid, in macrophages leads to peroxisome proliferator-activated receptor delta (PPARδ) release of the transcriptional repressor protein B-cell lymphoma 6 (BCL6). BCL6 suppresses Il23a transcription in microbe-exposed macrophages. The studies suggest dietary lipid modulation of the macrophage PPARδ/BCL6 transcriptional repressor complex is a key mechanism of fat-associated defects in intestinal damage repair and immune dysregulation. Overall, our findings provide new insights into dietary lipid contribution to intestinal disease progression and identify new potential therapeutic targets to decrease diet-associated risk for IBD.

Bacterial nanocellulose (BNC) is a promising dietary fiber with potential as a functional food additive. We evaluated BNC fibers (BNCf) in the Caenorhabditis elegans model to obtain insight into the BNCf's biointeraction with its gastrointestinal tract while reducing the variables of higher complex animals. BNCf were uptaken and excreted by worms without crossing the intestinal barrier, confirming its biosafety regarding survival rate, reproduction, and aging for concentrations up to 34 μg/ml BNCf. However, a slight decrease in the worms' length was detected. A possible nutrient shortage or stress produced by BNCf was discarded by measuring stress and chemotactic response pathways. Besides, we detected a lipid-lowering effect of BNCf in N2 C. elegans in normal and high-caloric diets. Oxidative damage was computed in N2 worms and Rac1/ced-10 mutants. The GTPase Rac1 is involved in neurological diseases, where its dysregulation enhances ROS production and neuronal damage. BNCf reduced the lipid oxidative markers produced by ROS species in this worm strain. Finally, we detected that BNCf activated the genetic expression of the immunological response and lipid catabolic process. These results strengthen the use of BNCf as a functional dietary fiber and encourage the potential treatment of neurological disease by modulating diet.

A limited number of bacteria are able to colonize the nuclei of eukaryotes. 'Candidatus Endonucleobacter' infects the nuclei of deep-sea mussels, where it replicates to ≥80,000 bacteria per nucleus and causes nuclei to swell to 50 times their original size. How these parasites are able to replicate and avoid apoptosis is not known. Dual RNA-sequencing transcriptomes of infected nuclei isolated using laser-capture microdissection revealed that 'Candidatus Endonucleobacter' does not obtain most of its nutrition from nuclear DNA or RNA. Instead, 'Candidatus Endonucleobacter' upregulates genes for importing and digesting sugars, lipids, amino acids and possibly mucin from its host. It likely prevents apoptosis of host cells by upregulating 7-13 inhibitors of apoptosis, proteins not previously seen in bacteria. Comparative phylogenetic analyses revealed that 'Ca. Endonucleobacter' acquired inhibitors of apoptosis through horizontal gene transfer from their hosts. Horizontal gene transfer from eukaryotes to bacteria is assumed to be rare, but may be more common than currently recognized.

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring compositional changes in the microbiome is crucial in identifying the functional relationships between these physiological changes. Caenorhabditis elegans has emerged as a powerful host system to examine the molecular drivers of host-microbiome interactions. With its transparent body plan and fluorescent-tagged natural microbes, the relative levels of microbes within the gut microbiome of an individual C. elegans animal can be easily quantified using a large particle sorter. Here we describe the procedures for the experimental setup of a microbiome, collection, and analysis of C. elegans populations in the desired life stage, operation, and maintenance of the sorter, and statistical analyses of the resulting datasets. We also discuss considerations for optimizing sorter settings based on the microbes of interest, the development of effective gating strategies for C. elegans life stages, and how to utilize sorter capabilities to enrich animal populations based on gut microbiome composition. Examples of potential applications will be presented as part of the protocol, including the potential for scalability to high-throughput applications.

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring compositional changes in the microbiome is crucial in identifying the functional relationships between these physiological changes. Caenorhabditis elegans has emerged as a powerful host system to examine the molecular drivers of host-microbiome interactions. With its transparent body plan and fluorescent-tagged natural microbes, the relative levels of microbes within the gut microbiome of an individual C. elegans animal can be easily quantified using a large particle sorter. Here we describe the procedures for the experimental setup of a microbiome, collection, and analysis of C. elegans populations in the desired life stage, operation, and maintenance of the sorter, and statistical analyses of the resulting datasets. We also discuss considerations for optimizing sorter settings based on the microbes of interest, the development of effective gating strategies for C. elegans life stages, and how to utilize sorter capabilities to enrich animal populations based on gut microbiome composition. Examples of potential applications will be presented as part of the protocol, including the potential for scalability to high-throughput applications.

Endozoicomonadaceae bacteria are widespread in many marine animals, and generally considered beneficial. Members of one clade, however, Candidatus Endonucleobacter, infect the nuclei of deep-sea mussels, where they replicate to ≥ 80,000 bacteria per nucleus and cause the nuclei to swell to 50 times their original size. How these parasites are able to persist in host nuclei without the cell undergoing apoptosis is not known. We show here that Ca. Endonucleobacter encodes and expresses 7-15 inhibitors of apoptosis (IAPs), proteins previously only known from animals and viruses. Dual RNA-seq transcriptomes of infected nuclei revealed parallel upregulation of Ca. Endonucleobacter IAPs and host caspases, suggesting an arms race between the parasite and host for control of apoptosis. Comparative phylogenetic analyses revealed that Ca. Endonucleobacter acquired IAPs repeatedly through horizontal gene transfer from their hosts in convergent acquisition, possibly mediated by herpes viruses that may infect both the parasite and the host.

Chronic exposure to high-fat diets (HFD) worsens intestinal disease pathology, but acute effects of HFD in tissue damage remain unclear. Here, we used short-term HFD feeding in a model of intestinal injury and found sustained damage with increased cecal dead neutrophil accumulation, along with dietary lipid accumulation. Neutrophil depletion rescued enhanced pathology. Macrophages from HFD-treated mice showed reduced capacity to engulf dead neutrophils. Macrophage clearance of dead neutrophils activates critical barrier repair and antiinflammatory pathways, including IL-10, which was lost after acute HFD feeding and intestinal injury. IL-10 overexpression restored intestinal repair after HFD feeding and intestinal injury. Macrophage exposure to lipids from the HFD prevented tethering and uptake of apoptotic cells and Il10 induction. Milk fat globule-EGF factor 8 (MFGE8) is a bridging molecule that facilitates macrophage uptake of dead cells. MFGE8 also facilitates lipid uptake, and we demonstrate that dietary lipids interfere with MFGE8-mediated macrophage apoptotic neutrophil uptake and subsequent Il10 production. Our findings demonstrate that HFD promotes intestinal pathology by interfering with macrophage clearance of dead neutrophils, leading to unresolved tissue damage.

The gut microbiota is essential for maintenance and repair of the intestinal epithelial barrier. As shifts in both intestinal epithelial barrier function and microbiota composition are found in inflammatory bowel disease patients, it is critical to understand the role of distinct bacteria in regulating barrier repair. We identified a mouse commensal E. coli isolate, GDAR2-2, that protects mice from Citrobacter rodentium infection and dextran sulfate sodium-induced colitis. Colonization with GDAR2-2 in mice resulted in expansion of CX3CR1+ mononuclear phagocytes, including CX3CR1+ macrophages/dendritic cells and monocytes, along with IL-22-secreting type 3 innate lymphoid cells and improved epithelial barrier function. In vitro co-culture of macrophages with GDAR2-2 resulted in IL-1β production. In vivo, protection after GDAR2-2 colonization was lost after depletion of CX3CR1+ MNPs, or blockade of IL-1β or IL-22. We further identified human commensal E. coli isolates that similarly protect mice from C. rodentium infection through CX3CR1+ MNP and IL-1β production. Together, these findings demonstrate an unexpected role for commensal bacteria in promoting IL-1β secretion to support intestinal barrier repair.

Microbiota interactions with host tissue support proper intestinal functions. In diseases such as inflammatory bowel disease, microbial shift along with epithelial dysfunctions are found. Understanding how intestinal epithelial functions are regulated by distinct commensal bacteria is key to improving outcomes in such diseases. Here, we identified a mouse commensal E. coli isolate, GDAR2-2, that promotes intestinal barrier repair in mouse colitis models including Citrobacter rodentium infection and dextran sulfate sodium-induced colitis. In mice, protection after GDAR2-2 colonization depends on CX3CR1+ mononuclear phagocytes (MNPs), which induce expansion of IL-22-secreting type 3 innate lymphoid cells. We detect upregulation of markers of transit amplifying cells, proliferation of which is downstream of IL-22 signaling. Protection is lost if IL-22 is blocked, indicating IL-22 driven epithelial regeneration is downstream of GDAR2-2 colonization. By co-culturing bone marrow derived macrophages with live GDAR2-2, we found GDAR2-2 promotes IL-1b production and the in vivo protection was lost after blockade of IL-1b. We further identified a subset of human commensal E. coli isolates that similarly induce CX3CR1+ MNP expansion and IL-1b mediated protection from C. rodentium infection in mice. This study reveals an unexpected role for IL-1b that promotes intestinal barrier repair and is induced by select commensal bacteria. Supported by NIH (AI125264, P30DK056338)

Host genetic landscapes can shape microbiome assembly in the animal gut by contributing to the establishment of distinct physiological environments. However, the genetic determinants contributing to the stability and variation of these microbiome types remain largely undefined. Here, we use the free-living nematode Caenorhabditis elegans to identify natural genetic variation among wild strains of C. elegans that drives assembly of distinct microbiomes. To achieve this, we first established a diverse model microbiome that represents the strain-level phylogenetic diversity naturally encountered by C. elegans in the wild. Using this community, we show that C. elegans utilizes immune, xenobiotic, and metabolic signaling pathways to favor the assembly of different microbiome types. Variations in these pathways were associated with enrichment for specific commensals, including the Alphaproteobacteria Ochrobactrum. Using RNAi and mutant strains, we showed that host selection for Ochrobactrum is mediated specifically by host insulin signaling pathways. Ochrobactrum recruitment is blunted in the absence of DAF-2/IGFR and modulated by the competitive action of insulin signaling transcription factors DAF-16/FOXO and PQM-1/SALL2. Further, the ability of C. elegans to enrich for Ochrobactrum as adults is correlated with faster animal growth rates and larger body size at the end of development. These results highlight a new role for the highly conserved insulin signaling pathways in the regulation of gut microbiome composition in C. elegans.

BACKGROUND:Skin-penetrating nematodes of the genus Strongyloides infect over 600 million people, posing a major global health burden. Their life cycle includes both a parasitic and free-living generation. During the parasitic generation, infective third-stage larvae (iL3s) actively engage in host seeking. During the free-living generation, the nematodes develop and reproduce on host feces. At different points during their life cycle, Strongyloides species encounter a wide variety of host-associated and environmental bacteria. However, the microbiome associated with Strongyloides species, and the behavioral and physiological interactions between Strongyloides species and bacteria, remain unclear. RESULTS:We first investigated the microbiome of the human parasite Strongyloides stercoralis using 16S-based amplicon sequencing. We found that S. stercoralis free-living adults have an associated microbiome consisting of specific fecal bacteria. We then investigated the behavioral responses of S. stercoralis and the closely related rat parasite Strongyloides ratti to an ecologically diverse panel of bacteria. We found that S. stercoralis and S. ratti showed similar responses to bacteria. The responses of both nematodes to bacteria varied dramatically across life stages: free-living adults were strongly attracted to most of the bacteria tested, while iL3s were attracted specifically to a narrow range of environmental bacteria. The behavioral responses to bacteria were dynamic, consisting of distinct short- and long-term behaviors. Finally, a comparison of the growth and reproduction of S. stercoralis free-living adults on different bacteria revealed that the bacterium Proteus mirabilis inhibits S. stercoralis egg hatching, and thereby greatly decreases parasite viability. CONCLUSIONS:Skin-penetrating nematodes encounter bacteria from various ecological niches throughout their life cycle. Our results demonstrate that bacteria function as key chemosensory cues for directing parasite movement in a life-stage-specific manner. Some bacterial genera may form essential associations with the nematodes, while others are detrimental and serve as a potential source of novel nematicides.

Hydrogen peroxide (H 2 O 2 ) is the most common chemical threat that organisms face. Here, we show that H 2 O 2 alters the bacterial food preference of Caenorhabditis elegans , enabling the nematodes to find a safe environment with food. H 2 O 2 induces the nematodes to leave food patches of laboratory and microbiome bacteria when those bacterial communities have insufficient H 2 O 2 -degrading capacity. The nematode’s behavior is directed by H 2 O 2 -sensing neurons that promote escape from H 2 O 2 and by bacteria-sensing neurons that promote attraction to bacteria. However, the input for H 2 O 2 -sensing neurons is removed by bacterial H 2 O 2 -degrading enzymes and the bacteria-sensing neurons’ perception of bacteria is prevented by H 2 O 2 . The resulting cross-attenuation provides a general mechanism that ensures the nematode’s behavior is faithful to the lethal threat of hydrogen peroxide, increasing the nematode’s chances of finding a niche that provides both food and protection from hydrogen peroxide.

ABSTRACTThe study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g. human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified naturalCaenorhabditis elegansmicrobiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of theC. elegansmodel system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions inC. elegans.Dataset accession numbersWhole genome sequencing data (PRJNA624308); microbiome sequencing [PRJEB37101 and PRJEB37035]; data supplement on the GSA Figshare Portal.

The gut microbiome is an important driver of host physiology and development. Altered abundance or membership of this microbe community can influence host health and disease progression, including the determination of host lifespan and healthspan. Here, we describe a robust pipeline to measure microbiome abundance and composition in the C. elegans gut that can be applied to examine the role of the microbiome on host aging or other physiologic processes.

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g., human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified naturalCaenorhabditis elegansmicrobiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of theC. elegansmodel system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions inC. elegans.

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g., human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans.

The work of Zeevi et al. (2019) in a recent issue of Nature shows that variations in gene content and organization between different strains of the same microbial species are widespread in the human gut microbiota and could be linked to many measures of health.

Most autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts ("Candidatus Thiobarba") of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that "Ca. Thiobarba" switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated "Ca. Thiobarba". Direct stable isotope fingerprinting showed that "Ca. Thiobarba" has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.