Application of Flow Vermimetry for Quantification and Analysis of the <em>Caenorhabditis elegans</em> Gut Microbiome

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring ...

Baylor College of Medicine

The initial acquisition and early development of the intestinal microbiome during infancy are important to human health across the lifespan. Mode of birth, antibiotic administration, environment of care, and nutrition have all been shown to play a role in the assembly of the intestinal microbiome during early life. For preterm infants, who are disproportionately at risk of inflammatory intestinal disease (i.e., necrotizing enterocolitis), a unique set of clinical factors influence the establishment of the microbiome. The purpose of this study was to establish the influence of nutritional exposures on the intestinal microbiome in a cohort of preterm infants early in life.

Influence of maternal breast milk ingestion on acquisition of the intestinal microbiome in preterm infants.

The nematode  is used as a central model system across biological disciplines. Surprisingly, almost all research with this worm is performed in the absence of its native microbiome, possibly affecting generality of the obtained results. In fact, the  microbiome had been unknown until recently. This review brings together results from the first three studies on  microbiomes, all published in 2016. Meta-analysis of the data demonstrates a considerable conservation in the composition of the microbial communities, despite the distinct geographical sample origins, study approaches, labs involved and perturbations during worm processing. The  microbiome is enriched and in some cases selective for distinct phylotypes compared to corresponding substrate samples (e.g., rotting fruits, decomposing plant matter, and compost soil). The dominant bacterial groups include several  (, and ) and . They are consistently joined by several rare putative keystone taxa like . The bacteria are able to enhance growth of nematode populations, as well as resistance to biotic and abiotic stressors, including high/low temperatures, osmotic stress, and pathogenic bacteria and fungi. The associated microbes thus appear to display a variety of effects beneficial for the worm. The characteristics of these effects, their relevance for  fitness, the presence of specific co-adaptations between microbiome members and the worm, and the molecular underpinnings of microbiome-host interactions represent promising areas of future research, for which the advantages of  as an experimental system should prove of particular value.

 as a Model for Microbiome Research.

Escherichia coli encodes two DNA ligases, ligase A, which is essential under normal laboratory growth conditions, and ligase B, which is not. Here we report potential functions of ligase B. We found that across the entire Enterobacteriaceae family, ligase B is highly conserved in both amino acid identity and synteny with genes associated with oxidative stress. Deletion of ligB sensitized E. coli to specific DNA damaging agents and antibiotics resulted in a weak mutator phenotype, and decreased biofilm formation. Overexpression of ligB caused a dramatic extension of lag phase that eventually resumed normal growth. The ligase function of ligase B was not required to mediate the extended lag phase, as overexpression of a ligase-deficient ligB mutant also blocked growth. Overexpression of ligB during logarithmic growth caused an immediate block of cell growth and DNA replication, and death of about half of cells. These data support a potential role for ligase B in the base excision repair pathway or the mismatch repair pathway.

Escherichia coli DNA ligase B may mitigate damage from oxidative stress.

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.

The Devil Is in the Microbial Genetic Details.

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.

High-Throughput Assessment of Changes in the Caenorhabditis elegans Gut Microbiome.

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 (, human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural  microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the  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 .

CeMbio - The  Microbiome Resource.

The free-living nematode Caenorhabditis elegans remains one of the most robust and flexible genetic systems for interrogating the complexities of animal biology. Targeted genetic manipulations, such as RNA interference (RNAi), CRISPR/Cas9- or array-based transgenesis, all depend on initial delivery of nucleic acids. Delivery of dsRNA by feeding can be effective, but the expression in Escherichia coli is not conducive to experiments intended to remain sterile or with defined microbial communities. Soaking-based delivery requires prolonged exposure of animals to high-material concentrations without a food source and is of limited throughput. Last, microinjection of individual animals can precisely deliver materials to animals' germlines, but is limited by the need to target and inject each animal one-by-one. Thus, we sought to address some of these challenges in nucleic acid delivery by developing a population-scale delivery method. We demonstrate efficient electroporation-mediated delivery of dsRNA throughout the worm and effective RNAi-based silencing, including in the germline. Finally, we show that guide RNA delivered by electroporation can be utilized by transgenic Cas9 expressing worms for population-scale genetic targeting. Together, these methods expand the scale and scope of genetic methodologies that can be applied to the C. elegans system.

Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation.

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.

Natural genetic variation drives microbiome selection in the Caenorhabditis elegans gut.

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.

Skin-penetrating nematodes exhibit life-stage-specific interactions with host-associated and environmental bacteria.

Hydrogen peroxide (H2O2) is the most common chemical threat that organisms face. Here, we show that H2O2 alters the bacterial food preference of Caenorhabditis elegans, enabling the nematodes to find a safe environment with food. H2O2 induces the nematodes to leave food patches of laboratory and microbiome bacteria when those bacterial communities have insufficient H2O2-degrading capacity. The nematode's behavior is directed by H2O2-sensing neurons that promote escape from H2O2 and by bacteria-sensing neurons that promote attraction to bacteria. However, the input for H2O2-sensing neurons is removed by bacterial H2O2-degrading enzymes and the bacteria-sensing neurons' perception of bacteria is prevented by H2O2. 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.

Modulation of sensory perception by hydrogen peroxide enables Caenorhabditis elegans to find a niche that provides both food and protection from hydrogen peroxide.

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  isolate, GDAR2-2, that protects mice from  infection and dextran sulfate sodium-induced colitis. Colonization with GDAR2-2 in mice resulted in expansion of CXCR1 mononuclear phagocytes, including CXCR1 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 CXCR1 MNPs, or blockade of IL-1β or IL-22. We further identified human commensal  isolates that similarly protect mice from  infection through CXCR1 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.

Interleukin-1β secretion induced by mucosa-associated gut commensal bacteria promotes intestinal barrier repair.

Editorial:  host-microbiome interactions: From medical to ecological and evolutionary model.

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.

Acute high-fat diet impairs macrophage-supported intestinal damage resolution.

The microbiome is increasingly receiving attention as an important modulator of host health and disease. However, while numerous mechanisms through which the microbiome influences its host have been identified, there is still a lack of approaches that allow to specifically modulate the abundance of individual microbes or microbial functions of interest. Moreover, current approaches for microbiome manipulation such as fecal transfers often entail a non-specific transfer of entire microbial communities with potentially unwanted side effects. To overcome this limitation, we here propose the concept of precision prebiotics that specifically modulate the abundance of a microbiome member species of interest. In a first step, we show that defining precision prebiotics by compounds that are only taken up by the target species but no other species in a community is usually not possible due to overlapping metabolic niches. Subsequently, we present a metabolic modeling network framework that allows us to define precision prebiotics for a two-member C. elegans microbiome model community comprising the immune-protective Pseudomonas lurida MYb11 and the persistent colonizer Ochrobactrum vermis MYb71. Thus, we predicted compounds that specifically boost the abundance of the host-beneficial MYb11, four of which were experimentally validated in vitro (L-serine, L-threonine, D-mannitol, and γ-aminobutyric acid). L-serine was further assessed in vivo, leading to an increase in MYb11 abundance also in the worm host. Overall, our findings demonstrate that constraint-based metabolic modeling is an effective tool for the design of precision prebiotics as an important cornerstone for future microbiome-targeted therapies.

Buck S. Samuel

Alkek Center for Metagenomics and Microbiome Research and Department of Molecular Virology and Microbiology,
Development,
Disease Models and Therapeutics Program,
Graduate School of Biomedical Sciences,
Development, Disease Models and Therapeutics Program, Graduate School of Biomedical Sciences

Baylor College of Medicine

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome