The Devil Is in the Microbial Genetic Details.

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.

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

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.

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

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.

Caenorhabditis elegans endorse bacterial nanocellulose fibers as functional dietary Fiber reducing lipid markers.

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.

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans 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 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.

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

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.

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

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-1ß production. In vivo, protection after GDAR2-2 colonization was lost after depletion of CX3CR1+ MNPs, or blockade of IL-1ß or IL-22. We further identified human commensal E. coli isolates that similarly protect mice from C. rodentium infection through CX3CR1+ MNP and IL-1ß production. Together, these findings demonstrate an unexpected role for commensal bacteria in promoting IL-1ß secretion to support intestinal barrier repair.

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

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.

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

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.

CeMbio - The Caenorhabditis elegans Microbiome Resource.

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.

Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria).

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.

A specific and widespread association between deep-sea Bathymodiolus mussels and a novel family of Epsilonproteobacteria.

Bathymodiolus mussels dominate animal communities at many hydrothermal vents and cold seeps. Essential to the mussels' ecological and evolutionary success is their association with symbiotic methane- and sulfur-oxidizing gammaproteobacteria, which provide them with nutrition. In addition to these well-known gammaproteobacterial endosymbionts, we found epsilonproteobacterial sequences in metatranscriptomes, metagenomes and 16S rRNA clone libraries as well as by polymerase chain reaction screening of Bathymodiolus species sampled from vents and seeps around the world. These epsilonproteobacterial sequences were closely related, indicating that the association is highly specific. The Bathymodiolus-associated epsilonproteobacterial 16S rRNA sequences were at most 87.6% identical to the closest cultured relative, and 91.2% identical to the closest sequences in public databases. This clade therefore represents a novel family within the Epsilonproteobacteria. Fluorescence in situ hybridization and transmission electron microscopy showed that the bacteria are filamentous epibionts associated with the gill epithelia in two Bathymodiolus species. In animals that host highly specific symbioses with one or a few types of endosymbionts, other less-abundant members of the microbiota can be easily overlooked. Our work highlights how widespread and specific associations with less-abundant microbes can be. Possibly, these microbes play an important role in the survival and health of their animal hosts.