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[
International Worm Meeting,
2019]
The gut microbiome both extends the capabilities of its host and alters its physiology. Host genetics, diet, environment, and microbe-microbe interactions influence microbiome form and function. In humans, microbiome community function is important for resilience to disease. Despite its importance, the essential functions that drive microbiome assembly and stability in remain largely unaddressed in any host. To address this challenge, we leverage the nematode Caenorhabditis elegans to explore how microbiomes of distinct scales and functional complexity assemble in different host genetic backgrounds. This system has several advantages, including (1) a simple microbiome that can be removed (bleaching) and manipulated in the lab; (2) highly conserved intestinal physiology and innate immunity; and (3) comparable microbial mechanisms for host gut persistence. Previous studies in the lab have identified host-dependent variation in the microbiome: around 40 C. elegans wild strains were exposed to a model microbiome (BIGbiome) and they form three distinct gut types that differ in dominant taxa [Ochrobactrum, Bacteroidetes or a mix]. To examine the essential functions for colonization of the C. elegans gut, we first sequenced more than 100 bacterial genomes of its microbiome to determine their functional potential. Next, bacteria in each C. elegans microbiome were mapped to their corresponding genomes and scaled by abundance (16S) to construct virtual metagenomes. In-silico metabolic reconstructions and comparative metagenomic analyses both indicate broad variation in potential functions and provide many candidate genes important for host association. Unique functions may also allow for host selection of specific taxa. Analyses of each of the genomes indicate a wide range of potential metabolic capacities from "generalists" (like Enterobacteria) that have genes for utilization of a wide range of carbohydrate substrates to "specialists" (like Ochrobactrum) that have a narrower nutrient usage potential. Interestingly, specialists thrive within the worm gut whereas generalists are less successful in a community context. To explore this further, we exposed C. elegans strains to microbiomes of increasing complexity in function and taxa diversity. Together, these studies highlight key molecular functions under host selection that may underlie assembly and stability of the microbiome.
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[
International Worm Meeting,
2021]
Teasing apart the dense network of molecular mechanisms that link digestive tract microbial community members to each other and their host is challenging in most systems due to complexity and tractability. Recently, Caenorhabditis elegans has emerged as a powerful model system to study host-microbe interactions. The simplicity of the C. elegans digestive tract, together with the nematode's genetic amenability, and the availability of relevant microbial collections make it ideal for the study of the fundamental mechanisms of host-microbiota interactions. To characterize those mechanisms, we focused on identifying genetic features important for bacteria colonizing the C. elegans gut. We developed "WormBiome" a pipeline that predicts the combined functional potential of defined C. elegans gut microbiome. The "Wormbiome" pipeline match predicted microbial abundance from 16S rRNA amplicon datasets to functional genomic annotations and output functional profiles for each microbial community present in the submitted dataset. Our functional annotations rely on curated metabolic (Metacyc) and functional annotations (KEGG) databases build on known C. elegans related bacteria. Then the pipeline predicts features significantly different between user-defined sample groups. We tested the pipeline using a defined and fully sequenced 12-member model microbiome (CeMbio) grown with and without N2 animals. With 10 replicates for each condition, we identified 1700 significantly different features, distributed across 180 KEGG and 63 Metacyc categories. The most abundant features belong to the lipid, amino acid, and cofactor metabolisms. Among genes predicted to be more abundant in worm-associated communities, we found the de novo synthesis of vitamin B12 and metabolic pathways for host-essential amino acids, such as proline, alanine, and arginine. We verified the pipeline's prediction by examining the impact of nutrient depletion on gut microbiome composition by selectively supplementing or removing amino acids individually or altogether. Our results show that a single change in amino acid can affect how bacteria interact with each other and promote the growth of certain community members and that complete removal of amino acids promotes colonization of metabolically flexible members of the microbiome like Ochrobactrum. This study establishes a robust framework for identifying microbial functions that govern affect host-microbe associations and beneficial interactions.
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[
International Worm Meeting,
2021]
Microbes shape many aspects of host physiology, development and predisposition to disease. Yet the complexity of host-associated communities (microbiomes) makes studying host-microbe interactions challenging. As such, we employed the Caenorhabditis elegans system to investigate molecular mediators of host-microbial interactions with a simplified 12-member microbiome (CeMbio). Previous studies showed the microbiome exerts significant influence on the physiology and development of C. elegans, but lack of molecular tools has limited the ability to identify the microbial factors responsible. To address this challenge, we developed resources to enable genetic manipulation of CeMbio strains. Thus far, we identified several genetic determinants of microbe-microbe interactions, host-specific association and impact on physiology in the dominant microbiome member Ochrobactrum. For genetic manipulation of the CeMbio strains, we utilized broad host range vectors to make an effective panel of fluorescent reporter strains. We used these strains to test pairwise interactions with other microbiome members both in vitro and within the C. elegans gut. Ochrobactrum growth was inhibited by three strains while synergistic with one strain in rich media. In the C. elegans gut, however, microbial interactions were often dramatically different. Notably, Myroides suppresses Ochrobactrum growth in vitro, while Ochrobactrum benefits from the presence of Myroides to colonize the gut. This suggests that host factors may be driving the enrichment for and interactions between members of its microbiome. To examine the molecular determinants of host association and competition, we developed tools for random transposon mutagenesis in Ochrobactrum and several CeMbio strains. We screened a 96-clone mutant library of Ochrobactrum for fitness changes relative to the wild-type in ability to colonize C. elegans hosts with and without Myroides. None of the mutants altered Myroides inhibition of Ochrobactrum in vitro, but several (12) mutants exhibited host association and inter-microbial competition defects. These mutants highlight specific metabolic pathways that Ochrobactrum relies on to colonize and compete for nutrients in association with C. elegans. Our work has demonstrated the ability to use broad host range molecular tools to manipulate CeMbio strains allowing us to visualize and identify molecular mechanisms underlying microbiome assembly and impact upon the C. elegans host.
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Chavez, Ivan, Bryant, Astra, Assie, Adrien, Samuel, Buck, Hallem, Elissa, Brown, Taylor
[
International Worm Meeting,
2021]
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 of their life cycle, Strongyloides species encounter bacteria from various ecological niches. However, the microbial interactions between Strongyloides and bacteria remain uncharacterized. 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 a distinct microbiome, suggesting that they selectively associate with 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 soil 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, greatly decreasing parasite viability. Our results identify bacteria that serve as key sensory cues for directing movement, as well as bacteria that decrease the parasite's reproductive fitness.