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[
International Worm Meeting,
2019]
We are interested in knowing how natural bacteria interact with the Caenorhabditis elegans intestine. Currently, many natural and clinically-relevant bacteria are thought to float in the lumen of the intestine without observing any direct interaction with internal epithelial cells. During our ecological sampling at SDSU and the San Diego Botanical Gardens, we found two C. elegans strains (LUA21 and LUA11) that have unknown bacteria adhering to the intestinal epithelial cells in the lumen of the intestine. Phenotypically, we note that the bacteria have a rod-like shape that grows perpendicularly along the inner sides of the lumen and are found throughout the intestine along the anterior-posterior axis. We approximate that 95-99% of the worms on a plate are colonized by this bacteria. Due to these phenotypes, we believe that this species of natural bacteria represents part of the microbiome of C. elegans in the wild. In order to identify this bacteria, we first cleaned the worm of any contaminating bacteria using a series of detergent washes and incubations with antibiotics. We then dissected the worm intestine and conducted PCR using universal 16S bacterial primers and identified a new species of bacteria in the Enterobacteriaceae family, close to the Buttiauxella genus. To confirm this identification we made a unique fluorescence in situ hybridization (FISH) probe and saw that the bacteria were bound to the intestine in a similar manner to what we saw by light microscopy. We verified the specificity of this probe by doing FISH on a wild C. briggsae strain (JU3205) colonized by a different intestinal adhering bacteria species. Early observations suggest that this Buttiauxella species in both LUA11 and LUA21 has a minimal impact on the growth rate of C. elegans, suggesting a potential commensal organism. In contrast, the JU3205 strain when infected in C. elegans, has a severe impact on the growth rate, suggesting a pathogenic effect. In conclusion, we have discovered and identified a new species of Buttiauxella bacteria that was found binding to the lumen of two different wild C. elegans strains, LUA21 and LUA11. In total, we have many natural bacteria that adhere to the lumen of C. elegans, which make them a great tool to explore the relationship between natural bacteria in the C. elegans diet and their symbiotic effects (i.e. pathogenic, commensal, beneficial). We expect this discovery to aid in a better understanding of direct host-bacteria interactions in the C. elegans intestine.
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[
International Worm Meeting,
2021]
From sampling of wild nematodes, we have found that they are naturally associated with a diverse array of microbes such as bacteria, viruses, fungi, and microsporidia. In Bangalore, India, a wild C. briggsae strain (LUAb1) was discovered with adhering bacteria that colonized within the intestinal lumen, causing the morphology of the worm to change and potentially affect its health and growth. Phenotypically, this microbe appears to attach perpendicular to epithelial cells in the intestinal lumen, giving it a bristle-like appearance. This microbe may be pathogenic, as colonized animals appear to grow slower and have severely reduced intestinal cell sizes. Similar phenotypes were seen when LUAb1 was transferred to C. elegans N2 via co-culture with the wild cognate C. briggsae strain. Given that the NGM plates containing these nematodes are visibly contaminated, it is not possible to associate pathogenic phenotypes with LUAb1. Therefore, to determine if LUAb1 is causing pathogenic phenotypes in C. elegans, we aimed to eliminate any associated microbes to focus on the interactions between LUAb1 and the host. We have created a protocol that reduces the presence of microbes that cause visible contamination on the plates of the worms and enrich for our non-culturable LUAb1. This protocol involved a series of antibiotic and SDS washes of dauer animals to preserve live bacteria in the lumen while eliminating external contamination. After this decontaminating procedure, plates containing LUAb1 colonized animals showed no visible contamination, and comparative FISH found no other bacteria in the lumen except for LUAb1. However, to identify any potential contaminating bacteria in the lumen, we will use 16S amplicon sequencing to compare LUAb1 colonized and uncolonized C. elegans. This procedure involves PCR amplification of bacterial variable regions of the 16S sequence using nested PCR and universal bacterial probes. Altogether, we have discovered and identified a new species of Enterobacteriaceae bacteria that can bind to the apical side of intestinal epithelia cells in C. briggsae and C. elegans. Our objective is to conduct whole genome analysis to inform our attempts to grow LUAb1 in vitro and to discover host and bacterial factors necessary for bacterial binding to the intestinal cells of the lumen. In fact, an intestinal GFP C. elegans strain allows for indirect visualization of bacteria colonization which can be used in a forward genetic screen.
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[
International Worm Meeting,
2021]
Functional and morphological parallels to the intestines of humans, as well as the variety of microbes that make up its natural microbiome, have led to the emergence of C. elegans as a model system to study host-microbe interactions in vivo. Through ecological sampling, we have identified three bacterial species that bind longitudinally to the intestinal epithelial cells of Caenorhabditis isolates. DIC microscopy imaging reveals these adhering bacteria bind in a hair-like pattern along the intestinal wall. We were interested in whether these bacteria had any effects on host fitness when monocolonized in the C. elegans lumen. Interestingly, we found adherent bacteria 1 (LUAb1), negatively affects the life span and brood size of C. elegans, whereas the two others, adherent bacteria 2 and 3 (LUAb2 and LUAb3), have a neutral effect on C. elegans fitness and are best classified as commensal bacteria. We conducted 16s rRNA sequencing by extracting worm intestines, conducting PCR with universal bacterial primers, and Sanger sequencing of the amplicon. This approach revealed all three bacterial isolates to be novel. LUAb1 and LUAb3 belong to the Enterobacteriaceae family, whereas LUAb2 belongs to the Alphaproteobacterial class. Fluorescence in situ hybridization (FISH) probes were designed to these three bacterial strains to allow for visualization in the intestines. We found LUAb1 colonized nearly 90% of the anterior-posterior intestinal length in more than 90% of the population. Although LUAb2 and LUAb3 colonized the intestines of the worm less than 90% of the intestinal length, the frequency of colonization is the same. Moreover, LUAb3 is the only strain currently culturable in vitro. Whole genome sequence of LUAb3 revealed it is not a new species, but rather a Gram-negative bacterium Lelliottia jeotgali. Absent from the complete L. jeotgali genome sequence, however, was a plasmid encoding a Type IV secretion system, pilins, and adhesins found in LUAb3. We plan to investigate if this plasmid facilitates adherence in the C. elegans lumen. To summarize, we have found three bacterial isolates that exhibit a directional binding phenotype in the intestinal epithelium of C. elegans. We plan to elucidate the host and bacterial receptors that facilitate this adherence mechanism through a forward genetic screen in C. elegans. This research could expand our current understanding of the C. elegans microbiome and inform host-microbe interaction studies in other animals.