Berg, Maureen et al. (2019) International Worm Meeting "Genetic control of Enterobactercommensal abundance and function in C. elegans."

Nelson, Lydia, Berg, Maureen, Shapira, Michael, Monnin, David, Cho, Juhyun

[International Worm Meeting, 2019]

The gut microbiota contributes to host health and fitness, and imbalances in its composition are associated with pathology. However, what shapes microbiota composition is not clear, in particular the role of genetic factors. Previous work in Caenorhabditis elegansdefined a characteristic worm gut microbiota significantly influenced by host genetics. Using synthetic communities and qPCR we subsequently explored the role of genes involved in immune and stress regulation in controlling abundance of core microbiota taxa, revealing a role for the TGF?/BMP pathway. Immune compromised TGF?/BMP mutants showed a bloom specifically of Enterobacterspecies. TGF? overexpression coupled to fluorescent imaging of tdTomato-expressing Enterobacterin turn indicated that TGF?/BMP-exerted control operated primarily in the anterior gut and depended on multi-tissue contributions. Enterobactercommensals are common in the worm gut, accelerating development and enhancing infection resistance. However, disruption of TGF?/BMP signaling turned a normally beneficial Enterobactercommensal to pathogenic. Interestingly, during normal aging worms demonstrated a similar bloom as in TGF?/BMP mutants, but this bloom was independent of the TGF?/BMP pathway. These results demonstrate specificity in gene-microbe interactions underlying gut microbial homeostasis and highlight age-dependent disruption of this homeostasis.

Berg, Maureen et al. (2015) International Worm Meeting "The C. elegans gut microbiota and its significance to its host."

Ho, Joshua, Parke, Caitlin, Alvarez-Cohen, Lisa, Shapira, Michael, Stenuit, Ben, Berg, Maureen

[International Worm Meeting, 2015]

The animal intestine is home to an abundance of microorganisms that play diverse roles in host health and disease. Characterizing the gut microbiota has become an active area of research, but the relative contribution of factors shaping microbiota composition is still unclear. Gut microbiota composition may be determined by environmental availability or shaped by host factors, enabling the selection of potentially beneficial taxa. Thus, it is of interest to understand the significance of those selected microbes, or those that are lost due to aberrant assembly, for their host. Using the genetically tractable Caenorhabditis elegans as a model host and 16S rDNA 'deep sequencing,' we are studying the assembly of the gut microbiota starting from diverse environmental communities. Comparisons of worm microbiotas to microbiotas in their soil environment revealed that worm microbiotas resembled each other, even when assembled from different microbial environments. Our results support a dominant contribution of the host niche in shaping the gut microbiota. Among the recurring taxa in the gut microbiota, we found Enterobacter and Pseudomonas, both of which provide services to their hosts. One resident, P. mendocina, is able to provide protection against the gram-negative pathogen P. aeruginosa by priming the p38-oxidative stress pathway, while E. cloacae can extend survival of worms infected with the gram-positive pathogen, Enterococcus faecalis. Assessing these interactions between gut isolates and their host will fill in a gap of our understanding of C. elegans interactions with microbes, and will help establish C. elegans as a model for studying host-microbiota interactions.

Berg, Maureen et al. (2015) International Worm Meeting "Assembly of the Caenorhabditis elegans gut microbiota is a deterministic process shaped by the host."

Shapira, Michael, Alvarez-Cohen, Lisa, Wang, Andrew, Ho, Joshua, Stenuit, Ben, Knight, Matthew, Berg, Maureen, Parke, Catelin

[International Worm Meeting, 2015]

Animals harbor gut microbiotas that play critical roles in the maintenance of host health, from digestion to immunity. Characterizing the gut microbiota has become an active area of research, but the relative contribution of factors shaping microbiota composition is still unclear. C. elegans could provide a useful model to study such questions, utilizing genetically homogenous populations to discern common patterns in microbiota composition beyond individual variation. However, to date, very little is known about the natural C. elegans microbiota. To fill this gap, we grew worms of the N2 wildtype strain in the lab in natural-like mini-environments of different composted soils, and used 16S rDNA deep sequencing to characterize the assembled microbiota of adult worms. Comparisons of worm microbiotas to microbiotas in their soil environment revealed that worm microbiotas resembled each other even when assembled from different microbial environments. This enabled defining a core gut microbiota that accounted for ca. 50% of gut microbes, and was primarily composed of members of the Enterobacteriaceae, Pseudomonadaceae, and Xanthomonadaceae families. Beyond the shared core, two distinct types of worm microbiotas were identified, distinguished by varying abundance of core taxa as well as the presence of auxiliary taxa. Ecological analyses indicated that the assembly rules for worm microbiotas differed from those in their soil habitat, and pointed at the importance of competitive interactions between gut-residing taxa. Our results support a dominant contribution of the host niche in shaping the gut microbiota, and suggest contributions from inter-microbial competition within the gut. The data presented provides a framework to better understand the worm natural history and its interactions with microbes.

Choi, Rebecca et al. (2021) International Worm Meeting "Age-dependent changes in C. elegans gut microbiome composition and their consequences"

Choi, Rebecca, Cho, Juhyun, Shapira, Michael, Baek, Song-Ah, Berg, Maureen, Monnin, David

[International Worm Meeting, 2021]

Aging involves a multi-tissue deterioration. Among other things, it affects the gut microbiome. Changes in gut microbiome composition, in turn, could contribute to age-related pathologies, as imbalances in microbial composition (dysbiosis) are associated with pathology. Identifying central themes in age-dependent microbiome changes is lagging, as is our understanding of their consequences. To examine the bidirectional relationships between host aging and the gut microbiome, we followed age-dependent changes in microbiome composition in worms raised on either natural-like compost microcosms or on well-defined synthetic microbiotas consisting of natural worm gut colonizers. Microbiome composition was analyzed using 16S next generation sequencing or with CFU counts of bacteria cultured on selective media. Additionally, gut colonization at different ages was evaluated using fluorescent microscopy of an RFP-expressing derivative of an Enterobacter commensal. Experiments in both compost microcosms and synthetic microbiotas revealed an expansion of the Enterobacteriaceae family in aging worms, a trend similarly observed in humans. Worms sampled during continuous growth on complex microbiotas showed similar age-dependent changes in their gut microbiomes as worms shifted to complex bacterial communities for a fixed time at advancing ages, suggesting that the aging intestinal niche is more important for determining changes in microbiome composition than the duration of exposure to bacterial communities. Further supporting this expansion, experiments with the RFP-expressing Enterobacter demonstrated increased age-dependent gut colonization. This particular Enterobacter commensal was previously shown to protect young worms from Enterococcus infection, but was found to bloom in immune mutants, reversing its contributions and causing increased pathogen susceptibility (Berg et al. 2019). Indeed, increased commensal colonization in aging worms abolished the commensal's beneficial effects and increased worm susceptibility to infection, as compared to age-matched worms raised on Escherichia coli. Current experiments are underway to examine whether rebalancing of the gut microbiota in aging worms could ameliorate host survival.

Hutchinson T et al. (1991) International C. elegans Meeting "MULTI-POINT MAPPING OF age-1."

Johnson TE, Lindsay D, Hutchinson T

[International C. elegans Meeting, 1991]

The fundamental mechanisms of aging are being explored through the analysis of genetic stocks that have postponed senescence. age-1(hx546) is a mutant allele that results in an average 40% increase in life span at 20 C. Associated with the increased life span is a 75 /O decrease in hermaphrodite self-fertility. The long-lived phenotype is closely linked to fer-15(b26), a temperature-sensitive allele on linkage group 11 causing defective spermatogenesis and the decrease in self-fertility. These genes are recessive and affect both males and hermaphrodites. We are currently mapping age-1 using a variety of strategies. One of these is to walk to age-1 from nearby transposable element (Tc1) generated RFLPs. These Tc1 RFLPs have been created by introgressing fer-15(+ Berg) and age-1(+ Berg) (as well as several centiMorgans of flanking Bergerac DNA) into a Bristol genetic background. This DNA contains at least 7 additional Tc1 elements. A total of 46 recombinants of this congenic crossed with a dpy-10 fer-15 age-1 unc-4 strain were isolated and assayed for Tc1 patterns, fer-15, self- fertility (number of offspring), and life span. However, it is problematic to assay Age in a Dpy or Unc background. Therefore, a further 62 recombinants were created by crossing a Dpy non-Unc congenic recombinant with a fer-15 age-1 unc-4 strain and by crossing an Unc non-Dpy congenic recombinant with a dpy-10 fer-15 age-1 strain. We chose those recombinants which were wild-type with respect to dpy- 10 and unc-4 and assayed them as before. fer15 and decreased self- fertility cosegregated and mapped to the middle of the region containing novel Tc1 bands. age-1 mapped further to the right, closer to unc-4 (see figure). This confirms the results of an earlier deficiency mapping analysis (Johnson et al., submitted). We are now physically mapping age-1 using a set of cosmids which starts at unc-4 and extends about 500 kb to the left. To localize age-1 we are screening the recombinants for non-Tc1 RFLPs. Presently, these strategies have brought us to within 200 kb of age- 1. The actual cloning of the gene will involve deficiency mapping of this region, transformation of mutants with age-1(+), and sequence analysis to identify the mutational events responsible for the alteration of life span. [See Figure]

Buechner M et al. (2000) Midwest Worm Meeting "Polycystic Kidney Disease Gene Homologues in C. elegans"

King KV, Buechner M, Branden M

[Midwest Worm Meeting, 2000]

Polycystic Kidney Disease (PKD) is an inherited condition caused by mutations in several genes, in which fluid-filled cysts form and slowly swell within tubular epithelia of several tissues, with the most drastic effects usually seen in the nephrons. Two mammalian genes, PKD1 and PKD2, cause a dominant condition when mutated. These two genes encode proteins with a similar transmembrane structure; it is thought that these proteins dimerize to form a Calcium channel. At least 3 similar genes have been found in humans: PKDREJ, PKDL, and PKD2L. It is unknown if mutations in these genes cause PKD; the PKDREJ protein is most strongly produced in the testes. C. elegans contains a single homologue of PKD1 and PKD2. The PKD1 homologue was identified as the product of the lov-1 gene (affecting male mating behaviour) by Maureen Barr & Paul Sternberg (Nature 401, 386, '99). We compare the sequence of the mammalian genes and find that the C. elegans homologue is most closely related to the PKDREJ protein. The LOV-1 protein is likely to have been duplicated, with the second gene evolving into the PKD1 gene, as the metanephric mammalian kidney evolved from the nervous system. In mice, mutations in the Tg737 gene cause recessive PKD. Recessive PKD exists in humans as well, but the affected genes have not been cloned. A Tg737 homologue exists in C. elegans; this gene shows much greater homology to its mammalian equivalent than do the PKD proteins. The role of CeTg737 in signal transduction may therefore be better conserved between worms and mammals, and its study should shed light on similarities between nematode neural function and mammalian kidney structure.

Yasuda H et al. (1991) International C. elegans Meeting "AN EQUALIZED GENE COPY cDNA LIBRARY OF C. ELEGANS"

Siddiqui SS, Yasuda H

[International C. elegans Meeting, 1991]

From several aspects it is worthwhile to equalize the abundance of individual cDNAs in a cDNA library. C. eleFans is well suited for this purpose, for its small genome size, small number and size of introns, small number of expressed repetitive sequences, which can interfere in the normalization step. As most of the structural genes are unique in C. elegans, we have developed a method for the normalization of a cDNA library, based on hybridization of cDNA with the genomic DNA first, a unidirectional cDNA library was constructed as per Okayama and Berg with lX10 7 cfu that we call 1st library. This library was infected with M13 helper phage, that produces single stranded circular cDNA molecules with only the anti-sense strand. Excess amount of the single strand cDNA library was hybridized with denatured C. elegans genomic DNA that was immobilized on Nylon membrane filters. After prolonged hybridization, filters were washed with high stringency, and hybridized single strand cDNA molecules were eluted from the filters. Finally, ampicillin resistant transformants were obtained from a direct transformation of E. coli host cells by the eluate, to form the 2nd library (7X10 (5) cfu) About 100 independent clones, from 2nd library were randomly picked up and each cDNA was used as a labeled probe. Genomic Southern analysis revealed that most of these clones were unique genes and all of them were differnt. Colony hybridization method was used to measure the frequency with which each gene is represented in the libraries. Most of the clones are equally represented in the 2nd library. This general strategy can be used for the whole genome or even a part of C. eleFans genome, i.e. permitting the construction of chromosome specific cDNA libraries, or a YAC specific library.

Alcantara, Alfredo Jr. et al. (2021) International Worm Meeting "Simulated Microgravity Impairs C. elegans Gut Immunity"

Higashibata, Akira, Higashitani, Atsushi, Lee, Jin Il, Alcantara, Alfredo Jr., Hashizume, Toko

[International Worm Meeting, 2021]

Humanity had been fascinated with space exploration and habitation. However, weak gravity force in outer space, termed as microgravity, has been known to alter normal human biology and induce health risks, such as muscle atrophy and compromised immunity. The underlying processes on how gravity is sensed by the body and how it affects immunity are still unclear. The nematode Caenorhabditis elegans, is a well-known model organism in the field of innate immunity and space research. Similar to humans, worm immune response signaling is regulating by MAPK and TGFbeta pathways. Previous microgravity experiments in C. elegans have shown an increase in p38 MAPK pmk-1 expression in simulated microgravity (Li, Wang, & Wang, 2018) and decreased expression of TGFbeta ligand dbl-1 in space (Harada et al., 2016). In this study, we have utilized Enterobacter cloacae carrying tdTomato fluorescence reporter to observe gut colonization in C. elegans under simulated microgravity using a 3D clinostat. E. cloacae is a known commensal in C. elegans' gut but is invasive in immunocompromised dbl-1 mutant worms (Berg et al., 2019). Our preliminary results have shown that simulated microgravity can induce E. cloacae gut colonization in wild type C. elegans and aggravated colonization in pmk-1 mutants, while there is no difference observed in dbl-1 mutants which have robust colonization in both normal earth gravity and simulated microgravity, which demonstrates potential involvement of these pathways. Our study aims to discover the underlying mechanism of gravity sensing and how it affects the immune signaling pathways. In addition, spaceflights are already scheduled to send C. elegans aboard the International Space Station (ISS) for actual space gravity experiments.

Horvitz HR et al. (2000) East Coast Worm Meeting "A screen for mutants defective in the specification of the programmed cell deaths of the male-specific CEM neurons"

Horvitz HR, Schwartz H

[East Coast Worm Meeting, 2000]

During wild-type hermaphrodite development, 131 somatic cells undergo programmed cell death. While many genes involved in the execution of cell death have been identified, the mechanisms that control the commitment of specific cells to undergo programmed cell death are poorly understood. To date, mutations in four genes, ces-1, -2, and -3 (cell death specification), and egl-1, have been found to affect specifically the deaths of particular cells. ces-1 and ces-2 encode transcription factors. Mutations in a transcriptional regulatory element of egl-1, which encodes a protein required for all somatic cell deaths, cause inappropriate expression of egl-1 in the HSNs in hermaphrodites, resulting in their deaths. We have performed a genetic screen for hermaphrodites in which the male-specific CEM neurons fail to undergo programmed cell death. The CEM neurons die during normal hermaphrodite development, but survive and differentiate in males. The reporter pkd-2::gfp (kindly provided by Maureen Barr and Paul Sternberg) expresses in the CEMs of males and in the CEMs of ced-3 hermaphrodites, which are defective in programmed cell death. By using the pkd-2::gfp reporter as a marker for CEM survival, we were able to screen efficiently for survival of a single cell using a dissecting microscope fitted with fluorescence optics. We expect this screen to yield mutations in the sex determination and programmed cell death pathways and hope it will also yield mutations in genes specifically required for the deaths of the CEM neurons in hermaphrodites. A screen of 60,000 mutagenized haploid genomes yielded at least 135 independent mutations that cause survival of the CEMs, including at least 50 that cause sexual transformation and at least 29 alleles of known cell-death genes. We are currently mapping the uncategorized mutations and placing them into complementation groups.

Kissoyan, K.A.B. et al. (2019) International Worm Meeting "Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group."

Dierking, K., Kissoyan, K.A.B.

[International Worm Meeting, 2019]

C. elegans is associated in nature with a species-rich, distinct microbiota, which was characterized only recently [1]. Our understanding of C. elegans microbiota function is thus still in its infancy. Here, we identify natural C. elegans microbiota isolates of the Pseudomonas fluorescens subgroup that increase C. elegans resistance to pathogen infection. We show that different Pseudomonas isolates provide paramount protection from infection with the natural C. elegans pathogen Bacillus thuringiensis through distinct mechanisms [2] . The P. lurida isolates MYb11 and MYb12 (members of the P. fluorescens subgroup) protect C. elegans against B. thuringiensis infection by directly inhibiting growth of the pathogen both in vitro and in vivo. Using genomic and biochemical approaches, we demonstrate that MYb11 and MYb12 produce massetolide E, a cyclic lipopeptide biosurfactant of the viscosin group, which is active against pathogenic B. thuringiensis. In contrast to MYb11 and MYb12, P. fluorescens MYb115-mediated protection involves increased resistance without inhibition of pathogen growth and most likely depends on indirect, host-mediated mechanisms. We are currently investigating the molecular basis of P. fluorescens MYb115-mediated protection using a multi-omics approach to identify C. elegans candidate genes involved in microbiota-mediated protection. Moreover, we are further exploring the antagonistic interactions between C. elegans microbiota and pathogens. This work provides new insight into the functional significance of the C. elegans natural microbiota and expands our knowledge of immune-protective mechanisms. 1. Zhang, F., Berg, M., Dierking, K., Felix, M.A., Shapira, M., Samuel, B.S., and Schulenburg, H. (2017). Caenorhabditis elegans as a model for microbiome research. Front. Microbiol. 8:485. 2. Kissoyan, K.A.B., Drechsler, M., Stange, E.-L., Zimmermann, J., Kaleta, C., Bode, H.B., and Dierking, K. (2019). Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr. Biol. 29.