Blackburn, Dana et al. (2021) International Worm Meeting "Genetic determinants of host-microbiome interactions in Caenorhabditis elegans"

Samuel, Buck, Assie, Adrien, Deutschbauer, Adam, Vidal Vilchis, Daniela, Blackburn, Dana

[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.

Moon, K. et al. (2019) International Worm Meeting "Gait selection of Caenorhabditis elegans is regulated by mechanosensitive DEG/ENaC channels."

Moon, K., Kim, K., Kim, J., Yeon, J.

[International Worm Meeting, 2019]

Animals change their locomotion or gaits in response to environmental condition. In vertebrates, gait transition has been shown to be mediated by monoamines, which is conserved across many species including the nematode Caenorhabditis elegans (Vidal-Gadea et al., 2011). However, molecular mechanisms of gait transition are still unclear. C. elegans exhibits two gaits, swimming in liquids and crawling on dense gels. C. elegans genome contains evolutionarily conserved 28 DEG/ENaC channels, of which functions may be involved in mechanosensory transduction and locomotion (Goodman and Schwarz, 2003). We first hypothesized that mechanosensitive channels could act as a gait transition initiator and examined crawl-to-swim transition phenotype in DEG/ENaC mutants. We found that while acd-5 mutants show normal crawling, transition from crawling to swimming upon liquid exposure is defective, suggesting roles of ACD-5 in gait transition. We are currently generating acd-5 rescue lines and examining expression pattern of acd-5.

Walhout AJM et al. (1998) East Coast Worm Meeting "Towards a comprehensive C. elegans protein interaction map: identification of "Interaction-Sequence Tags""

Kreutzer MA, Vidal MI, van den Heuvel SJL, Walhout AJM, Wong WL

[East Coast Worm Meeting, 1998]

The complete genome sequence of C. elegans should be available by the end of this year. It is estimated that the sequence will reveal approximately 13,000 ORFs, most of which correspond to predicted gene-products whose functions have yet to be determined. Hence, in addition to the powerful genetic and biochemical approaches used conventionally, it will be important to develop high-throughput strategies to help addressing the biological role of these gene-products. Since many cellular processes depend on protein-protein interactions, one approach used to elucidate functions consists in identifying physical interactions between proteins. Thus, our long-term goal is to generate a comprehensive protein interaction map for C. elegans, as a way to help characterizing regulatory networks and protein complexes in this model organism (see the abstract by Vidal et al.). We have selected the yeast two-hybrid system to identify potential interactions since on the one hand, its effectiveness has been demonstrated widely, and on the other, it is readily automatable. In this system, interaction between X and Y proteins, expressed as fusions with a DNA binding domain (DB) and an activation domain (AD) respectively, reconstitutes a functional transcription factor which in turn leads to a selectable phenotype. Each potential interaction is referred to as an Interaction Sequence Tag (IST), defined as a pair of short sequences obtained from cDNAs encoding interacting proteins in the two-hybrid system. We intend to release ISTs into AceDB in a format similar to the introduction of ESTs. We will describe the following aspects of the project: -the choice of the first subset of DB-X hybrid proteins used as "baits", -the semi-automated method used to generate the DB-X fusions, -the design and construction of an AD-cDNA library, -the semi-automated two-hybrid selections used to identify ISTs, and -the potential significance of the initial ISTs identified. Since ISTs merely represent hypotheses of interactions, we are also developing high throughput strategies to assess the biological significance of the potential interactions directly in worms (see the abstract by Vidal et al.). The combination of both the IST information available in AceDB and the functional assessment in vivo should be helpful in generating a comprehensive interaction map for C. elegans.

N Mandhana et al. (2004) European Worm Meeting "DEVELOPMENT OF CHIP CHIP FOR C. ELEGANS FOR FINDING IN VIVO BINDING SITES OF CHROMATIN FACTORS"

M Vidal, J Ahringer, D Dupuy, N Mandhana

[European Worm Meeting, 2004]

Chromatin-modifying complexes are important for transcriptional control, but their roles in the regulation of development remains poorly understood. We are interested in elucidating how chromatin regulatory complexes function in developmental decisions. The synthetic multivulva (synMuv) genes negatively regulate vulval induction and a sub-set of these genes are components of histone deacetylase (HDAC) complexes (e.g., Rb and NuRD). HDACs are are thought to be recruited to target genes by sequence specific transcription factors, where they locally deacetylate histone H3 tails, and bring about transcriptional repression. We are trying to understand how chromatin-remodelling complexes are integrated into signalling and developmental pathways. To this end, we are currently constructing a C. elegans promoter DNA array which will be used in ChIP-chip (Chromatin immunoprecipitation with DNA Microarray) experiments to identify binding sites for chromatin factors and sequence specific binding proteins. We are spotting ~9000 intergenic PCR fragments generated for the promoterome project in the Vidal Lab. These PCR products are amplified upstream DNA sequences and cover approximately 1.5 - 2.2 kp long sequences from the translation start site. We are also comparing the gene expression profiles of synMuv mutants using the Afffymetrics gene system to look for change of gene expression in these mutants compared to wildtype.

Topper, Stephen M. et al. (2013) International Worm Meeting "Dopaminergic control of gait switching in C. elegans."

Pierce-Shimomura, Jonathan, Aguilar, Sara, Topper, Stephen M., Young, Layla

[International Worm Meeting, 2013]

The ability to switch between different forms of locomotion is critical to many aspects of survival, whether it is switching from walking to running to evade predators, or switching to a slower gait to obtain food. However, the neuronal mechanisms of gait switching are not well understood. We recently showed C. elegans, displays distinct crawl and swim gaits, mediated by dopamine and serotonin, respectively (Vidal-Gadea et al., 2011). Further investigation into the role of dopamine signaling in the transition to crawl has indicated the D1-like dopamine receptor dop-4 as a key component. Laser microablation of dop-4-expressing neurons has revealed single neurons that are required for the transition to crawl. Optogenetic activation of dop-4-expressing neurons via the light-activated GPCR optoXRB2 induces crawl-like behavior in swimming worms. Finally, expression of dop-4 in specific subsets of dop-4 neurons is sufficient to rescue the swim to crawl transition. We are now combining noninvasive imaging of these neurons in freely moving worms with optogenetics to dissect how dopamine controls gait switching at the circuit level. A genetic analysis dopaminergic locomotory in C. elegans may reveal fundamental principles on gait switching with implications for many fields, including the study of human ailments such as Parkinson's disease.

Neeraj Mandhana et al. (2005) International Worm Meeting "Development of ChIP-chip in C. elegans for finding in vivo binding sites of chromatin factors"

Denis Dupuy, Marc Vidal, Julie Ahringer, Neeraj Mandhana

[International Worm Meeting, 2005]

Chromatin-modifying complexes are important for transcriptional control, but their role in the regulation of development remains poorly understood. We aim to understand how chromatin-remodelling complexes are integrated into signalling and developmental pathways. The synthetic multivulva (synMuv) genes negatively regulate vulval induction and a sub-set of these genes encode components of the NuRD histone deacetylase and other chromatin modifying complexes. To try to find targets of these complexes and to investigate their modes of interactions, we are developing ChIP-chip (Chromatin immunoprecipitation with DNA Microarray) methodology for the worm. This will allow us to identify globally genes associated with each complex at different times in development. We have spotted ~9000 C. elegans promoter PCR fragments from the Vidal Lab Promoterome version 1.1" on our arrays. This gives us ~50% coverage of C. elegans proximal promoter regions and 15% coverage of the C. elegans genome. We are now optimizing the chromatin IP protocol. Besides studying the binding of chromatin factors, the ChIP-chip methodology will be of obvious use in generating in vivo genome-wide binding data for sequence specific DNA binding factors. In addition to ChIP-chip, we are also comparing transcription profiles of synMuv mutants using Afffymetrics chips to look for differentially expressed genes between mutant and wildtype worms.

Thamsen, Maike et al. (2009) International Worm Meeting "Monitoring oxidative stress in aging C.elegans."

Jakob, Ursula, Thamsen, Maike, Knoefler, Daniela

[International Worm Meeting, 2009]

Aging is a complex physiological process and numerous aging theories have been proposed. One of the leading models is the free radical theory of aging, which suggests that the accumulation of reactive oxygen species (ROS), like superoxide (O2-) and hydrogen peroxide (H2O2), causes protein, lipid and DNA damage and leads to the observed age-related decline of cells and tissues. To directly monitor the onset and extent of oxidative stress during the lifespan of C. elegans, we utilize two complementary approaches. In the first approach, we use the fluorescent H2O2-sensor protein HyPer (Belousov, 2006) to evaluate the accumulation of endogenous H2O2 in the muscle cells of C. elegans. This ratiometric sensor protein has two excitation maxima, which substantially change in the presence of H2O2. With this tool, we are now able to determine and monitor endogenous H2O2 levels over the whole life span of a worm population. In the second approach, we use a highly quantitative mass spectrometry based thiol trapping technique termed OxICAT to identify the protein targets of oxidative stress in aging animals. This technique allows us not only to detect and quantify oxidative thiol modifications in hundreds of different proteins in a single experiment but enables us to identify the affected proteins and to define their redox-sensitive cysteine(s). Using this technique we were able to determine the redox status of numerous C. elegans proteins and monitor their age-related changes. The combination of these techniques provides us now with valuable insights into the underlying mechanism of aging and into the role that oxidative stress plays in this process. Reference: Belousov, V.V., et al., Nat Methods, 2006. 3(4). Maike Thamsen and Daniela Knoefler contributed equally to the work for this abstract.

Vidal MI (1997) International C. elegans Meeting "EARLY STEPS IN THE CONSTRUCTION OF A PROTEIN-PROTEIN INTERACTION DATABASE FOR C. ELEGANS"

Vidal MI

[International C. elegans Meeting, 1997]

Large-scale projects of genome and Expressed Sequence Tags (ESTs) sequencing are extremely valuable. One of the next challenges is to determine how the predicted ORFs resulting from such projects can be functionally linked to each other. Since protein-protein interactions are critical to such a wide range of processes, I propose to functionally link ORFs by identifying interaction partners and relate these interactions to biological questions. Automated high-throughput strategies will be presented to generate worm "Interaction Sequence Tags" (ISTs). ISTs are defined as pairs of short sequences obtained from cDNAs encoding interacting proteins in the yeast two-hybrid system. ISTs will be released in the form of databases publicly available on the Internet. In addition, automated high-throughput strategies will also be presented to generate !interaction defective alleles!. These alleles are defined as single amino-acid changes that specifically affect protein-protein interaction and can be selected in the reverse two-hybrid system (Vidal et al, 1996, PNAS, 93, 10315-10320 and 10321-10326). Such interaction defective alleles will be made available to the worm community. They could be tested for their ability to rescue particular phenotypes when reintroduced in the relevant worm loss-of-function mutants. This work should help developing the tools and the strategies needed to eventually generate and interpret a comprehensive protein-protein interaction map based on both physical and genetic interactions.

Min Jiang et al. (2005) International Worm Meeting "Identification of molecular markers for aging in C. elegans"

Min Jiang, Yelena Budovskaya, Kendall Wu, Stuart K Kim

[International Worm Meeting, 2005]

Aging is among the most complicated biological processes. We have taken a functional genomics approach to study aging in C. elegans by performing an extensive set of DNA microarray experiments to identify large set of genes whose expression changes significantly under a variety of conditions associated with aging: such as normal aging, in the dauer state, and in several longevity mutants such as daf-2, age-1, daf-16 and age-1; daf-16. Our goal is to identify molecular markers for aging based on these DNA microarray data. We have found 233 genes that show consistent expression changes during normal aging (Cur. Bio. 2002, 12, 1566-73), in the dauer state (Development 2003, 130, 621-34) and in longevity mutants such as age-1 and daf-16. This result suggests that there may be common molecular mechanisms underlying normal aging, the dauer state, and extended longevity in insulin-like pathway mutants. We have started to examine expression of GFP reporters corresponding to these age-regulated genes. We used GFP constructs generated by the Vidal lab and GFP lines generated by the Baillie and Moerman labs, and so far have obtained 81 GFP transgenic lines. Our preliminary GFP expression data confirm the patterns of age regulation revealed by the DNA microarray experiments; for example, GFP expression of mtl-1(metallothionein) and sod-3 (manganese superoxide dismutase) decrease in old worms is consistent with the DNA microarray data (Cur. Bio. 2002, 12, 1566-73).

Neve, Isaiah et al. (2015) International Worm Meeting "Modified OP50 E. coli expressing dsRNA elicite a robust RNA interference response in C. elegans."

Wang, Meng, Sowa, Jessica, Neve, Isaiah

[International Worm Meeting, 2015]

In the majority of research laboratories using C. elegans as a model, they sustain their animals on the B strain E. coli OP50. This strain is easy to manipulate in the laboratory, and has provided us with a key platform for defining many genetic pathways. Simultaneously, the series of revolutionary realizations which culminated in the 2006 Nobel Prize, quickly allowed the technique of dsRNA feeding to incite RNA interference (RNAi) to become a key technique in many laboratories. Two major resources are available to the RNAi wielding researcher, the Ahringer and Vidal libraries respectively. These libraries house thousands of E. coli clones with unique plasmids expressing dsRNA corresponding to individual C. elegans genes. Countless experimental insights have been facilitated using these resources. However, both libraries are housed in the HT115 E. coli K12 strain background. Several recent studies (Brooks et al., 2009; Soukas et al., 2009; Pang & Curran 2014) have revealed the importance of bacterial environment in eliciting certain genetically induced phenotypes. With the realization that gene-environment interactions may play a key role in the elucidation of genetic pathways, we have generated an OP50 strain capable of dependably housing and robustly expressing dsRNA plasmid vectors. Furthermore, when C. elegans are fed this OP50 strain, they activate a substantial RNAi response against the target gene of interest. We anticipate that this resource will allow the further characterization of gene-environment specific relationships. .