Gupta, Anjali et al. (2019) International Worm Meeting "Caenorhabditis elegans-bacteria interactions: Neuronal Control of Innate Immune Response."

Singh, Varsha, Varma, Manoj M, Gupta, Anjali

[International Worm Meeting, 2019]

The survival of an organism depends on its ability to sense potential threats and execute appropriate defense mechanisms. Pattern recognition receptors allow multicellular organisms to sense microbe-associated molecular patterns and mount an effective immune response. It is not clear how Caenorhabditis elegans recognizes pathogenic microbes in the absence of classical pattern recognition pathways. Thus, we asked if sensory neurons of C. elegans allow it to mount pathogen specific immune response. Exposure of C. elegans to Gram-positive bacterium Enterococcus faecalis versus a Gram-negative bacterium Pseudomonas aeruginosa showed predominantly pathogen-specific signatures. Using nematodes defective in sensory perception, osm-6, we show that neuronal sensing is essential to mount pathogen specific immune response. We show that sensory neurons utilize neuropeptide dependent signalling mechanisms to optimize immune effector production. We identify a set of E. faecalis specific immune effectors and another set of P. aeruginosa induced immune effector under the control of ciliated neurons of C. elegans. In all, this study delineates essential role of sensory perception in the regulation of pathogen-specific immunity in C. elegans.

Amon S et al. (2017) Data Brief "Multi-species alignments of C. elegans lin-11 intronic sequences and putative transcriptional regulators."

Amon S, Gupta BP

[Data Brief, 2017]

This data article contains multi-species alignments of the regulatory region of C. elegans LIM-HOX gene lin-11 and lists of transcription factors that are predicted to bind to lin-11 enhancers and regulate expression in amphid neurons. For further details and experimental findings please refer to the article by Amon and Gupta in Developmental Biology (S. Amon, B.P. Gupta, 2017) [1].

R Venkatesh, Siddharth et al. (2021) International Worm Meeting "Sensory Neurons Regulate Innate Immune Responses in Caenorhabditis elegans"

Gupta, Anjali, R Venkatesh, Siddharth, Singh, Varsha

[International Worm Meeting, 2021]

Caenorhabditis elegans, a bacterivorous forager, often encounters pathogenic microbes in its habitat. Upon encountering pathogens, the nematode either avoids them to minimise chances of infection, or induces an immune response to combat infection. Despite being equipped with only an innate immune system, recent studies have shown that the worm can induce selective responses against specific pathogens. To mount such responses, the worm has to recognise and differentiate among these pathogens. We hypothesised that the recognition of pathogens, induction of an immune response and its subsequent regulation is likely performed by the sensory neurons, since they are the primary site of sensory perception in the worm. We created stable genetic neuronal ablation lines of the amphid sensory neurons and tested for their roles during infections with Pseudomonas aeruginosa and Enterococcus faecalis. 9 out of the 11 neuron pairs tested showed involvement in regulating immune responses against these pathogens. We found 6 of these pairs to either have a P. aeruginosa or an E. faecalis specific function, while the remaining 3 pairs were involved in mounting an immune response against both pathogens. Only one of these 3 pairs showed a very strong involvement in mounting an effective immune response; we found this neuron to negatively regulate innate immunity in C. elegans. This neuron also suppresses immune responses against Salmonella enterica, Staphylococcus aureus and Cryptococcus neoformans, and might thus have a broader role in immune-homeostasis. RNASeq analysis of worms lacking this neuron showed an upregulation of several immune-related genes even at the basal level, along with a set of infection-inducible genes. In particular, we found a number of bZIP family transcription factors to be upregulated in these worms upon infection. Knockdown of three of the bZIP family transcription factors neutralised the enhanced resitance displayed by these neuron ablated worms against all tested pathogens. In conclusion, our study illustrates the roles of a chemosensory neuron that negatively regulates immune responses by downregulating bZIP family transcription factors, to maintain immune-homeostasis. With increasing evidences suggesting roles for the nervous system in immune regulation in humans and other higher vertebrates, the findings of our study could help us better understand neuroimmune regulation in higher animals.

Dasgupta, Madhumanti et al. (2019) International Worm Meeting "Nuclear hormone receptor NHR-49 shapes immune-metabolic response of Caenorhabditis elegans to Enterococcus faecalis infection."

Singh, Varsha, Gupta, Anjali, Javed, Salil, Shashikanth, Meghana, Dasgupta, Madhumanti, Bojanala, Nagagireesh

[International Worm Meeting, 2019]

Immune responses to pathogenic microbes include activation of resistance and tolerance mechanisms in the host both of which are energetically expensive. In this study, we show that C. elegans fed on Gram positive bacterium, Enterococcus faecalis rapidly utilize lipid droplets, the major energy reserve in the nematode. Feeding on E. faecalis causes developmental arrest in C. elegans larvae and growth arrest in adults, compared to E. coli diet. We find that nematode's early response to infection entails an increase in expression of 10 genes involved in lipid breakdown as early as 8 hours of exposure, suggesting that neutral lipids are utilized as energy source during E. faecalis infection. Feeding on this pathogen also leads to a concomitant decrease in expression of lipid synthesis genes in C. elegans. We also show that lipid droplets play a protective role in C. elegans during infection. NHR-49, a PPAR? ortholog, is required for E. faecalis induced beta-oxidation of fatty acids and immune effector production and thus regulates an immuno-metabolic axis required for survival of the nematode on E. faecalis.

(2021) Development "The people behind the papers - Clement Dubois, Shivam Gupta, Andrew Mugler and Marie-Anne Felix."

[Development, 2021]

Cell migration needs to be precisely regulated during development so that cells stop in the right position. A new paper in Development investigates the robustness of neuroblast migration in the <i>C. elegans</i> larva in the face of both genetic and environmental variation. To hear more about the story, we met the paper's four authors: Clement Dubois and Shivam Gupta, and their respective supervisors Andrew Mugler (currently Assistant Professor at the Department of Physics and Astronomy at the University of Pittsburgh, where his lab recently moved from Purdue University) and Marie-Anne Felix (Principal Investigator at Institut de Biologie de l'Ecole Normale Superieure in Paris and Research Director at CNRS).

Mallick, Avijit et al. (2019) International Worm Meeting "Axin-like scaffolding protein PRY-1 regulates lipid metabolism and lifespan in C. elegans."

Mallick, Avijit, Gupta, Bhagwati, Ranawade, Ayush

[International Worm Meeting, 2019]

Our lab is working on the C. elegans Axin-like scaffolding protein PRY-1 for its involvement in reproductive system development and aging [1, 2]. PRY-1 has been studied mainly in the context of canonical WNT signaling to regulate developmental processes. Since Axin family members also interact with WNT-independent pathways, pry-1 serves as a useful genetic tool to investigate the conserved mechanism of function of Axin proteins. During the process of investigating pry-1 role in worms, we found that pry-1 mutants have reduced lipid levels as well as a drastic reduction in lifespan. In agreement with the lowered lipid content, mutant worms also have smaller brood size and poor L1 survival. To understand how pry-1 regulates these processes, we set out to identify interacting genes using a genome-wide RNA-seq experiment in pry-1(mu38) strain. As expected from the phenotypes, the transcriptome profiling of pry-1 mutant revealed differentially expressed genes associated with aging and lipid metabolism, such as vitellogenins (yolk lipoproteins, vit), fatty acid desaturases, lipases, and fatty acid transporters. The expression of vits was found to be significantly upregulated in animals lacking pry-1 function. Additionally, knockdown of vit genes in a pry-1 mutant background restored lipid levels, suggesting that vitellogenins contribute to PRY-1 role in lipid metabolic processes. The reduced levels of lipids in pry-1 mutants led us to investigate changes in lipid metabolic pathways. Our Gas Chromatography-Mass Spectrometry analysis, combined by transcriptomic and other gene expression data, provided evidence that fatty acid synthesis is significantly reduced in pry-1 mutants. Consistent with this, an exogenous supply of oleic acid (mono-unsaturated fatty acid) restored lipids in somatic tissues of the mutant worms. We also found that sbp-1, one of the transcription factors involved in the regulation of desaturases is significantly downregulated in animals lacking pry-1 function. Since nhr-49 and nhr-80, two nuclear hormone receptor family members, are known regulators of fatty acid synthesis, we examined their role in pry-1 signaling. Our genetic interaction studies suggest that both these transcription factors act in parallel to sbp-1-mediated pry-1 pathway. Current research in the lab focuses on the mechanism of pry-1 function in lipid synthesis and its potential role in lifespan maintenance. References: [1] Ranawade A, Mallick A and Gupta BP (2018). PRY-1/Axin signaling regulates lipid metabolism in Caenorhabditis elegans. PLoS ONE 13(11):e0206540. [2] Seetharaman A, Cumbo P, Bojanala N and Gupta BP (2010). Conserved mechanism of Wnt signaling function in the specification of vulval precursor fates in C. elegans and C. briggsae. Dev Bio. 346(1):128-139.

Andrea M Skantar et al. (2001) International C. elegans Meeting "Molecular Analysis of daf-21, a multi-faceted Hsp90 gene, from Plant-parasitic Nematodes"

Lynn K Carta, Andrea M Skantar

[International C. elegans Meeting, 2001]

Many plant-parasitic nematodes hatch out of their eggs as a developmentally arrested pre-infective juveniles (L2) that share functional similarity to C. elegans dauers. Nothing is known about the genetic programming of development in these nematodes or how it may be linked to environmental cues. Nevertheless, it is likely that plant-parasitic nematodes use molecules similar to those defined by the C. elegans dauer pathway to regulate developmental arrest, lifespan, and chemosensation. The long-range goal of this research is to determine the molecular mechanisms controlling plant-parasitic nematode development in order to discover new targets for safe, specific and biologically based methods for controlling these damaging pathogens. The daf-21 gene from C. elegans encodes a molecular chaperone of the Hsp90 class (Birnby, et al., 2000). Mutation of daf-21 is dauer-constitutive, and has been shown to cause defects in chemosensory responses mediated by several classes of chemosensory neurons. The precise function of daf-21 in the dauer pathway is unclear. However, Hsp90 chaperones are known to refold denatured or misfolded proteins, especially under conditions of stress. In C. elegans , transcription of Hsp90 is increased 15-fold in dauers versus non-dauers, which would be consistent with this type of protective function (Dalley and Golomb, 1992). Under non-stress conditions, Hsp90 has been shown to guide the proper folding of specific target proteins, including nuclear hormone receptors and protein kinases. Possible targets of Hsp90 in C. elegans include the daf-11 transmembrane guanylate cyclase, the daf-5 putative Sno transcription factor, and the daf-12 nuclear hormone receptor. Additionally, the C. elegans aryl hydrocarbon receptor, AHR-1, has been shown to bind to Hsp90 (Powell-Coffman, et al ., 1998), suggesting daf-21 involvement in the response to environmental pollutants. We have focused on the cloning and characterization of Hsp90 ( daf-21 ) from several species of plant-parasitic nematodes of economic concern to agriculture. We have obtained full-length cDNA and partial genomic sequence from the soybean cyst nematode, Heterodera glycines , and are in the process of examining expression of the gene and its protein product. In addition, we have developed a set of PCR primers that amplify a portion of the Hsp90 gene from single nematode extracts. This technique has enabled us to isolate partial Hsp90 sequence from a wide range of agriculturally important nematodes, including cyst, root-knot, and lesion nematodes. We have identified variation in intron number and position in this amplification segment within the root-knot nematodes. This variation may comprise a useful diagnostic for several nematodes of agricultural concern, so we are now expanding this study to include more taxa. Hsp90 is a highly conserved protein whose sequence has been extensively used in determining the evolutionary relationships between eubacteria, archaebacteria, and eukaryotes (Gupta, 1998). Mutations in Hsp90 have been shown to unmask hidden genetic variation in fruit flies (Rutherford and Lindquist,1998) and therefore may play a critical role in regulating the evolutionary potential of an organism. Whether this phenomenon holds true for nematodes is unclear, however, we hope to gain insight into the function of plant-parasitic nematode Hsp90&#146;s by examining the sequences from a phylogenetic perspective. Birnby, et al., 2000. Genetics 155:85-104. Dalley and Golomb, 1992. Developmental Biology 151: 80-90. Powell-Coffman, et al., 1998. PNAS,95:2844-2849. Gupta, 1998. Microbiology and Molecular Biology Reviews. 62:1435-1491. Rutherford and Lindquist, 1998. Nature 396: 336-342.

Amon, Siavash et al. (2011) International Worm Meeting "Dissections of lin-11 neuronal enhancers in C. elegans and C. briggsae."

Amon, Siavash, Gupta, Bhagwati

[International Worm Meeting, 2011]

The LIM homeodomain transcription factor lin-11 plays an important role in the morphogenesis of the egg-laying system and differentiation of several neurons. We are dissecting the enhancer regions of lin-11 to understand its mechanism of transcriptional regulation. Our lab had earlier identified conserved vulva and uterine modules that are located in the 5' UTR (Marri and Gupta, 2009). The uterine module was shown to be regulated by fos-1/fos and two LIN-12/Notch pathway targets lag-1/Su(H) and egl-43/evi1. To identify the neuronal enhancers of lin-11 we are now focusing on its intronic regions. We have found that two of the lin-11 introns (intron 3 and intron 7) activate reporter gene expression in a subset of olfactory and chemosensory neurons. This suggests that the neuronal expression of lin-11 utilizes regulatory inputs distinct from those in the vulva and uterus. The corresponding introns in C. briggsae show similar expression profiles suggesting that the mechanism of lin-11 regulation is likely to be evolutionarily conserved. We did four-way sequence comparison of the two introns using C. briggsae, C. remanei, C. brenneri lin-11 orthologs and found limited conservation in some regions. It is likely that these regions are involved in regulating lin-11 expression. We are using bioinformatics to identify putative transcription factor binding sites, especially within the conserved sequences. The dissection of lin-11 introns will help us understand the signaling network of lin-11 and how it regulates neuronal differentiation. In parallel, we are also generating chimeric LIN-11 protein by swapping LIM and Homeodomain regions from other LIM-HOX family members to understand domain specificity in different cell types. These experiments will provide a better understanding of lin-11 function in nematodes and how it regulates developmental processes in multiple tissues.

Cohen, Sarah M et al. MicroPubl Biol

Sternberg, Paul W, Cohen, Sarah M

[MicroPubl Biol, 2019]

Genomic editing of the roundworm Caenorhabditis elegans using the CRISPR/Cas9 system has allowed for widespread creation of null mutants vital for scientific understanding of this model organism. A closely-related nematode species, Caenorhabditis briggsae, is emerging as an alternative model organism to better understand how findings in C. elegans can broaden and develop the larger field of nematology (Gupta et al. 2007). To that end, we have developed an effective and efficient co-conversion CRISPR/Cas9 system for use in C. briggsae using the gene dpy-10. We modified the universal STOP-IN cassette method, as described by Wang et al. (2018), for use in C. briggsae (Wang et al. 2018). Using the wildtype AF16 strain, we tested the method by choosing the gene Cbr-dpy-10 because of its readily observed predicted phenotype. Cbr-dpy-10 is a predicted one-to-one ortholog of C. elegans dpy-10, which encodes a protein important for cuticle development and has a phenotype characterized by short, fat animals relative to wild type (Brenner 1974). A universal STOP-IN allele of Cbr-dpy-10, sy1387, was generated and confirmed by genotype sequencing. The expected phenotype was subsequently observed, consistent with the creation of a null allele (Figure 1). Surprisingly, sy1387 is dominant. Injections of 20 animals produced 15 successful injections; 247 animals from these 15 injections (F1) were singled out and allowed to self-fertilize to produce F2. 81% of the F1 Dumpy progeny were homozygous as evidenced by their segregation of only Dumpy progeny one of these candidates became sy1387. 2% of the F1s did not produce progeny. 17% of the 247 F1 Dumpy progeny were heterozygotes and had a mixture of Dumpy and non-Dumpy progeny; these non-Dumpy F2s only produced non-Dumpy progeny. This discovery of a dominant mutation will allow for more effective use of this as a co-conversion marker when screening for other mutations. We used Cbr-dpy-10 as a potential co-CRISPR marker for a second target that will be described elsewhere. We used PCR to detect insertion of a STOP-IN cassette at this other locus; we screened 39 Dumpy strains to obtain 11 candidates for our target gene, from which we have three STOP-IN alleles.

Baytek, G. et al. (2019) International Worm Meeting "The impact of sumoylation on MRG-1's role in germline safeguarding."

Popp, O., Tursun, B., ul Fatima, N., Mertins, P., Bulut, S., Wolfram, T., Baytek, G.

[International Worm Meeting, 2019]

Direct reprogramming of cellular identities by ectopically-expressed transcription factors (TFs) is highly regulated by a variety of factors including post-translational modifications. In a recent RNAi screen to identify reprogramming barriers in C. elegans, we found that the conserved chromatin regulator MRG-1 safeguards germ cells against ectopic fate induction and conversion to neuron-like cells (Hajduskova and Baytek et al., 2018). In order to reveal whether MRG-1 requires other interacting proteins to protect the germ cell identity, we established a robust Immunoprecipitation protocol followed by Mass Spectrometry (IP-MS). In addition to using the N2 background with specific MRG-1 antibodies, we generated a 3xHA knock-in using CRISPR to obtain a confident set of MRG-1-interacting proteins upon comparing anti-MRG-1 and 3xHA antibody-based IP-MS results. Consistently, SMO-1 was one the most robust interactors of MRG-1 and further protein-biochemical analysis revealed that MRG-1 is post-translationally modified by sumoylation. Interestingly, smo-1 and mrg-1 mutants display defects in related pathways including DNA repair (Psakhye and Jentsch, 2012 ; Bleuyard et al. 2017), repressing X-linked genes (Meyer et al 2010; Dombecki et al. 2011; Xu et al. 2012), chromosome pairing (Cheng et al., 2006; Fujita et al. 2002), and germ cell proliferation (Jones et al, 2002; Gupta et al. 2015). Furthermore, we revealed that MRG-1 associates predominantly with genomic sites that carry the histone modification H3K36me3 and SUMO has been shown to enforce distinct chromatin states to protect cell identities in other species (Cossec et al. 2018). We therefore analyzed the impact of sumoylation on the DNA-binding pattern of MRG-1 and performed ChIP-seq upon smo-1 depletion. Since the lack of MRG-1 sumoylation appears to affect the genomic MRG-1 binding pattern, we mutated lysine residues of MRG-1 that are predicted as sumoylation sites. Overall, our findings indicate that sumoylation impacts MRG-1's function in regulating chromatin in order to protect germ cell identity, which might be a conserved mechanism to post-translationally regulate also the MRG-1 homolog MRG15 in other species.