Singh, Varsha et al. (2011) International Worm Meeting "Neuronal control of stress response and innate immunity in C. elegans."

Singh, Varsha, Aballay, Alejandro, Sun, Jingru

[International Worm Meeting, 2011]

Heat Shock Protein (HSP) and Unfolded Protein Response (UPR) genes are upregulated in response to various stresses including heat shock, oxidative stress and ER stress. In C. elegans, HSP and UPR genes are also required for an adequate immune response to pathogens. Recent studies have shown that thermosensory neurons can regulate HSP gene induction at the organismal level. Increasing evidence also indicates that the nervous system of C. elegans controls avoidance to certain pathogens and activation of innate immune pathways. To identify neurons capable of controlling the activation of HSP or UPR genes as well as innate immune responses to pathogens, we screened mutants, in genes involved in the function of sensory neurons, for expression of stress proteins in response to bacterial pathogens and their pathogen susceptibility. Mutation in thermosensory neurons affected not only the expression of stress proteins but also pathogen susceptibility. Additional sensory neurons suppressed innate immunity while others enhanced innate immunity.

Singh, Varsha et al. (2009) International Worm Meeting "Over-activation of FOXO transcription factor, DAF-16, causes pathogen susceptibility in Caenorhabditis elegans."

Aballay, Alejandro, Singh, Varsha

[International Worm Meeting, 2009]

DAF-16 is a FOXO transcription factor involved in regulation of metabolism and stress response in C. elegans. Previous studies have shown that overexpression of DAF-16 increases lifespan and resistance to pathogens . Here we report that hyper-activation of DAF-16 may lead to deleterious consequences during infection with bacterial pathogens. We also provide evidence that a stress-inducible Heat Shock Factor (HSF)-1 is utilized to control DAF-16 activitation by promoting its export from nucleus. Effect of HSF-1 appears to be mediated through Hsp70 which is also induced and translocated to nucleus during stress. Aquaporin upregulation is partly responsible for enhanced susceptibility to pathogens. Susceptibility due to DAF-16 activation could also be rescued by increasing osmolarilty of the exogenous medium indicating that osmotic balance is an important aspect of infection. In summary, while overexpression of DAF-16 is beneficial, its sustained activation leads to enhanced susceptibility to pathogens. C. elegans utilizes chaperones to promote DAF-16 nuclear export in a timely manner and prevent susceptibility.

Singh, Varsha et al. (2021) International Worm Meeting "Neuronal control of lipid metabolism by STR-2 G protein-coupled receptor promotes longevity in C. elegans"

Dixit, Anubhuti, Shashikanth, Meghana, Koushika, Sandhya, Singh, Varsha, Modi, Souvik, Sandhu, Anjali, Watts, Jennifer

[International Worm Meeting, 2021]

The G proteincoupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. We find that C. elegans longevity is regulated by a chemosensory GPCR STR2, expressed in AWC and ASI amphid sensory neurons. STR2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR2 regulates expression of delta9 desaturases, fat5, fat6 and fat7, and of diacylglycerol acyltransferase dgat2. Rescue of the STR2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat5, dgat2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wildtype levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.

Varsha Singh et al. (2007) International Worm Meeting "Activation of Innate Immunity to bacterial pathogens through Heat Shock pathway."

Alejandro Aballay, Varsha Singh

[International Worm Meeting, 2007]

Using C. elegans as a model host for infection with bacterial pathogens, we have shown that a heat-inducible pathway regulated by Heat Shock transcription Factor (HSF)-1 is required for innate immunity to Gram negative and Gram positive bacterial pathogens. HSF-1 contributes to immunity through Heat Shock Proteins (HSPs) and is independent of a previously reported p38 MAPK pathway which is also required for immunity. Increased resistance of insulin-like signaling pathway mutant, daf-2, is dependent on HSF-1. Decline in heat shock response also correlates with immunosenescence in C. elegans suggesting that activation of HSF-1 may be critical for a sustained immune response. In mammalian cells, ERK MAPK is required for deactivation of HSF-1 during recovery after heat stress. Therefore, lack of ERK MAPK would be expected to lead to prolonged activation of HSF-1 and increased resistance to pathogens. Consistent with that, we find that inactivation of erk by RNAi or mutation leads to increased resistance to pathogens in C. elegans. The enhanced resistance to pathogens (erp) phenotype of erk RNAi animals can be seen in wild type and daf-2 mutant but not in the hsf-1 mutant confirming that HSF-1 is responsible for protective effects of erk knockdown. We will present our approach to identify additional regulators of HSF-1 activation.

Prakash, Deep et al. (2021) International Worm Meeting "Pseudomonas aeruginosa Associated Volatiles Drive Chemotactic Behaviour and Immune Response In C. elegans"

Prakash, Deep, Danggen, Kaling, Singh, Varsha

[International Worm Meeting, 2021]

Olfaction plays an important role in threat perception in higher organisms. To understand the role of olfaction in host survival, we studied interaction of C. elegans with Pseudomonas aeruginosa, a bacterium commonly found in the same ecological niche as this nematode. P. aeruginosa acts as a pathogen against C. elegans, colonizing its gut and reducing its survival. It is known that C. elegans actively detects and avoids P. aeruginosa (Zhang et al. Nature 2005). We have identified two volatiles produced by P. aeruginosa that elicit a behavioural response in C. elegans. One of the chemicals is an attractant and the other is a repellent for C. elegans. The amphid neurons AWA, AWB and AWC are mainly responsible for detecting volatile odorants. While AWA and AWC detect attractants, AWB is responsible for detecting repellents. By studying calcium dynamics in olfactory neurons, we have identified specific neurons which detect volatiles from P. aeruginosa. Through qPCR analysis, we have observed induction of P. aeruginosa specific immune response genes upon exposure to the volatile repellent. The induction of immune response genes is dependent on functional olfactory neurons. Further, pre-exposure of worms to the identified repellent also increases their survival on Pseudomonas aeruginosa lawn. Keywords: Host-microbe interactions, Olfaction, Chemotaxis, Immune response, Pseudomonas aeruginosa, Volatiles

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.

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.

Sun, Jingru et al. (2011) International Worm Meeting "G-protein coupled receptor OCTR-1 controls innate immunity by regulating non-canonical unfolded protein response genes."

Sun, Jingru, Aballay, Alejandro, Kajino-Sakamoto, Rie, Singh, Varsha

[International Worm Meeting, 2011]

The endoplasmic reticulum (ER) has developed specific signaling pathways called the unfolded protein response (UPR) to cope with ER stress and restore ER homeostasis. Recent studies indicate that the increased demand on protein folding in the ER, that may occur during bacterial infections, must be successfully alleviated by canonical and non-canonical UPR pathways for a complete immune response to be mounted. We found that the nervous system controls the activity of a non-canonical UPR pathway required for innate immunity in Caenorhabditis elegans. OCTR-1, a G-protein coupled receptor, functions in ASH and ASI neurons to actively suppress innate immune responses by down-regulating the expression of non-canonical UPR genes pqn/abu in non-neuronal tissues. Pseudomonas aeruginosa exposure up-regulates pqn/abu genes to levels comparable to those induced by treatment with the ER stressor tunicamycin. Our studies highlight pathways that may play important roles at dealing with pathogens directly and with the damages caused by the infection.

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.

Huang, Huiyan et al. (2013) International Worm Meeting "A genetic screen for Notch downstream targets regulating C. elegans sleep."

Zhu, Chen-Tsen, Hart, Anne, Huang, Huiyan

[International Worm Meeting, 2013]

The conserved Notch signaling pathway plays well-defined roles in cell fate determination during development. Recently, Notch has also been implicated in non-developmental neuronal processes across species, including invertebrate sleep. In C. elegans, the level of Notch activity affects both quality and quantity of lethargus quiescence, a sleep-like behavior coinciding with larval molts. Also, transiently overexpressing the Notch co-ligand, OSM-11, is sufficient to drive inappropriate quiescence in adult animals, which can be suppressed by loss of either Notch receptor or downstream players in Notch signaling (Singh et al., 2011). To find pertinent transcriptional targets of the Notch pathway and to gain insight into regulation of sleep, we undertook a classical suppressor screen to identify suppressors of inappropriate quiescence in adult hsp::osm-11 animals. In this screen, 2122 mutant lines were assessed; 79 independent isolates suppressed the OSM-11-induced inappropriate quiescence. To exclude mutations with pervasive effects on locomotion, we examined endogenous L4/adult lethargus quiescence in these 79 strains using both an adaption of the Multi-Worm Tracker (Swierczek et al., 2011) and the microfluidic chamber-based assay (Singh et al., 2011). Twenty-seven strains had defects in endogenous lethargus quiescence. We are in the process of identifying the corresponding genes by whole genome sequencing. As Notch signaling also modulates sleep behavior in Drosophila (Seugnet et al., 2011), we are confident that the genes identified in this screen will be conserved regulators of arousal and sleep across species.