Poorna Viswanathan et al. (2007) International Worm Meeting "Virulence characteristics of diverse B.cenocepacia isolates and the identification of virulence factors using the C.elegans model for pathogenicity."

Todd Ciche, Poorna Viswanathan, Martha Mulks, Luke Gray

[International Worm Meeting, 2007]

The C.elegans model for pathogenic host-bacteria interactions has been used extensively to study virulence many pathogens including Pseudomonas aeruginosa, an important opportunistic pathogen for cystic fibrosis patients. P.aeruginosa kills C.elegans by producing toxins on PGS media (fast killing) or by infecting nematodes on NGM media (slow killing). Burkholderia cenocepacia is a ubiquitous organism that is also an important opportunistic pathogen for CF patients. We used the nematode model to determine virulence characteristics of clinical, agricultural and environmental isolates and as an animal host to study virulence in select strains of B.cenocepacia. The virulence characteristics of some B.cenocepacia are similar to P.aeruginosa(e.g. Midwest clone PC184), however some strains cause rapid mortality on NGM (e.g. AU1054, HI2424, and MI onion field isolate 6RT-130) and PGS, while other strains kill slowly on PGS or do not kill at all. B.cenocepaciaAU1054 is a Mid-Atlantic CF-patient isolate, that is pathogenic to onions, virulent to worms and has anti-fungal properties. Because of this broad host range, we focused on this strain to understand its mechanism of C.elegans killing and to identify its virulence genes by transposon mutagenesis. We have used GFP-labeled B.cenocepaciaAU1054 in our experiments. B.cenocepaciaAU1054 appears to kill C. elegans by infections and by producing toxins. To identify the virulence genes, we used transposon mutagenesis and screened for mutants that did not kill C.elegans at 72 h on NGM plates. We have four mutants that appear to be avirulent to worms. Work is in progress to identify the gene(s) that have been interrupted by the transposon in these mutants, to determine if these genes encode general or specific virulence factors and to characterize the virulence of AU1054 to C.elegans. In summary, C.elegans has proved useful for assessing virulence characteristics of diverse B.cenocepacia strains and to identify virulence factors present in select strains.

Antol, Weronika et al. (2022) MicroPubl Biol

Sychta, Karolina, Prokop, Zofia M., Antol, Weronika, Palka, Joanna K., Dudek, Katarzyna

[MicroPubl Biol, 2022]

Caenorhabditis elegans is a model species, increasingly used in experimental evolution studies to investigate such major topics as: maintenance of genetic variation, host-pathogen interaction and coevolution, mutations, life history, evolution of reproductive systems, sexual selection (Gray and Cutter, 2014; Teotnio, Estes, Phillips, and Baer, 2017). Its reproductive system in the wild, known as androdioecy, involves mostly self-fertilization of hermaphrodites and occasionally outcrossing with males, which are generally rare (Stewart and Phillips, 2002). This system can be experimentally changed to dioecy, i.e., obligatory outcrossing, through genetic manipulations (see Table I in Anderson, Morran, and Phillips, 2010; Gray and Cutter, 2014).

Marciniak SJ et al. (2008) Trends Cell Biol "Intracellular serpins, firewalls and tissue necrosis."

Marciniak SJ, Lomas DA

[Trends Cell Biol, 2008]

Luke and colleagues have recently attributed a new role to a member of the serpin superfamily of serine proteinase inhibitors. They have used Caenorhabditis elegans to show that an intracellular serpin is crucial for maintaining lysosomal integrity. We examine the role of this firewall in preventing necrosis and attempt to integrate this with current theories of stress-induced protein degradation. We discuss how mutant serpins cause disease either through polymerization or now, perhaps, by unleashing necrosis.

Chalasani, Sreekanth H. et al. (2009) International Worm Meeting "AWC sensory neurons release two neurotransmitters to regulate exploratory behavior upon removal from food."

Albrecht, Dirk, Bargmann, Cornelia I., Chalasani, Sreekanth H., Kato, Saul

[International Worm Meeting, 2009]

Animals increase their pirouette frequency in response to removal from food stimulus for a period of 15 min. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that AWC sensory neurons become active in response to removal of stimulus, releasing two neurotransmitters (glutamate and a neuropeptide NLP-1). The released glutamate acts to activate AIB and inhibit AIY interneurons, promoting reversals (Chalasani et al 2007). In contrast to glutamate, AWC-released NLP-1 acts on AIA interneurons to suppress reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. AWC calcium responses are modulated in these neurotransmitter mutants, suggesting that feedback pathways affect AWC neuronal activity. References: Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., and Bargmann, C. I. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70. Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Avery L (1995) Worm Breeder's Gazette "Effects of Conus venoms on the pharynx"

Avery L

[Worm Breeder's Gazette, 1995]

Cone snails are predatory snails. They harpoon their prey, which may be small fish, marine worms, or other snails, then inject a paralyzing venom through the hollow harpoon (Olivera et al, Science 249: 257). The venom is a complex mixture of tiny peptide toxins, different in each species. Several of these toxins have been shown to act on cell-surface molecules important for nervous system function such as the N-type Ca++ channel or the voltage-gated Na+ channel (Gray et al, Ann Rev Biochem 57: 665).

Chalasani S H et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Neuropeptide feedback modifies odor induced responses in Caenorhabditis elegans olfactory neurons"

Bargmann C I, Kato S, Chalasani S H, Albrecht D R

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Neural circuits transform sensory signals to generate behaviors on timescales from seconds to hours. In some C.elegans behaviors, sensory inputs lead to long lasting and complex behavioral outputs. Animals that have been removed from food spend about 15 minutes exploring a local area by interrupting long forward movements with reversals and turns (Wakabayashi et al., 2004, Gray et al 2005). AWC sensory neurons regulate this behavior by releasing two neurotransmitters, glutamate (promoting turns) and NLP-1 (inhibiting turns). AWC sensory neuron released glutamate activates AIB and inhibits AIY and AIA interneurons (Chalasani et al 2007). In contrast to glutamate, AWC neuron released NLP-1 acts on AIA interneurons to suppress reversals, indicating that turn frequencies are regulated by at least two opposing systems. AWC calcium responses are modulated in these neurotransmitter mutants suggesting that multiple pathways can influence AWC dependent behavior and neuronal activity. ReferencesChalasani, S. H., et. al. Dissecting a neural circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70 (2007).Gray, J.M., et. al. A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191 (2005).Wakabayashi, T., et. al. Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111 (2004).

Sreekanth Chalasani et al. (2007) International Worm Meeting "Functional analysis of a neural circuit controlling turning behavior in Caenorhabditis elegans."

Cornelia Bargmann, Jesse Gray, Makoto Tsunozaki, Sreekanth Chalasani, Nikolas Chronis

[International Worm Meeting, 2007]

C. elegans increase its frequency of reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999) after removal of a food stimulus. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that the sensory neuron AWC releases two neurotransmitters (glutamate and a neuropeptide, NLP-1) when the worm is removed from food. The released glutamate acts to activate AIB and inhibit AIY, promoting reversals. Strains with different reversal frequencies can be generated by manipulating the level of glutamate receptors on interneurons AIB and AIY. Decreasing receptor expression leads to fewer reversals, and increasing receptor expression results in more reversals than in wild-type. The AWC released neuropeptide NLP-1 serves to reduce reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. Consistent with behavioral responses, AWC and AIB respond (by increasing calcium concentration) to removal of stimulus. We plan to extend the imaging studies to other neurons in the circuit. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. References: Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Pierce-Shimomura, J.T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci 19, 9557-9569. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Sreekanth H Chalasani et al. (2005) International Worm Meeting "Molecular analysis of a neural circuit for navigation in Caenorhabditis elegans."

Sreekanth H Chalasani, Nikolas Chronis, Jesse M Gray, Cornelia I Bargmann

[International Worm Meeting, 2005]

Navigation in C.elegans is achieved by sustained forward movement that is interrupted with reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999). We are interested in the neural circuit that controls the frequency of reversals and turns during exploratory behavior. After worms are taken off bacterial food, they exhibit an initial local search with a high frequency of pirouettes. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY interneurons inhibit pirouettes. (Tsalik and Hobert 2003, Wakabayashi et al 2004, Gray et al 2005). How is activity transmitted through this neuronal circuit? The neurotransmitters glutamate and dopamine regulate turning frequency (Hills et al 2004). We found that the vesicular glutamate transporter EAT-4 is essential for the generation of pirouettes after removal from food. Using cell-specific rescue of eat-4 mutants, we show that both AWC and ASK sensory neurons can release glutamate to stimulate pirouettes. The released glutamate appears to be sensed by a glutamate-gated chloride channel (GLC-3) that inhibits the AIY interneurons, and the glutamate-gated cation channel GLR-1, which stimulates the AIB interneurons. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. We are currently attempting to image neuronal activity in these neurons using genetically encoded calcium sensors. References: Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Hills, T., Brockie, P.J., and Maricq, A.V. (2004). Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans. J. Neurosci 24, 1217-1225. Pierce-Shimomura, T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci 19, 9557-9569. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Makoto Tsunozaki et al. (2005) International Worm Meeting "A mutation in ODR-11 reverses AWC-mediated butanone responses from attraction to avoidance"

Makoto Tsunozaki, Cori Bargmann

[International Worm Meeting, 2005]

When wild-type C. elegans is exposed to an odor, the identity of the cell that senses the odor specifies its behavioral response. The AWA and AWC neurons mediate attraction, and AWB mediates an avoidance response. Ectopic expression, in AWB, of a receptor for a normally attractive odor can reprogram the animals response to the ligand from attraction to avoidance (Troemel et al., 1997). Here we describe a mutant, odr-11(ky713), in which AWC mediates an avoidance response to a normally attractive odor, butanone. odr-11 animals are defective in chemotaxis to AWC-sensed odors but have normal responses to AWA- and AWB-sensed odors. Mutant animals avoid butanone and show reduced chemotaxis to benzaldehyde, isoamylalcohol, and 2,3-pentanedione. A ceh-36 mutation eliminates AWC neurons, and butanone avoidance is absent in odr-11;ceh-36 mutants. Mutations in odr-1, a transmembrane guanylyl cyclase, and tax-4, a cyclic nucleotide-gated channel subunit, also suppress butanone avoidance in odr-11. These and other double mutant phenotypes suggest that butanone avoidance in odr-11 mutants requires cGMP signaling in the AWC neurons. We are also using an odor flow assay to quantitatively analyze the behavior of wild-type and odr-11 animals in response to temporal changes in olfactory stimuli. Wild-type animals respond to a step increase in butanone concentration by suppressing reversals, while they respond to a step decrease by increasing the frequency of reversals. These observations are consistent with the biased random-walk model for chemotaxis (Pierce-Shimomura et al., 1999). We are currently examining odr-11 responses. The removal-from-food assay measures AWC-regulated behavioral responses (Gray et al., 2005). Wild-type animals have a high frequency of reversals immediately after removal from food, and gradually suppress reversals over 30 minutes. odr-11 animals show a higher frequency, relative to wild type, of reversals in the earlier times off of food, and the subsequent decline in reversal frequency is faster. We are testing the idea that ODR-11 regulates the time course of behavioral state changes in response to sensory stimuli. References: Troemel et al. (1997) Cell 91:161. Pierce-Shimomura et al. (1999) J. Neurosci. 19:9557. Gray et al. (2005) PNAS 102:3184.

Galles, Celina et al. MicroPubl Biol

Galles, Celina, De Mendoza, Diego, Perez, Gaston M

[MicroPubl Biol, 2018]

daf-7 expression in ASI neurons is increased when nematodes are grown in the presence of endocannabinoid 2-arachidonoyl glycerol (2-AG). Left upper panel: representative image of reporter strain FK181 ksIs2 [Pdaf-7::GFP+rol-6(su1006)] captured under GFP epifluorescence microscopy. Right upper panel: same individual showing bright field microscopy together with GFP signal. Gray/black triangles mark ASI neurons expressing daf-7. Scale bars correspond to 0.03 mm. Lower panel: point plot of the adjusted GFP intensity/ASI neuron values gathered from worms treated either with the carrier solvent or with 2-AG. 2-AG treatment significantly increases daf-7 expression in ASI neurons. (*) indicates statistically significant difference with solvent control condition; the difference in the median values of the relative adjusted GFP fluorescence/ASI neuron between the two groups is greater than would be expected by chance; there is a statistically significant difference between solvent and 2-AG (Mann-Whitney Rank Sum Test, p=<0.001). The number of independent experiments carried out was three. The total number of ASI neurons analyzed was 70 for each condition.