Immediate activation of chemosensory neuron gene expression by bacterial metabolites is selectively induced by distinct cyclic GMP-dependent pathways in C. elegans

Dynamic gene expression in neurons shapes fundamental processes of the nervous systems of animals. But how different stimuli that activate the same neuron can lead to distinct transcriptional responses remains unclear. We have been studying how microbial metabolites modulate gene expression in chemosensory neurons of Caenorhabditis elegans. Considering the diverse environmental stimuli that can activate chemosensory neurons of C. elegans, we have sought to understand how specific transcriptional responses can be generated in these neurons in response to distinct cues. We have focused on the mechanism of rapid (<6 min) and selective transcriptional induction of daf-7, a gene encoding a TGF-β ligand that promotes bacterial lawn avoidance, in the ASJ chemosensory neurons in response to the pathogenic bacterium Pseudomonas aeruginosa. Here, we define the involvement of two distinct cyclic GMP (cGMP)-dependent pathways that are required for daf-7 expression in the ASJ neuron pair in response to P. aeruginosa. We show that a calcium-independent pathway dependent on the cGMP-dependent protein kinase G (PKG) EGL-4, and a canonical calcium-dependent signaling pathway dependent on the activity of a cyclic nucleotide-gated channel subunit CNG-2, function in parallel to activate rapid, selective transcription of daf-7 in response to P. aeruginosa metabolites. Our data suggest a requirement for PKG in promoting the fast, selective early transcription of neuronal genes in shaping responses to distinct microbial stimuli in a pair of chemosensory neurons of C. elegans. Author Summary The nervous systems of animals carry out the crucial roles of sensing and interpreting the external environment. When the free-living microscopic roundworm C. elegans is exposed to the pathogenic bacteria Pseudomonas aeruginosa, sensory neurons detect metabolites produced by the pathogen and induce expression of the gene for a neuroendocrine ligand called DAF-7. In turn, activity of DAF-7 is required for the full avoidance response to the P. aeruginosa, allowing the animals to reduce bacterial load and survive longer. Here, we systematically dissect the molecular pathway between the sensation of P. aeruginosa metabolites and the expression of daf-7 in a pair of C. elegans sensory neurons. We show that the intracellular signaling molecule cyclic GMP is a key signaling intermediate. In addition, we show that there are calcium-dependent and calcium-independent pathways that are both required to engage daf-7 expression, highlighting an organizational principle that allows neurons to distinguish between various stimuli.


stimuli.
Introduction 50 Chemosensory systems of animals transduce external chemical stimuli into neuronal signals, 51 with diverse roles in animal physiology (1-3). A challenge for chemosensory systems is to detect 52 and process a wide diversity of environmental information to generate corresponding appropriate 53 neuronal and behavioral responses. Whereas neurons utilize electrical impulses in rapid data 54 transmission, changes in gene expression serve as a mechanism for transducing information over 55 a longer time scale. Activity-dependent transcription of immediate-early genes has been shown 56 to involve the activation of calcium-dependent signal transduction converging on CREB (4). Our  Interactions with microbes, in a number of forms such as parasitism, symbiosis, predation, and 62 exploitation, have shaped the evolution of animals. There has been an increasing appreciation for 63 the role of the nervous system in recognizing and responding to microbes in the environment. 64 Disgust, for example in response to rotting food, elicits avoidance behavior (7). At the cellular Pdaf-7::gfp expression in the ASJ neurons ( Figure 1F, G, J), suggestive that CNG-2 functions in 119 a cell-autonomous manner in the ASJ neurons to mediate daf-7 upregulation in response to 120 Pseudomonas infection.

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We previously showed that exposure to the P. aeruginosa secondary metabolite, phenazine-1-123 carboxamide (PCN), results not only in the induction of daf-7 expression in the ASJ neuron pair, 124 but also in a rapid increase of calcium levels in the ASJ neurons (6). The molecular identity of 125 CNG-2 and relevant literature on the chemosensory apparatus in the ASJ neurons (13) led us to 126 test whether CNG-2 might be an integral part of the cation channel that is responsible for the 127 observed calcium influx. We observed that the influx of calcium ions in the ASJ neurons that is 128 observed upon exposure of wild-type animals to the P. aeruginosa metabolite phenazine-1-129 carboxamide was abrogated in cng-2 animals ( Figure 1M, N).

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We next examined mutants carrying mutations in the genes cmk-1, the C. elegans homolog of 132 calcium/calmodulin-dependent kinase CaMKI/IV, and crh-1, the C. elegans homolog of the 133 transcription factor CREB, and we observed that both genes are required for daf-7 expression in 134 the ASJ neurons ( Figure 1H, I, K, L). We note that both cmk-1 and crh-1 mutants also showed 135 minimal expression of daf-7 in the ASI neurons ( Figure 1H, I, K, L), raising the possibility of 136 additional pleiotropic effects on the development and/or physiology of the nervous systems of 137 these mutants. Nevertheless, expressing crh-1 cDNA in only the ASJ neurons was able to rescue 138 daf-7 expression in crh-1 mutants (Figure 1-supplement 1B), indicating CRH-1 is likely to 139 function cell-autonomously in the ASJ neurons to regulate daf-7 expression in response to P. 140 aeruginosa. These data implicate a canonical calcium-dependent signaling pathway downstream of CNG-2 that converges on CRH-1 to activate daf-7 expression in response to phenazine-1carboxamide.
143 144 cGMP-dependent signal transduction activates daf-7 expression in the ASJ neurons 145 The requirements for components of a cyclic nucleotide-gated channel, CNG-2/TAX-2/TAX-4, 146 and DAF-11, a guanylate cyclase, in the induction of Pdaf-7::gfp expression in response to P. 147 aeruginosa metabolites suggested the involvement of cGMP-dependent signaling. We 148 demonstrated that mutations in the gcy-12 gene, encoding another guanylate cyclase subunit, also 149 caused markedly reduced Pdaf-7::gfp expression in the ASJ neurons in response to P. 150 aeruginosa, but did not abolish Pdaf-7::gfp expression in the ASI neuron pair as was observed  Figure 2B), suggestive that the PDEs function redundantly in the ASJ neurons. We also 162 examined the effect of addition of a cell-permeable, non-hydrolysable analog of cGMP, pCPT-cGMP, to wild-type animals in the absence of P. aeruginosa, and we observed the marked 164 induction of expression of daf-7 in the ASJ neurons ( Figure 2C).

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The cGMP-dependent protein kinase G EGL-4 upregulates daf-7 expression in ASJ 167 neurons in response to P. aeruginosa 168 The involvement of cGMP-and calcium-dependent signaling support a role for canonical 169 activity-dependent signaling pathways in the induction of daf-7 expression in response to P. 170 aeruginosa metabolites. However, prior studies have shown that multiple stimuli including low 171 pH, E. coli supernatant, sodium chloride, temperature changes, and even water can cause calcium 172 influx in the ASJ neurons without the robust upregulation of daf-7 observed in the presence of P. 173 aeruginosa (15-17). We sought to define additional, calcium-independent mechanisms that 174 might be involved in the selective transcriptional response to P. aeruginosa metabolites.

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The dependence of daf-7 expression in the ASJ neurons on cGMP led us to consider the 177 involvement of the cGMP-dependent protein kinase G (PKG), EGL-4. EGL-4 has been 178 implicated in various phenotypes including egg-laying behavior, chemosensory behavior, sleep-179 like state, satiety signaling, and aversive learning behaviors (19-29). We observed that 180 presumptive loss-of-function egl-4(n478) and egl-4(n479) mutants exhibited a lack of Pdaf-181 7::gfp expression in the ASJ neurons in response to P. aeruginosa ( Figure 3A-C, E). Expression 182 of egl-4 cDNA in the ASJ neurons was sufficient to rescue daf-7 expression on P. aeruginosa 183 ( Figure 3D, E). A gain-of-function allele, egl-4(ad450), exhibited detectable expression of daf-7 184 expression in the ASJ neurons even in the absence of P. aeruginosa ( Figure 4A). In order to 185 examine whether EGL-4 functioned in a calcium-dependent or calcium-independent manner, we examined how egl-4 loss-of-function affected the influx of calcium into the ASJ neurons 187 observed upon exposure to phenazine-1-carboxamide. We found that unlike cng-2(tm4267) 188 mutants, egl-4(n479) mutants showed a wild-type calcium level increase in ASJ neurons upon 189 exposure to phenazine-1-carboxamide ( Figure 3F, G). To better define the respective roles and interaction between cng-2 and egl-4, we again utilized 201 the gain-of-function allele of egl-4, ad450. In the egl-4(ad450) cng-2(qd254) double mutant, the 202 expression we observed in the egl-4(ad450) mutant was abolished ( Figure 4A), which suggested 203 that basal CNG-2-dependent calcium-dependent signaling in the absence of P. aeruginosa is 204 required for the observed daf-7 expression. We further sought to gain clarity regarding the 205 pathway involving cng-2 and egl-4 by seeing how each of the mutants might change their daf-7 206 expression in response to the addition of pCPT-cGMP. We first tested mutants of the 207 heterotrimeric G-protein gpa-2(pk16) and gpa-3(pk35). These proteins are thought to act in the 208 initial steps of the chemosensory cascade by associating with the presumptive receptor for P. aeruginosa metabolites, and we have previously shown that the gpa-2 gpa-3 double mutant is 210 defective in daf-7 transcription in response to P. aeruginosa (6). Consistent with this prediction, 211 adding pCPT-cGMP to gpa-2 gpa-3 double mutants elicited the same induction of Pdaf-7::gfp 212 expression in the ASJ neurons as observed in wild-type animals ( Figure 4B). However, when 213 pCPT-cGMP was added to cng-2 and egl-4 mutants, the response was markedly attenuated or The neuroendocrine TGF-beta ligand, DAF-7, is rapidly transcribed in the ASJ neurons upon 220 exposure to P. aeruginosa metabolites (6). In this study, we have identified and characterized

C. elegans Strains
All animals were maintained and fed as previously described (36). The animals were incubated at 20°C unless any For experiments quantifying the level of Pdaf-7::gfp on the Pseudomonas aeruginosa strain PA14, bacteria was 324 cultured overnight in 3 mL LB broth at 37°C, and the following day 7 μl was seeded onto 3.5cm slow-killing assay 325 (SKA) plates as described previously (14). The seeded plates were maintained at 37°C overnight and then 326 transferred to room temperature, where they were kept additional two days before experiments. To preemptively rid 327 animals of bacterial contamination, gravids were bleached to get a large amount of eggs. Animals were loaded onto 328 PA14 at stage L4 and then were kept at 25°C for 14-16 hours before quantification. For assays using pCPT-cGMP, 329 pCPT-cGMP was added to SKA plates in mixed DMSO and water, with the resulting concentration in plates to be 5 330 mM. Plates were left overnight for the chemical to diffuse. The next day, 5 μl inoculate of E. coli strain OP50 was 331 seeded to the middle, and plates were kept in room temperature overnight before experiments commenced. Animals 332 were similarly egg-prepped for this condition as noted above. L4s were loaded onto the center of the SKA plates and 333 kept at 20°C for 17-20 hrs before quantification.

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The animals were immobilized and exposed to soluble compound in a controlled manner using a microfluidics chip 350 as previously described (38). Imaging was carried out at 40x with a Zeiss Axiovert S100 inverted microscope 351 equipped with an Andor iXon EMCCD camera. Stimulus was given at noted concentrations. Phenazine-1-