Toll-like Receptor Signaling Promotes Development and Function of Sensory Neurons Required for a C. elegans Pathogen-Avoidance Behavior

Abstract

Toll-like receptors (TLRs) play critical roles in innate immunity in many animal species. The sole TLR of C. elegans--TOL-1--is required for a pathogen-avoidance behavior, yet how it promotes this behavior is unknown. We show that for pathogen avoidance TOL-1 signaling is required in the chemosensory BAG neurons, where it regulates gene expression and is necessary for their chemosensory function. Genetic studies revealed that TOL-1 acts together with many conserved components of TLR signaling. BAG neurons are activated by carbon dioxide (CO₂), and we found that this modality is required for pathogen avoidance. TLR signaling can therefore mediate host responses to microbes through an unexpected mechanism: by promoting the development and function of chemosensory neurons that surveil the metabolic activity of environmental microbes.

Figure 1. The p38 MAP kinase PMK-3 is required for proper gene expression by chemosensory BAG neurons. A

Overlays of differential interference contrast (DIC) and fluorescence micrographs of wild-type and pmk-3(wz31) mutants carrying a marker of differentiated BAG cells, P_gcy-33::GFP. White arrows indicate the BAG neuron nucleus. B. Structure of the pmk-3 locus showing four alleles isolated by our screen - wz27, wz31, wz34 and wz36 – and a deletion allele ok169. C. Expression of BAG neuron markers in the wild type and pmk-3 mutants, normalized to wild-type levels. Promoters from the indicated genes were used to drive expression of either GFP or mCherry (see methods). D. Percent animals expressing P_flp-17::GFP in the wild type, pmk-3(wz31) and pmk-3(wz31) mutants expressing pmk-3 specifically in BAG. Data from three independent transgenic lines are shown. E. Percent animals expressing P_flp-17::GFP in the wild type and pmk-3(wz31) mutants carrying a transgene for heat-shock-inducible expression of pmk-3 (P_hsp-16.41::pmk-3) that had been heat-shocked at the indicated developmental stages. Bar graph data are plotted as means ± SEM. N ≥ 20 for all measurements. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons. P values for these and other comparisons in subsequent figures are presented in Supplementary Table 1.

Figure 2. PMK-3 is required for function of the CO₂-sensing BAG neurons

A. A schematic of a behavior assay for BAG neuron function. B. CO₂-avoidance indices of the wild type, gcy-9(tm2816), pmk-3(ok169), pmk-3(wz31), pmk-1(km25), pmk-2(qd284) and pmk-3(wz31) mutants carrying a BAG cell-specific rescuing transgene. C. CO₂-avoidance indices of the wild type, and pmk-3(wz31) mutants carrying a transgene for heat-shock-inducible expression of pmk-3 (P_hsp-16.41::pmk-3) that had been heat-shocked at the indicated developmental stages. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.

Figure 3. The TAK homolog MOM-4 and the TLR TOL-1 are required for the function of CO₂-sensing BAG neurons

A. CO₂-avoidance indices of the wild type, dlk-1(ju475), sek-1(km4), mom-4(gk563) and mom-4(gk563) mutants carrying a transgene for BAG-specific expression of mom-4. B. CO₂-avoidance assays of the wild type, tol-1(nr2033) and tol-1(nr2013) mutants, animals carrying a transgene for BAG cell-specific RNAi of tol-1 and tol-1(nr2013) mutants carrying a transgene for BAG cell-specific expression of tol-1. C. Fluorescence micrograph showing a single optical section of an animal carrying a P_tol-1::GFP transcriptional reporter and the BAG cell-specific reporter P_flp-17::dsRed. This section did not capture strong GFP expression in URY neurons (see Supplemental Figure 3). D. Expression of transgene markers of differentiated BAG neurons in the wild type and animals carrying a transgene for BAG cell-specific RNAi of tol-1, normalized to wild-type levels, which are re-plotted from Figure 1C. Promoters from the indicated genes were used to drive expression of either GFP or mCherry. For these bar graphs data are plotted as means ± SEM. N ≥ 20 for all measurements. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.

Figure 4. PMK-3 functions in a Toll-like receptor signaling pathway

A. CO₂-avoidance indices of the wild type, tol-1(nr2013), mom-4(gk563) and pmk-3(wz31) mutants with and without a transgene driving mkk-4(gf) specifically in BAG cells. B. Avoidance indices of pmk-3(wz31) mutants and tol-1(RNAi knockdown specifically in BAG cells) animals with and without the ikb-1 deletion allele nr2027. C. CO₂-avoidance indices of the wild type and two other TLR signaling mutants: trf-1(nr2014) and pik-1(gk345127) with and without a transgene driving mkk-4(gf) specifically in the BAG neurons. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons. D. A model of TLR signaling that acts in BAG cells to promote CO₂-sensing.

Figure 5. Toll-like receptor signaling is required for chemotransduction in CO₂-sensing BAG neurons

A. Calcium responses of wild-type BAG neurons to CO₂ stimuli. B. Calcium responses of tol-1(nr2013) mutant BAG cells to CO₂ stimuli. Responses to 10% CO₂ were measured using the ratiometric calcium indicator YC3.60. Individual responses are plotted in light gray, while the black line shows average responses. In each plot stimulus presentation is indicated as a black bar. N = 11 for wild-type measurements and N = 15 for tol-1 mutant measurements. C. Peak ratio changes of wild-type and tol-1 mutant BAG cells. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.

Figure 6. C. elegans avoidance of the pathogen S. marcescens requires the CO₂-sensing modality of BAG neurons

A. A schematic of pathogen-avoidance behavior and the calculation of a dwelling index. B. Dwelling indices for the wild type, tol-1(nr2033), pmk-3(wz31), pmk-3(wz31) mutants carrying a BAG-specific rescuing construct, tol-1 RNAi in BAG cells and tol-1(nr2033) mutants carrying a BAG cell-specific rescuing construct on non-pathogenic E. coli lawns and S. marcescens lawns. C. Dwelling indices of animals lacking BAG cells, gcy-9(tm2816) mutants, which fail to detect CO₂ (14), and gcy-31(ok296) mutants, which fail to detect hypoxia (17), on E. coli and S. marcescens lawns. D. Dwelling indices of the wild type on lawns of living or heat-killed S. marcescens (Db10), or the attenuated strain Db1140. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.

Figure 7. Microbial CO₂ acts together with other factors to trigger avoidance behavior

A. Schematic showing delivery of S. marcescens odorant to animals dwelling on non-pathogenic E. coli. B. Dwelling indices of the wild type and gcy-9(tm2816) mutants on E. coli after one hour of exposure to air, artificial atmospheres containing CO₂, and S. marcescens odorant containing 1% CO₂. C. Calcium responses of wild-type BAG neurons to 1% CO₂ (N = 10) or S. marcescens odorant containing 1% CO₂ (N = 10), and responses of gcy-9(tm2816) mutant BAG cells to S. marcescens odorant containing 1% CO₂, (N = 8). Individual responses are plotted in light gray, while the black line shows average responses. In all plots stimulus presentation is indicated as a black bar. D. A model for the role of BAG neurons in pathogen-evoked avoidance behavior. Asterisks indicate statistical significance at P ≤ 0.05 for the indicated comparisons.