The Signaling Pathway of Caenorhabditis elegans Mediates Chemotaxis Response to the Attractant 2-Heptanone in a Trojan Horse-like Pathogenesis

Abstract

The nematode Caenorhabditis elegans exhibits behavioral responses to a wide range of odorants associated with food and pathogens. A previous study described a Trojan Horse-like strategy of pathogenesis whereby the bacterium Bacillus nematocida B16 emits the volatile organic compound 2-heptanone to trap C. elegans for successful infection. Here, we further explored the receptor for 2-heptanone as well as the pathway involved in signal transduction in C. elegans Our experiments showed that 2-heptanone sensing depended on the function of AWC neurons and a GPCR encoded by str-2 Consistent with the above observation, the HEK293 cells expressing STR-2 on their surfaces showed a transient elevation in intracellular Ca²⁺ levels after 2-heptanone applications. After combining the assays of RNA interference and gene mutants, we also identified the Gα subunits and their downstream components in the olfactory signal cascade that are necessary for responding to 2-heptanone, including Gα subunits of egl-30 and gpa-3, phospholipase C of plc-1and egl-8, and the calcium channel of cmk-1 and cal-1. Our work demonstrates for the first time that an integrated signaling pathway for 2-heptanone response in C. elegans involves recognition by GPCR STR-2, activation by Gα subunits of egl-30/gpa-3 and transfer to the PLC pathway, indicating that a potentially novel olfactory pathway exists in AWC neurons. Meanwhile, since 2-heptanone, a metabolite from the pathogenic bacterium B. nematocida B16, can be sensed by C. elegans and thus strongly attract its host, our current work also suggested coevolution between the pathogenic microorganism and the chemosensory system in C. elegans.

Keywords: 2-heptanone; Caenorhabditis elegans (C. elegans); G protein-coupled receptor (GPCR); bacterial pathogenesis; chemotaxis; chemotaxis response; co-evolution; microbial pathogens; olfactory pathway; signal transduction.

FIGURE 1.

Chemotaxisto 2-heptanone depends on the chemosensory neuron AWC^on and the GPCR STR-2 expressed on it. A, schematic diagram of chemotaxis assay. A thin layer of agar in a Petri dish was used as a substrate for chemotaxis, since C. elegans moves efficiently over the agar surface. About 100 washed adult animals were placed near the center of a 10-cm assay plate with the attractant that was generally diluted by ethanol at one side of the plate and the control (ethanol) at the opposite side. The numbers of animals within the attractant area and the control area were counted at 1 h. A chemotaxis index was calculated as (animal numbers at attractant-animal numbers at counter-attractant)/total animal number in assay. B, chemotaxis assay of 2-heptanone on the wild-type, odr-1(n1936), and odr-7(ky4) animals at 1:10 or 1:100 dilutions. C, chemotaxis assay of 2-heptanone on the worms with the str-2 (ok3148) and sra-13 (zh13) mutations. D, response of str-2 (ok3148) animals to benzaldehyde and 2-ethyl alcohol. E, chemotaxis assay to 2-heptanone using the worm lines of ceh-36 (ks86), nsy-1 (ok593), and inx-19 (ky634). The worms with ceh-36 and inx-19 mutations represented the obviously decreased sensing of 2-heptanone. The statistical differences were analyzed using one-way ANOVA, n.s., p ≥ 0.05; ***, p < 0.01.

FIGURE 2.

The expression levels of STR-2 in AWC neurons can be induced by 2-heptanone. A, animals under white light. B, expression of gene str-2 without 2-heptanone. C, expression of gene str-2 induced by 2-heptanone. Arrows indicated the neurons of AWC, and the arrowheads marked ASI neurons. The changes of fluorescence were taken at 45 min after 2-heptanone application.

FIGURE 3.

The transient elevation of Ca²⁺ within HEK293T cells transfected with the plasmids encoding myc-STR-2. A, STR-2 expression in mammalian cell line HEK293T. For immunofluorescence, the primary antibodies were mAb against Myc epitopes, and the secondary antibody was a PE-conjugated goat anti-mouse (H+L) (red). DAPI was used to stain the cell nucleus. a and b, represented the Myc-str-2-transfected cells HEK 293T cells under the fluorescence microscope and confocal microscope, respectively. B, images of Ca²⁺ measurements for Myc-tag3c-transfected and Myc-str-2-transfected cells with or without the addition of 2-heptanone. C, graphs of time-dependent changes in intracellular Ca²⁺ for Myc-tag3c-transfected and Myc-str-2-transfected cells. The addition of 7.2 × 10⁻² mol/liter 2-heptanone was performed at the time point of 90 s. D, quantitative analyzing of fluorescence intensity for each 800 cells of both pCMV-str-2 and the negative control pCMV-tag3C. E, cells expressing myc-str-2 still exhibited little change of intracellular Ca²⁺ when adding benzaldehyde. The statistical differences were analyzed using one-way ANOVA, n.s., p ≥ 0.05; ***, p < 0.01.

FIGURE 4.

G protein α subunit regulates the chemotaxis to 2-heptanone. A, Gα-mutant animals have different chemotaxis to 2-heptanone, and the genes of gpa-3, odr-3, and egl-30 seriously decreased the chemotaxis to 2-heptanone. B, Odr-3 mutants were defective in chemotaxis to 2-heptanone, but animals of odr-3RNAi responded to 2-heptanone normally. C, qPCR validated the decreased the expression of odr-3 in the worms of odr-3RNAi. D, most of the chemotaxis to 2-heptanone was rescued in the transgenic animals of Psrsx-3::egl-30. E, effects of gene gpa-3 and egl-30 were cumulative and affected the chemotaxis for 2-heptanone more severely. The statistical differences were analyzed using one-way ANOVA, n.s., p ≥ 0.05; **, <0.05; ***, p < 0.01.

FIGURE 5.

Effects of the PLC pathway on sensing 2-heptanone. A, genes in the PLC pathway, including egl-8, plc-1, itr-1, cmk-1, and cal-1, affected the response of adult worms to 2-heptanone. B, changes of Ca²⁺ concentration in the AWC neuron when the adult worms sensed the attractant 2-heptanone. The statistical differences were analyzed using one-way ANOVA, n.s., p ≥ 0.05; ***, p < 0.01.

FIGURE 6.

The genes in the cGMP pathway have effects on the olfactory signal transduction of sensing 2-heptanone via maintaining the expression level of GPCR gene str-2. A, gene mutations located on the cGMP pathway, including odr-1, daf-11, tax-2, and tax-4, led to the deficiencies in response to 2-heptanone. B, result from the fluorescence of odr-1::RFP showed that the addition of the molecule of 2-heptanone could not induce the expressional increase of gene odr-1. C, expression of GPCR STR-2 was dependent on the intact of gene odr-1. The fluorescence of str-2::GFP was fairly weak under the background of odr-1 RNAi regardless of the level of 2-heptanone. D, cGMP pathway has effect on sensing 2-heptanone via maintaining the expression level of GPCR gene str-2. Additional cGMP rescued the expression level of gene str-2 under the background of odr-1 RNAi. The changes of fluorescence were taken at 45 min after 2-heptanone application. The statistical differences were analyzed using one-way ANOVA, ***, p < 0.01.

FIGURE 7.

A potentially novel olfactory signal pathway existing in AWC olfactory neurons. The pathway of sensing 2-heptanone was affected by genes located on the PLC and cGMP pathways. Between them, the genes in the PLC pathway, such as phospholipase C of plc-1and egl-8, calcium channel of cmk-1and cal-1, were expected to be responsible for transducing the specific olfactory signals of 2-heptatone. However, the genes of cGMP pathway seemed to play more important roles in maintaining str-2 expression to influence the olfactory signal transduction.