Behavioral and immune responses to infection require Gαq- RhoA signaling in C. elegans

Abstract

Following pathogen infection the hosts' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection.

Figure 1. EGL-30 (Gαq) signaling is required in different tissues for behavioral and immune responses to infection.

The locomotion rate of wild type and unc-29(e1072) animals was increased following infection with M. nematophilum (A). No increase was observed in egl-30(ad805) loss-of-function mutants (A). Synaptic release of endogenous acetylcholine was measured by determining the onset of paralysis induced by the acetylcholine esterase inhibitor aldicarb. Infection of wild type animals with M. nematophilum resulted in a faster onset of aldicarb-induced paralysis relative to wild type controls grown on E. coli, suggesting an increase in the levels of ACh release following infection (B). In contrast egl-30(ad805) was resistant to aldicarb and infection of these animals did not increase ACh release (B). Mutations in egl-30(ad805) significantly decreased the percentage of Dar animals observed following M. nematophilum infection although bacteria, labeled in green using the nucleic acid stain SYTO13, still attached to the anal opening (C, D and G) (rectal opening is indicated with an arrow in C and D). These animals were severely constipated and the intestinal distention is indicated by a double-headed arrow (D). Expression of EGL-30 (Gαq) in the rectal epithelium (F kindly drawn by H. Chamberlin) using a 1.3 Kb fragment of the egl-5 promoter (egl-5p::EGL-30; egl-30(ad805)) was sufficient to rescue the Dar response following infection (G) however these animals remained resistant to aldicarb and ACh release was not increased following infection (E). In contrast cholinergic motorneuron expression of EGL-30 (Gαq) from the unc-17 promoter (MN::EGL-30) rescued increases in locomotion (A) and ACh release following infection (E) but not the Dar response (G). P values between 0.05 and 0.001 (*), and P values of 0.001 or less (**).

Figure 2. UNC-73 (Trio) is required in rectal epithelial cells for the Dar response to infection.

Viable RhoGEF mutants were infected with M. nematophilum and the percentage of Dar animals scored. Mutations in unc-73(ce362), and ect-2(ku427), but not other RhoGEF's, significantly decreased the percentage of Dar animals (A). The UNC-73 gene contains two RhoGEF domains, one specific for Rac (RhoGEF1) and the other specific for Rho (RhoGEF2) (B). Animals with mutations that prevented Rac activation (unc-73(e936) and (ok936)) had a wild-type Dar response whereas mutations in RhoGEF2 (unc-73(ce362) and (ox317)) significantly decreased the percentage of Dar animals (C). Expression of UNC-73 isoforms E or D1 using heat shock at L1, L2/L3 and L3/L4 stage (hs::UNC-73E) or rectal epithelial (egl-5p::UNC-73D1::GFP), but not neuronal (n::UNC-73E), expression rescued the Dar phenotype in unc-73(ce362) animals (D). Although unc-73(ce362) animals failed to produce a Dar response M. nematophilum bacteria, labeled using the nucleic acid stain SYTO13, still attached to the anal opening (E), the rectal opening is indicated with an arrow).

Figure 3. UNC-73 (Trio) is required in neurons for the behavioral response to infection.

Animals carrying a mutation in unc-73(ce362) did not significantly change their locomotion rate when infected by M. nematophilum and this effect was rescued by expression of UNC-73E from a pan-neuronal promoter (n::UNC-73E) (A). unc-73(ce362) mutants were slightly resistant to aldicarb when grown on E. coli (B). ACh release was not increased in these animals following infection (B). Expression of UNC-73 in neurons (n::UNC-73E), but not in the rectal epithelial cells (egl-5p::UNC-73D1GFP), was sufficient to rescue the increase in ACh release upon infection by M. nematophilum of unc-73(ce362) mutants (C). P values between 0.05 and 0.001 (*), and P values of 0.001 or less (**).

Figure 4. Rho signaling in the adult rectal epithelium causes tail swelling and changes cell morphology.

Inhibition of Rho in a subset of rectal epithelial cells using the Rho inhibitor C3 Transferase expressed from the bus-1 promoter (bus-1p::C3T) significantly decreased the percentage of Dar animals (A). Expression of RHO-1* in adult animals using a heat shock-inducible promoter triggers the Dar response (B and D), in the absence of heat shock animals expressing hs::RHO-1* were wild type (B and C). Cell specific expression of RHO-1* in the rectal epithelial cells (egl-5p::RHO-1*); but not in neurons (n::RHO-1*) or muscle (muscle::RHO-1*), also resulted in tail swelling (B and E). Rectal opening is indicated with an arrow. § indicates 0%. Expression of mCherry together with RHO- 1* in the rectal epithelium using the 1.3 Kb egl-5 promoter fragment (J and K) or infection of animals expressing mCherry from the same promoter (H and I) results in changes in the morphology of the epithelial cells when compared to wild-type controls (F and G). Rectal opening is indicated with an arrow. Rectal epithelial cell boundaries are indicated with a dotted line. P values between 0.05 and 0.001 (*), and P values of 0.001 or less (**).

Figure 5. Gαq-Rho GEF Trio-Rho Signaling is required for aversion to pathogenic M. nematophilum.

Animals were placed equidistant from a two lawns of bacteria (A vs B) and the number of animals on lawns A and B were counted at 30 minutes (solid bars) and at 4 hours (hatched bars). The preference ratio shown is given by the formula [animals at A- animals at B/animals (A+B)]. Wildtype animals have no preference between E. coli (OP50) and pathogenic M. nematophilum at 30 minutes, but at 4 hours they have a strong preference for OP50 E. coli. This preference is abolished if the strain of M. nematophilum is avirulent (UV336) or if animals have a mutation in unc-73 or egl-30. Expression of EGL-30 in the motorneurons (MN::EGL-30) or of UNC-73 in all neurons (N::UNC-73) rescued the preference for OP50 in egl-30 and unc-73 mutants respectively. P values between 0.05 and 0.001 (*), and P values of 0.001 or less (**).

Figure 6. LET-60 (Ras) activation is sufficient to cause tail swelling and is required for the Dar response to infection.

Two different let-60 (Ras) reduction-of-function mutations, n2021 and sy93, significantly decreased the Dar response upon infection with M. nematophilum (A). This decrease was not observed using ras-1(gk237) and ras-2(ok628) mutants that showed a wild-type response to infection (A). Although decreased tail swelling was observed in let-60(n2021) animals infected with M. nematophilum bacteria, labeled using the nucleic acid stain SYTO13, still attached to the anal opening (B). let-60(n2021) animals were slightly hypersensitive to aldicarb when grown on E. coli OP50 and ACh release was increased in these animals following infection with M. nematophilum (C). Cell specific expression of constitutively active MEK- 2(S223E, S227D) (egl-5p::MEK-2*) (D) or constitutively active LET-60(G12V) (egl- 5p::LET-60*) (E) in the rectal epithelial cells using a 1.3 Kb egl-5 promoter fragment resulted in tail swelling that phenocopied the Dar phenotype observed following infection and RHO-1* activation. Arrows in D and E indicate the rectal opening. P values between 0.05 and 0.001 (*), and P values of 0.001 or less (**).

Figure 7. Gαq-Rho GEF Trio-Rho Signaling and Ras converge on Raf to regulate morphology during the immune response to infection.

The simplest explanation of our results is that following pathogen infection RHO-1 is activated in the rectal epithelial cells by multiple upstream regulators including EGL-30 (Gαq) and UNC-73 (Trio). Together with Ras, Rho signaling converges on Raf to activate the MAPK pathway. Activation of these pathways, together with at least one other, in the rectal epithelial cells leads to the changes in morphology that occur as part of the immune response.