Identification of a Conserved, Orphan G Protein-Coupled Receptor Required for Efficient Pathogen Clearance in Caenorhabditis elegans

Abstract

G protein-coupled receptors contribute to host defense across the animal kingdom, transducing many signals involved in both vertebrate and invertebrate immune responses. While it has become well established that the nematode worm Caenorhabditis elegans triggers innate immune responses following infection with numerous bacterial, fungal, and viral pathogens, the mechanisms by which C. elegans recognizes these pathogens have remained somewhat more elusive. C. elegans G protein-coupled receptors have been implicated in recognizing pathogen-associated damage and activating downstream host immune responses. Here we identify and characterize a novel G protein-coupled receptor required to regulate the C. elegans response to infection with Microbacterium nematophilum We show that this receptor, which we designate pathogen clearance-defective receptor 1 (PCDR-1), is required for efficient pathogen clearance following infection. PCDR-1 acts upstream of multiple G proteins, including the C. elegans Gαq ortholog, EGL-30, in rectal epithelial cells to promote pathogen clearance via a novel mechanism.

Keywords: Caenorhabditis elegans; G protein-coupled receptor; Microbacterium nematophilum; pathogen clearance.

FIG 1

PCDR-1 is a G protein-coupled receptor required for efficient pathogen clearance. (A) Genomic structure of pcdr-1. pcdr-1(gk1000) and pcdr-1(gk1122) are partially overlapping deletions in the 3′ end of pcdr-1. The genomic region and pcdr-1p promoter used for rescue experiments are also indicated. (B) Predicted protein sequence of PCDR-1. Predicted transmembrane domains are highlighted in gray. Regions deleted in pcdr-1(gk1000) are indicated by a dotted line. Regions deleted in pcdr-1(gk1122) are indicated by a solid line. Regions deleted in both pcdr-1(gk1000) and pcdr-1(gk1122) are indicated by a wavy line. (C to G) Animals were infected with M. nematophilum, and pathogen attached to the rectal opening of adult animals was labeled with the nucleic acid stain SYTO13. (C) SYTO13-labeled M. nematophilum was cleared from the rectal opening of wild-type animals, and 50% of animals remained colonized 90 min after transfer to assay plates. The clearance of labeled pathogen was significantly decreased in strains containing deletions in pcdr-1 [pcdr-1(gk1122) and pcdr-1(gk1000)]. (D) No significant differences in the amounts of SYTO13-labeled M. nematophilum bacteria attached to the rectal opening were observed between strains. AU, arbitrary units. (E) Pathogen clearance rates were significantly decreased in pcdr-1(gk1122)/(gk1000) transheterozygotes but not in animals heterozygous for either pcdr-1(gk1122) or pcdr-1(gk1000). (F and G) Rescue experiments were performed using the fosmid WRM0618bH06, which covers the coding region of pcdr-1 and 2.8 kb upstream of the predicted pcdr-1 ATG as well as 4 other downstream genes (see Materials and Methods for details). Expression of WRM0618bH06 in the pcdr-1(gk1122) (F) and pcdr-1(gk1000) (G) strains rescues pathogen clearance defects. Indeed, rescued animals clear pathogen infections significantly faster than wild-type animals. PCDR-1 cDNA, expressed under the control of the 2.8-kb region upstream of the predicted pcdr-1 ATG (pcdr-1p), also rescued the pathogen clearance defects of pcdr-1(gk1122) (F) and pcdr-1(gk1000) (G) animals. * indicates significance relative to the wild type. # indicates significance relative to the pcdr-1(gk1122) (C and F) or pcdr-1(gk1000) (G) strain (see Materials and Methods for details of statistical analysis). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 2

pcdr-1(gk1122) and pcdr-1(gk1000) are not null alleles. (A) Schematic of PCDR-1 highlighting primers used for RT-PCR and pcdr-1(gk1122) and pcdr-1(gk1000) deletions. Details of primers are given in Table 1. (B, top 3 panels) RT-PCR for PCDR-1 in wild-type, pcdr-1(gk1122), and pcdr-1(gk1000) strains. NTC, no-template control. (Bottom) ACT-1 was used as a positive control.

FIG 3

PCDR-1 is expressed in neurons, vulval muscle, and rectal epithelial cells. GFP was expressed under the control of the 2.8-kb region upstream of the predicted pcdr-1 ATG (pcdr-1p) (B, E, H and K) in order to determine the cellular expression pattern of PCDR-1. This was injected into wild-type animals with acr-2p::mCherry (A, D, and G) or egl-5p::mCherry (J). (A to I) We observed GFP expression in cholinergic (orange arrow) and noncholinergic (green arrow) neurons in the ventral nerve cord (A to C), in several neurons in the head (D to F), and in the vulval muscle (G to I). (J to L) We also observed GFP expression in nonneuronal cells in the tail, which colocalized with the egl-5p::mCherry rectal epithelial marker (the rectal opening is indicated with an arrow). (M) qRT-PCR was used to determine whether PCDR-1 expression was regulated by infection with M. nematophilum. No significant difference in PCDR-1 expression was observed between infected and uninfected wild-type animals. n.s., not significant (P > 0.05).

FIG 4

PCDR-1 is required in rectal epithelial cells for efficient pathogen clearance. To determine where PCDR-1 was required to mediate pathogen clearance, we performed cell-specific rescue experiments by expressing the PCDR-1 cDNA in neurons (using the panneuronal rab-3 promoter [n::PCDR-1]) or rectal epithelial cells (using a 1.3-kb fragment of the egl-5 promoter [re::PCDR-1]). Partial rescue of pcdr-1(gk1122) (A), but not pcdr-1(gk1000) (B), was observed at 60 min when PCDR-1 was expressed neuronally. We were able to fully rescue the pathogen clearance defect of pcdr-1(gk1122) (A) and pcdr-1(gk1000) (B) strains by expressing PCDR-1 cDNA in the rectal epithelium. * indicates significance relative to wild type. # indicates significance relative to the pcdr-1(gk1122) (A) or pcdr-1(gk1000) (B) strain (see Materials and Methods for details of statistical analysis). *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

FIG 5

PCDR-1 acts upstream of, and in parallel with, EGL-30(Gαq) to mediate efficient pathogen clearance. Animals were infected with M. nematophilum, and the percentage of animals retaining SYTO13-labeled pathogen was scored 90 min after transfer to unseeded NGM plates. The percentage of animals retaining SYTO13-labeled pathogen was increased for the pcdr-1(gk1122) strain and slightly decreased for the egl-30(js126gf) strain compared to wild-type controls. pcdr-1(gk1122);egl-30(js126gf) animals retained significantly more SYTO13-labeled pathogen than egl-30(js126gf) animals but significantly less than pcdr-1(gk1122) animals. The percentage of pcdr-1(gk1122);let-60(n1046gf) animals retaining labeled pathogen was not significant different from that of pcdr-1(gk1122) animals. See Materials and Methods for details of statistical analysis. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant (P > 0.05).

FIG 6

PCDR-1 has a minor effect on cellular immune responses to M. nematophilum infection. Adult wild-type, pcdr-1(gk1122), and pcdr-1(gk1000) animals were infected as described in Materials and Methods. (A) The percentage of animals with the Dar phenotype was decreased very slightly from 98% in wild-type animals to 94.8% in pcdr-1(gk1122) and 84.9% in pcdr-1(gk1000) animals. (B and C) To determine whether PCDR-1 could regulate the expression of the antimicrobial peptide F53A9.8, we performed qRT-PCR on infected and uninfected wild-type, pcdr-1(gk1122), and pcdr-1(gk1000) animals as described in Materials and Methods. Expression of F53A9.8 was increased following infection of wild-type animals, and a similar increase was observed in pcdr-1(gk1122) (B) and pcdr-1(gk1000) (C) animals. See Materials and Methods for details of statistical analysis. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., not significant (P > 0.05).

FIG 7

PCDR-1 does not regulate changes in defecation during infection. (A) To determine whether a defect in defecation behavior was responsible for the pathogen clearance defect of pcdr-1(gk1122) and pcdr-1(gk1000) animals, we measured the defecation rate (as the interval between pBocs). Infection with M. nematophilum decreased the interval between defecation cycles from 60 s to 47 s in wild-type animals, and a similar decrease was observed in pcdr-1(gk1122) and pcdr-1(gk1000) animals. (B) In C. elegans, defecation is achieved by a series of muscle contractions (pBoc, aBoc, and Exp) that occur in a highly stereotyped manner (37). Following infection of wild-type animals, we observed that the final step in this cycle (the Exp, or expulsion, step) was frequently missing. This phenotype was also observed in pcdr-1(gk1122) and pcdr-1(gk1000) animals. * indicates significance relative to uninfected controls. See Materials and Methods for details of statistical analysis. ****, P ≤ 0.0001; n.s., not significant (P > 0.05).

FIG 8

Defective pathogen avoidance in PCDR-1 does not fully account for pathogen clearance defects. (A) Pathogen avoidance was assessed by scoring the percentage of animals not on M. nematophilum-contaminated lawns. Sixty-one percent of wild-type animals avoided contaminated lawns; however, both pcdr-1(gk1122) and pcdr-1(gk1000) animals were defective in this pathogen avoidance response. Expression of WRM0618bH06 rescued the avoidance defect of pcdr-1(gk1000) animals but failed to rescue the pathogen avoidance defect of pcdr-1(gk1122) animals. (B and C) To determine whether pathogen clearance rates were still decreased in pcdr-1(gk1000) and pcdr-1(gk1122) animals when they were unable to avoid pathogen-contaminated lawns, we modified our infection assays by spreading bacteria to the edge of the plates (full-plate assay). Pathogen clearance rates were decreased in wild-type animals in this full-plate assay, and we observed a similar decrease in pcdr-1(gk1000) animals (C). We also observed a small decrease in pathogen clearance rates of pcdr-1(gk1122) animals grown on full plate, although this was not significant (B). * indicates significance relative to the wild type. # indicates significance relative to pcdr-1(gk1000) standard plates (A and C) (see Materials and Methods for details of statistical analysis). **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.