IGLR-2, a Leucine-Rich Repeat Domain Containing Protein, Is Required for the Host Defense in Caenorhabditis elegans

Abstract

Enterohemorrhagic Escherichia coli (EHEC), a human pathogen, also infects Caenorhabditis elegans. We demonstrated previously that C. elegans activates the p38 MAPK innate immune pathway to defend against EHEC infection. However, whether a C. elegans pattern recognition receptor (PRR) exists to regulate the immune pathway remains unknown. PRRs identified in other metazoans contain several conserved domains, including the leucine-rich repeat (LRR). By screening a focused RNAi library, we identified the IGLR-2, a transmembrane protein containing the LRR domain, as a potential immune regulator in C. elegans. Our data showed that iglr-2 regulates the host susceptibility to EHEC infection. Moreover, iglr-2 is required for pathogen avoidance to EHEC. The iglr-2 overexpressed strain, which was more resistant to EHEC originally, showed hypersusceptibility to EHEC upon knockdown of the p38 MAPK pathway. Together, our data suggested that iglr-2 plays an important role in C. elegans to defend EHEC by regulating pathogen-avoidance behavior and the p38 MAPK pathway.

Keywords: Caenorhabditis elegans; enterohemorrhagic Escherichia coli; iglr-2; innate immunity; p38 MAPK pathway.

Figure 1

RNAi screening identified iglr-2 is required for host defense in C. elegans. (A) The doughnut chart represents the number of selected genes containing specific proteins/domains that are considered as PRRs and the number of RNAi bacteria clones in RNAi library constructed from Ahringer RNAi library or Worm ORFeome library. The outer ring represents 382 selected genes considered as potential PRRs. The inner ring represents 315 RNAi clones. Sixty-seven genes are deficient in both Ahringer RNAi library or Worm ORFeome library. CTLDs, C-type lectin-like domains; LBP, Lipid binding protein; LRR, Leucine-rich repeat domain; LysM, Lysin motif domain; PGRP, Peptidoglycan recognition protein. (B) Knockdown of iglr-2 by RNAi silencing resulted in N2 animals hypersensitive to E. coli O157:H7 strain EDL933 infection compared to that of L4440 (P < 0.001). ***P < 0.001 by the Mantel–Cox log-rank test. Survival curves represent the sum of animals in multiple experiments.

Figure 2

iglr-2 null mutants created by CRISPR-Cas9 genome editing are hypersusceptible to EHEC. (A, B) Top: Diagram of iglr-2 isoform transcript. Position and mutation as indicated. Colored filled boxes are exons. Boxes in red indicate the leucine-rich repeat (LRR) domain region. Boxes in blue indicate immunoglobulin-like (Ig-like) domains region. Boxes in green indicate the transmembrane region. Lines are introns. Blank regions are untranslated. Bottom: DNA sequence of iglr-2 mutants compared to N2 wild type. (C) Mutation at LRR domain of iglr-2 contributed C. elegans hypersusceptible to E. coli O157:H7 strain EDL933 infection. [N2 vs iglr-2 (wf275) P < 0.001, N2 vs iglr-2 (wf295) P < 0.001]. (D) Survival curves of two iglr-2 transmembrane domain mutants fed with EHEC exhibited a significant sensitive phenotype compared to that of N2 [iglr-2 (wf297) P < 0.001, iglr-2 (wf298) P < 0.001] animals at 20°C. ***P < 0.001 by the Mantel–Cox log-rank test. Survival curves represent the sum of animals in multiple experiments.

Figure 3

Overexpression of iglr-2 confers C. elegans resistance to EHEC. (A) iglr-2 transcriptional level is significantly up-regulated upon EHEC infection. The iglr-2 mRNA expression level of N2 worms infected with E. coli O157:H7 strain EDL933 at 20°C was measured by qRT-PCR analysis at 12 h p.i (hours post infection) or 24 h p.i. Results were the average of three biological replicates and were normalized to the expression level of the nhr-23 control gene. Expression is relative to E. coli OP50. The error bars represent the standard deviation. **P < 0.01 and ***P < 0.001 by the unpaired t-test, respectively. (B) The mRNA expression level of iglr-2 in iglr-2 overexpressed strains was measured by qRT-PCR analysis. cDNA was extracted from N2 and two overexpressed strains, iglr-2 o/e-1 and iglr-2 o/e-2, which were fed with OP50 and grown to young adult stage or gravid adult stage at 20°C. Results were normalized to the expression level of the nhr-23 control gene. Expression is relative to wild-type N2. ***P < 0.001 by the unpaired t-test. (C) Survival curves of two iglr-2 overexpressed strains feeding on EHEC exhibited a significant resistant phenotype compared to that of N2 (iglr-2 o/e-1 P < 0.001, iglr-2 o/e-2 P < 0.001) at 20°C. ***P < 0.001 by the Mantel–Cox log-rank test. Survival curves represent the sum of animals in multiple experiments.

Figure 4

iglr-2 is expressed in neurons and intestine. Fluorescence images show iglr-2p::mCherry or iglr-2p::mCherry::H2B reporter strains exhibiting mCherry fluorescent signal in both neuronal and anterior/posterior intestinal cells at the young adult stage. (A–I) Representative images of YQ305 [iglr-2p::mCherry] transgenic animals are shown. (A–C) Anterior part of C. elegans YQ305 strain is shown. Arrows indicate the neuronal cells of head and anterior part of intestinal cells in (C). (D–F) Middle part of C. elegans YQ305. (G–I) Posterior part of C. elegans YQ305 strain is shown. Arrows indicate the posterior part of intestinal cells in (I). (A, D, G) represent differential interference contrast (DIC) images of the proximal, middle, and distal parts of C. elegans, respectively. (B, E, H) mCherry fluorescence images of iglr-2p::mCherry animals. (C, F, I) Merged images of iglr-2p::mCherry animals. (J–R) Representative images of YQ326 [iglr-2p::mCherry::H2B] transgenic animals are shown. (J–L) Anterior part of C. elegans YQ326 strain is shown. Arrows indicate the neuronal nuclei of head and anterior part of intestinal nuclei in (L). (M–O) Middle part of C. elegans YQ326. (P–R) Posterior part of C. elegans YQ326 strain is shown. Arrows indicate the posterior part of intestinal nuclei in (R). (J, M, P) represent differential interference contrast (DIC) images of the proximal, middle, and distal parts of C. elegans, respectively. (K, N, Q) mCherry fluorescence images of iglr-2p::mCherry animals. (L, O, R) Merged images of iglr-2p::mCherry animals. All the scale bars represent 50 µm.

Figure 5

iglr-2 may act in the neuron and intestine to defend against EHEC. (A) Survival curve of RNAi-sensitized strain NL2099 rrf-3(pk1426) showed that iglr-2 silencing in whole-body cell resulted in C. elegans being more sensitive to EHEC infection compared to that of empty vector L4440 control (P < 0.001). (B) Survival curve of neuron-specific RNA knockdown strains TU3401 showed that iglr-2 silencing in neuron cells resulted in C. elegans is more sensitive to EHEC compared to that of empty vector L4440 (P < 0.001). (C) Survival curve of intestine-specific RNAi knockdown strain VP303 showed iglr-2 silencing in intestine cells resulted in C. elegans being more sensitive to EHEC infection compared to that of empty vector L4440 control (P < 0.001). (D) Specific knockdown of iglr-2 in muscle cells by muscle-specific RNAi knockdown strain WM118 showed a similar survival curve compared to that of empty vector L4440 control to EHEC infection (P = 0.3725). ***P < 0.001 by the Mantel–Cox log-rank test. Survival curves represent the sum of animals in multiple experiments.

Figure 6

iglr-2 is required for bacterial avoidance behavior to EHEC. (A) Occupancy index of wild-type N2, iglr-2 mutants, and iglr-2 overexpressed strains on E. coli OP50 bacterial lawn for 16 h was analyzed. iglr-2 mutant strain lost the ability to avoid OP50 compared to that of wild-type N2 by unpaired t-test (***, N2 vs. iglr-2 (wf275), P < 0.001, N2 vs iglr-2 o/e-1 P = 0.173). (B) Occupancy index of wild-type N2, iglr-2 mutants and iglr-2 overexpressed strains on EHEC bacterial lawn for 16 h was scored. iglr-2 mutants displayed higher occupancy on EHEC compared to that of wild-type N2 by unpaired t-test. *** indicates N2 vs. iglr-2 (wf275) P < 0.001 and ns (no significance) indicates N2 vs iglr-2 o/e-1 P = 0.871. (C) Occupancy index of wild-type N2, iglr-2 mutants, and iglr-2 overexpressed strains on P. aeruginosa PA14 bacterial lawn for 16 h was scored. iglr-2 mutants and overexpressed strain showed a similar avoidance behavior index compared to that of wild-type N2 by unpaired t-test [iglr-2 (wf275) P = 0.140, iglr-2 o/e-1 P = 0.289]. ns indicates no significance. Each experiment conducted on three biological replicates, and results were the average of experimental replicates. Error bars represent SD.

Figure 7

PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway acts as downstream signal of iglr-2. (A) RNAi-mediated knockdown of pmk-1 in iglr-2 overexpressed animal resulted in hypersusceptibility to EHEC compared to empty vector control, L4440 (P < 0.001). ***P < 0.001 by the Mantel–Cox log-rank test. (B) Survival curves of iglr-2 mutant RNAi against pmk-1 were comparable to EHEC compared to empty vector control, L4440 (P = 0.22) (C) Survival of nsy-1(ums8) gain-of-function allele and iglr-2(wf295) double mutant strain infected with EHEC at 20°C was examined. nsy-1(ums8);iglr-2(wf295) animals lived longer than iglr-2(wf295) (P < 0.0001) when fed with EHEC at 20°C but exhibited a similar survival compared to that of wild-type (WT) worms (P = 0.835). nsy-1(ums8) gain-of-function strain was significantly resistant to EHEC infection compared to that of WT (P < 0.001). ***P < 0.001 by the Mantel–Cox log-rank test. (D) Survival analysis of iglr-2(wf295) supplemented with 5 µM RPW-24 upon EHEC infection at 20°C was examined. Treatment of RPW-24 increased the survival of iglr-2(wf295) animals compared to that of DMSO control. (P < 0.001). Survival curves represent the sum of animals in multiple experiments. (E) Representative images of K08D8.5::GFP transgenic animals infected with EHEC were shown. K08D8.5::GFP wild type and iglr-2 mutant background worms were treated with EHEC for 8 h and imaged. Merged images indicate GFP overlaid with DIC. GFP images are presented inverted signal of GFP. Scale bars represent 100 µm. (F) Relative fluorescence expression of K08D8.5::GFP transgenic worms infected with EHEC for 8 h. Each dot represents the relative GFP expression of single animal to the mean of wild-type control. **P < 0.01 by the unpaired t-test. Error bars represent SD.

Figure 8

Model graphic of iglr-2 defense against EHEC in C. elegans. IGLR-2, an transmembrane protein containing immunoglobulin-like and leucine rich repeat domains, is involved in C. elegans’ avoidance behavior to EHEC and triggering downstream p38 MAPK pathway which might function in neuronal cell and intestinal cell.