ADARs regulate cuticle collagen expression and promote survival to pathogen infection

Abstract

Background: In all organisms, the innate immune system defends against pathogens through basal expression of molecules that provide critical barriers to invasion and inducible expression of effectors that combat infection. The adenosine deaminase that act on RNA (ADAR) family of RNA-binding proteins has been reported to influence innate immunity in metazoans. However, studies on the susceptibility of ADAR mutant animals to infection are largely lacking.

Results: Here, by analyzing adr-1 and adr-2 null mutants in well-established slow-killing assays, we find that both Caenorhabditis elegans ADARs are important for organismal survival to gram-negative and gram-positive bacteria, all of which are pathogenic to humans. Furthermore, our high-throughput sequencing and genetic analysis reveal that ADR-1 and ADR-2 function in the same pathway to regulate collagen expression. Consistent with this finding, our scanning electron microscopy studies indicate adr-1;adr-2 mutant animals also have altered cuticle morphology prior to pathogen exposure.

Conclusions: Our data uncover a critical role of the C. elegans ADAR family of RNA-binding proteins in promoting cuticular collagen expression, which represents a new post-transcriptional regulatory node that influences the extracellular matrix. In addition, we provide the first evidence that ADAR mutant animals have altered susceptibility to infection with several opportunistic human pathogens, suggesting a broader role of ADARs in altering physical barriers to infection to influence innate immunity.

Keywords: C. elegans; Double-stranded RNA (dsRNA); Innate immunity; P. aeruginosa; Post-transcriptional regulation; RNA editing; RNA modification; RNA-binding protein.

Fig. 1

ADAR mutant worms are susceptible to Pseudomonas infection. A–D Representative survival curve (of three biological replicates) for the indicated animals subjected to the slow-killing assay and scored for survival in response to P. aeruginosa strain PA14. Statistical significance determined using OASIS. p < 0.001 (***), p < 0.001 (**) and p < 0.05 (*). Survival assay replicates are provided in Additional file 1: Fig: S1A-F

Fig. 2

Adr mutant animals exhibit normal avoidance and feeding behavior to P. aeruginosa. A Lawn occupancy percentage was calculated by counting the number of worms of the indicated strains in the lawn and outside the lawn, which was summed and then divided by the total number of worms. Each data point represents the average of three technical replicates performed at the indicated time and all experiments were performed in three biological replicates. Error bars represent the standard error of the mean (SEM). Statistical significance determined using two-way ANOVA Tukey’s multiple comparisons test. p value of each of the mutants relative to WT was not significant (p > 0.05) for any of the timepoints. B Each dot in the graph represents the average pumping rate for three technical replicates obtained in two independent experiments. Error bars represent SEM. Statistical significance determined using unpaired Mann Whitney test, with no significant differences observed between wildtype and the adr mutant animals (p > 0.05). C qRT-PCR quantification of the level of the indicated genes relative to gpd-3 and normalized to the ratios obtained for OP50. The mean of three biological replicates was plotted. Error bars represent SEM. Statistical significance determined using a two-way ANOVA Sidak’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.005, ns indicates no significant difference (p > 0.05). D Equivalent amounts of lysate from animals with V5 and 3xFLAG epitope tags on ADR-1 and ADR-2, respectively, exposed to P. aeruginosa (PA14) ( +) or the control E. coli (OP50) ( −) for 7 h were subjected to SDS-PAGE and immunoblotting with V5 (ADR-1), FLAG (ADR-2), and Actin antibodies. Raw blot images of all three replicates are provided in Additional file 6: Fig. S6

Fig. 3

ADR-1 RNA binding is required for survival to P. aeruginosa. A Representative survival curve (of three biological replicates) for the indicated animals subjected to the slow-killing assay and scored for survival in response to P. aeruginosa strain PA14. Statistical significance determined using OASIS, where p < 0.001 (***), and ns indicates p > 0.05. Survival assay replicates are provided in Additional file 7: Fig. S7. Dots represent individual genes that are down- or upregulated or not significantly differentially expressed (black) in RNA-seq data from WT animals compared to adr-1(-) (maroon) (B) or ADR-1 dsRBD1 mutant animals (brown). D Average log₂ fold change (x-axis) is plotted against the negative log₁₀ p-values (y-axis). Genes considered significantly differentially expressed exhibited log₂ fold change of |0.5| (light gray dotted vertical lines) and p < 0.05 [ log₁₀ p-value of 1.3, solid black horizontal line]. C, F qRT-PCR quantification of the level of the indicated genes relative to gpd-3 and normalized to the ratios obtained for WT PA14 and WT OP50 in (C and F), respectively. The mean of three biological replicates was plotted. Error bars represent SEM. Statistical significance was determined using a two-way ANOVA Sidak’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p ≤ 0.0001, ns indicates no significant difference (p > 0.05). E Overlap of genes misregulated in (B and D)

Fig. 4

Altered gene expression in adr mutant animals. A–C Dots represent individual genes down- or upregulated or not significantly differentially expressed (black) in RNA-seq data from WT animals compared to adr-1(-) (purple) (A), adr-1(-);adr-2(-) (green) (B) and adr-2(-) (yellow) grown on OP50. Average log₂ fold change (x-axis) is plotted against the negative log₁₀ p-values (y-axis). Genes considered significantly differentially expressed exhibited log₂ fold change of |0.5| (light gray dotted vertical lines) and p ≤ 0.05 [log₁₀ p-value of 1.3, solid black horizontal line]. D Overlap of genes downregulated in (A, B and C)

Fig. 5

Loss of adrs results in altered cuticle structure and enhanced susceptibility to several pathogenic bacterial species. A Representative SEM images of the indicated strains after exposure to the bacterial strains indicated. Images were captured and categorized by blinded individuals (see methods) with 15 images available for wildtype animals fed OP50, 10 images available for wildtype animals exposed to PA14, 15 images available for adr-1(-); adr-2(-) animals fed OP50 and 10 adr-1(-); adr-2(-) animals exposed to PA14. Scale bar of image is 20 µm. B, C Representative survival curve (of three biological replicates) for the indicated animals subjected to the slow-killing assay and scored for survival in response to S. aureus (B) and S. enterica (C). Statistical significance was determined using OASIS, ****p < 0.0001, ***p < 0.001. Survival assay biological replicates are provided in Additional file 18: Fig. S18A-D