Autophagy protects C. elegans against necrosis during Pseudomonas aeruginosa infection

Abstract

Autophagy, a conserved pathway that delivers intracellular materials into lysosomes for degradation, is involved in development, aging, and a variety of diseases. Accumulating evidence demonstrates that autophagy plays a protective role against infectious diseases by diminishing intracellular pathogens, including bacteria, viruses, and parasites. However, the mechanism by which autophagy regulates innate immunity remains largely unknown. Here, we show that autophagy is involved in host defense against a pathogenic bacterium Pseudomonas aeruginosa in the metazoan Caenorhabditis elegans. P. aeruginosa infection induces autophagy via a conserved extracellular signal-regulated kinase (ERK). Intriguingly, impairment of autophagy does not influence the intestinal accumulation of P. aeruginosa, but instead induces intestinal necrosis. Inhibition of necrosis results in the survival of autophagy-deficient worms after P. aeruginosa infection. These findings reveal a previously unidentified role for autophagy in protection against necrosis triggered by pathogenic bacteria in C. elegans and implicate that such a function of autophagy may be conserved through the inflammatory response in diverse organisms.

Fig. 1.

Autophagy mediates host defense against P. aeruginosa in C. elegans. (A) Representative images of autophagosomes (GFP::LGG-1 puncta) in the seam cells and intestinal cells of worms exposed to P. aeruginosa PA14 for 12 h. The numbers of GFP::LGG-1 puncta in the seam cells and intestinal cells were counted (Right). These results are mean ± SD of three independent experiments performed in triplicate. **P < 0.01 versus OP50+empty vector (EV). The arrow denotes a representative autophagosome. (Scale bars: seam cells, 10 μm; intestinal cells, 20 μm.) (B) The levels of PE-conjugated LGG-1–GFP and LGG-1–GFP were measured by Western blotting. The blot shown here is typical of three independent experiments. P < 0.05, PA14 versus OP50; P < 0.01, PA14+bec-1 RNAi versus PA14+EV. (C) bec-1 RNAi significantly reduced survival of worms exposed to PA14. P < 0.01 versus EV. (D) Numbers of colony-forming units of PA14 were measured in worms subjected to bec-1 RNAi. These results are mean ± SD of three independent experiments performed in triplicate. *P < 0.05 versus WT+EV. (E) Fluorescence of worms exposed to P. aeruginosa PA14 expressing GFP for 24 h. The image is representative of three independent experiments. Right shows quantification of GFP levels. *P < 0.05 versus WT+EV. (Scale bars: 50 μm.)

Fig. 2.

The ERK pathway is required for autophagy during P. aeruginosa infection. (A and B) The numbers of GFP::LGG-1 puncta were counted in the seam cells (A) and intestinal cells (B) of worms exposed to P. aeruginosa PA14. (C and D) Mutations in the components of the ERK pathway suppressed autophagy in the seam cells (C) and intestinal cells (D) of worms exposed to PA14. These results are mean ± SD of three independent experiments performed in triplicate. **P < 0.01 versus control (EV or WT). (E) Mutations in the components of the ERK pathway reduced survival of worms exposed to PA14. P < 0.01 versus WT.

Fig. 3.

The ERK pathway is activated after P. aeruginosa infection. (A) The phosphorylation of ERK was elevated in WT worms, but not in mek-2(n1989) and mpk-1(n2521) mutants, after P. aeruginosa PA14 infection for 12 h. U0126 (25 μM), an inhibitor of MEK-2, attenuated the phosphorylation of ERK. The blot is typical of three independent experiments. P < 0.01, PA14 versus OP50; P < 0.05, U0126+PA14 versus PA14. (B) The lin-3 mRNA levels were up-regulated in WT worms exposed to PA14. These results are mean ± SD of three independent experiments performed in triplicate. *P < 0.05 versus OP50. (C) Expression of lin-3::GFP was up-regulated in WT worms exposed to PA14. Right shows quantification of GFP levels. *P < 0.05 versus OP50. (Scale bars: 20 μm.) (D) Mutations in lin-3 and let-23 attenuated the phosphorylation of ERK after PA14 infection for 12 h. The blot is typical of three experiments. P < 0.05 versus WT. (E) Mutations in lin-3 and let-23 suppressed autophagy in worms exposed to PA14. These results are mean ± SD of three independent experiments performed in triplicate. **P < 0.01 versus WT. (F) Mutations in lin-3 and let-23 reduced survival of worms exposed to PA14. P < 0.01 versus WT.

Fig. 4.

CDC-48.2 is a downstream effector of ERK. (A) A mutation in cdc-48.2(tm659), but not cdc-48.1(tm544), suppressed autophagy induced by P. aeruginosa PA14 infection. These results are mean ± SD of three independent experiments performed in triplicate. **P < 0.01 versus WT. (B) The ratio of PE-conjugated LGG-1–GFP and LGG-1–GFP was reduced in cdc-48.2(tm659) mutants after PA14 infection. The blot shown here is typical of three independent experiments. P < 0.05, cdc-48.2(tm659)+PA14 versus WT+PA14. (C) Schematic representation of the generated CDC-48.2 site-directed mutants. (D and E) Expression of CDC-48.2(Thr699Glu) protein rescued P. aeruginosa infection-mediated autophagy in the seam cells (D) and intestinal cells (E) in cdc-48.2(tm659) and mpk-1(n2521) mutants. These results are mean ± SD of three independent experiments performed in triplicate. **P < 0.01 versus EV. (F) Expression of CDC-48.2(Thr699Glu) protein partially rescued the immune-deficient phenotype of mpk-1(n2521) mutants. P < 0.01 versus mpk-1(n2521)+EV.

Fig. 5.

Necrosis is induced in autophagy-deficient worms. (A) Necrosis in the intestine occurred in mpk-1(n2521), cdc-48.2(tm659) mutants, or worms subjected to bec-1 RNAi in the presence of P. aeruginosa PA14. DIC images of worms and fluorescence microscopy of acridine orange (AO)- (Upper) and (Z-FR)₂-R110– (Lower) labeled intestine of worms are shown. Enlarged vacuoles are indicated by arrows. (B) DAPI-stained nuclei of intestine were reduced after PA14 infection. The lower panel shows quantification of the number of nuclei. These results are mean ± SD of three independent experiments performed in triplicate. *P < 0.05 versus WT. Nuclei are indicated by arrows. (C and D) Knockdown of necrosis-related genes (asp-4 and clp-1) by RNAi partially inhibited the immune-deficient phenotype of mpk-1(n2521) mutants (C) or worms subjected to bec-1 RNAi (D) after PA14 infection. *P < 0.05 versus mpk-1(n2521) or bec-1 RNAi. (Scale bars: A, 20 μm; B, 25 μm.)