The Invertebrate Lysozyme Effector ILYS-3 Is Systemically Activated in Response to Danger Signals and Confers Antimicrobial Protection in C. elegans
Fig 5
ilys-3 is required in the pharynx and in the intestine to prevent bacterial burden in the gut lumen and to protect against M. nematophilum.
2. (A) Images of a wild-type, ilys-3 and ilys-3; eEx752 one-day old adults fed for 24 hour on E. coli expressing GFP. Live bacteria cells are seen in the pharyngeal (arrow) but not intestinal lumen (arrowhead). (i-ii) N2. (i) Composite DIC and GFP fluorescence image. (ii) Green channel. (iii-iv) ilys-3 deletion mutants accumulate live bacteria in the gut lumen and exhibit impaired ability to disrupt bacteria. (iii) Composite DIC and GFP fluorescence image (iv) Green channel. (v-vi) Overexpression of ILYS-3 in ilys-3 with eEx752 array rescues luminal bacterial accumulation in an animal of the same chronological age. (v) Composite DIC and GFP fluorescence image. (vi) Green channel. (B) Overlays of DIC and epifluorescence images of one-day-old adults of WT, ilys-3 or ilys-3; eEx752 exposed to SYTO 13-labeled CBX102 cells for 2 hours. (i) Fluorescence image of an N2 animal showing few stained CBX102 cells, indicative of non-viable bacteria. (ii) Gut lumen of an ilys-3 animal with high accumulation of live CBX102 cells that fluoresced bright green due to SYTO 13. (iii) ilys-3; eEx752 transgenic displaying reduced luminal bacterial accumulation. (C) The effect of ilys-3 knockout on passage of live bacteria into the gut lumen. Bacterial load was calculated using a colony-forming units (CFU) count assay. N2 and ilys-3 mutants were exposed as L4 larvae to E. coli::GFP for 24 hours. Each symbol represents the average bacterial load obtained from pools of 10 animals. Thick horizontal bars represent the median of three independent experiments (n = 270 animals/ group analyzed). Asterisk indicates the results of a two-tailed unpaired t-test, with Welch's correction, comparing values of colony forming units/10 worms on ilys-3 versus N2 (* p = 0.0338, 95% CI). (D- E) Effect of ILYS-3 overexpression on survival rates of OP50-fed N2, ilys-3(ok3222), ilys-3; eEx752, ilys-3; eEx754, and +; eEx754 cultured at 20°C. P value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 3 independent experiments with an average of 100 animals analyzed each time. Data in bar graphs depict means ± standard deviation. (D) Lifespan analysis showing that ILYS-3 overexpression extends lifespan in ilys-3 mutants. (E) Average lifespan plot showing that the decreased average lifespan of ilys-3 deletion mutants is restored to WT levels in ILYS-3 overexpressing animals carrying eEx752 or eEx754 arrays (*** p < 0.0001). (F) Counts of CFU isolated from one-day old adult animals, fed for 24 hour on CBX102. Each symbol represents the average bacterial load obtained from three biological replicates. Asterisk indicates the results of a two-tailed unpaired t-test, with Welch's correction, comparing values of CFU/10 worms on ilys-3 versus N2 (* p = 0.0232) and ilys-3 versus eEx752 (* p = 0.0269), with a statistical confidence p value of <0.05 for each of the three repeats. (G-H) Effect of ILYS-3 overexpression on survival rates of N2 and ilys-3, upon exposure to CBX102. Transgenes used were eEx752 or eEx754. P value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 3 independent trials. Data in bar graphs depict means ± standard deviation. (G) Lifespan curves. (H) Loss of ilys-3 decreases lifespan in animals exposed to CBX102, but ILYS-3 overexpression enhances their survival during infection by this pathogen.