Excessive activation of SKN-1 by <i>wdr-23(RNAi)</i> impairs pathogen resistance.

<p>(<b>A</b>) Robust up-regulation of <i>Pgcs-1</i>::GFP and GST-4::GFP in 1 d adult <i>wdr-23(RNAi)</i> worms. (<b>B</b>) Pathogen resistance of <i>wdr-23(RNAi)</i>-fed N2 and <i>skn-1(zu135)</i> mutant worms. N2;<i>wdr-23(RNAi)</i> exhibited increased susceptibility to <i>P. aeruginosa</i> infection (p<0.0001). <i>skn-1(zu135)</i> mutant nematodes fed by <i>wdr-23(RNAi)</i> showed no significant difference in survival on PA14 (p = 0.1992). Killing assay was performed with at least 90 1-day old adult animals in each condition. Please note that data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat-1002673-g001" target="_blank">Figures 1A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat-1002673-g006" target="_blank">6B</a> were derived from the same set of experiments. (<b>C</b>) <i>wdr-23(RNAi)</i> treatment increased (p<0.0001, both at 3 mM and 5 mM H₂O₂, respectively), while <i>skn-1 RNAi</i> treatment decreased oxidative tolerance to H₂O₂ (p<0.0001 at 3 mM H₂O, p<0.05 at 5 mM H₂O₂). Worms were treated with 3 mM or 5 mM H₂O₂ for 1 hour, and 24 h after challenge survival was scored. Data were combined from three experiments with 120 animals in average for each group. EV: empty vector RNAi.</p

SKN-1 is required for bacterial pathogen resistance.

<p>(<b>A, B</b>) Increased susceptibility to <i>Pseudomonas aeruginosa</i> PA14 occurs in both <i>skn-1(zu135)</i> mutant (p<0.0001) and <i>skn-1(RNAi)</i> nematodes (p<0.0001). Killing assays were performed with at least 90 young adult animals in each condition. EV: empty vector RNAi.</p

Involvement of SKN-1 in immunosenescence.

<p>(<b>A</b>) Representative epifluorescence images showing the decreased induction of the <i>gcs-1</i> promoter. L3 larvae, 4 d/9 d adult <i>Pgcs-1::gfp</i> worms were exposed to PA14 for 24 h. (<b>B</b>) Quantification of the epifluorescence images of panel (A). Epifluorescence images are representatives of two independent experiments. EV: empty vector RNAi. (<b>C</b>) Pathogen resistance of young adult (1 day-old), 4 day-old and 9 day-old adult N2 and <i>skn-1(zu135)</i> mutant animals. <i>skn-1(zu135)</i> mutant worms exhibited significantly increased susceptibility to PA14 compared to N2 wild-type animals at all ages (p<0.0001). 1 day-old adult <i>skn-1(zu135)</i> worms show similar pathogen resistance to 4 day-old N2 worms (p = 0.1429) (middle graph). Killing assays were performed with 3 parallel plates in each condition in 2 independent trials. (<b>D</b>) Venn diagram showing the distribution of age-regulated SKN-1 target genes. Data were analyzed by finding the overlaps between micro-array databases containing the genes down-regulated at least 10-fold in 15 d adult compared to 6 d adult wild-type animals <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673-Youngman1" target="_blank">[27]</a> and SKN-1 dependent genes under non-stress <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673-Oliveira1" target="_blank">[29]</a> or oxidative stress conditions <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673-Park1" target="_blank">[30]</a> using expression data from Wormbase <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673-Yook1" target="_blank">[31]</a>. Please note that the majority of genes belong to those regulated by PA14 infection. 10 of 46 genes could be assigned to none of the groups. For the detailed gene list please refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673.s006" target="_blank">Table S2</a>.</p

Reduced IIS and oxidative preconditioning require SKN-1 for enhanced pathogen resistance.

<p>(<b>A</b>) <i>daf-2(e1370)</i> mutant nematodes exhibited increased resistance to <i>P. aeruginosa</i>, compared to that of wild-type N2 worms (p<0.0001). <i>skn-1(RNAi)</i> treatment of <i>daf-2(e1370)</i> animals increased the susceptibility to <i>P. aeruginosa</i> infection (p<0.0001). Killing assays were performed with at least 90 young 1-day old adult animals in each condition. (<b>B</b>) H₂O₂ pretreatment increased survival on PA14 in a concentration-dependent manner. Survival curves of N2 wild-type worms treated with various concentrations of H₂O₂ in liquid NGM: 1 mM (p = 0.425), 1.5 mM (p<0.0001) and 2 mM (p<0.0001) 12 h prior to the killing assay are shown. Killing assay was performed with 90 3-day old adult animals in each condition. (<b>C</b>) Oxidative preconditioning-induced pathogen resistance was impaired in the absence of SKN-1 or DAF-16. Increase in survival was less pronounced in either <i>skn-1(zu135)</i> (p = 0.0156) or <i>daf-16(mu86)</i> mutant (p = 0.0304), than in wild-type animals (p<0.0001). Survival curves of the same genetic background were compared in the absence and presence of H₂O₂. Data were combined from at least two experiments with 89 animals in average for each group. EV: empty vector RNAi.</p

<i>P. aeruginosa</i> infection activates SKN-1.

<p>(<b>A</b>) Representative epifluorescence image demonstrating the translocation of SKN-1::GFP in the <i>Is007</i>[SKN-1::GFP] strain to intestinal nuclei in L3 larvae, fed by the empty vector or <i>skn-1</i> dsRNA, upon a 5-hour exposure to <i>P. aeruginosa</i> PA14. Note that the intestinal tissue displays autofluorescence, and in the ASI neurons SKN-1::GFP is not silenced by <i>skn-1</i> RNAi treatment. (<b>B</b>) Quantification of SKN-1 nuclear translocation from data shown on panel (A). SKN-1::GFP-positive nuclei were counted in the intestine of 78 animals. “Low” refers to animals in which SKN-1::GFP was detected in less than 5 intestinal nuclei, while “high” indicates that SKN-1::GFP signal was present in more than 15 intestinal nuclei. (<b>C</b>) Representative epifluorescence microscopic image showing intestinal expression of <i>Pgcs-1</i>::GFP and GST-4::GFP in L3 larvae upon a 24-hour PA14 exposure. Images of control animals incubated on OP50 bacteria are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673.s002" target="_blank">Figure S2</a>. (<b>D</b>) Quantification of reporter expression demonstrating the SKN-1-dependence of the response. Data were obtained from panel (C) completed with the data of <i>skn-1(RNAi)</i> animals. Microscopic images are representatives of 3 independent experiments. EV: empty vector RNAi.</p

The pathogen response-specific TIR-1 and p38 MAPK PMK-1 are required for SKN-1 activation upon <i>P. aeruginosa</i> infection.

<p>(<b>A</b>) Representative epifluorescence microscopic images showing the expression of <i>Pgcs-1</i>::GFP in <i>pmk-1(km25)</i> mutants as well as in the p38 MAPK phosphatase <i>vhp-1(RNAi)</i>, and the Toll/IL-1 resistance (TIR) domain protein <i>tir-1(RNAi)</i> animals in response to <i>P. aeruginosa</i> infection. L3 larvae were exposed to PA14 for 24 hours. Microscopic images are representatives from 3 independent experiments. (<b>B</b>) Quantification of reporter expression from data shown on panel (A) completed with data of control animals fed by OP50 for 24 h. (<b>C</b>) Quantification of SKN-1 nuclear translocation in <i>tir-1(RNAi)</i> L3 larvae upon 5 h PA14 exposure. Representative epifluorescence images of <i>tir(RNAi)</i> L3 larvae are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat.1002673.s003" target="_blank">Figure S3</a>. Please note that data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat-1002673-g002" target="_blank">Figure 2B</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002673#ppat-1002673-g003" target="_blank">3C</a> were derived from the same set of experiments. (<b>D</b>) Suggested model of SKN-1 activation during <i>P. aeruginosa</i> infection. Upon exposure to PA14, the TIR-1/PMK-1 pathway is indispensable but insufficient to elicit SKN-1 transactivation. We propose a second, unknown factor/pathway that is required to activate SKN-1. Whether the two pathways act in parallel or consecutively is unclear. Solid arrows indicate a direct, while dashed arrows indicate an indirect/unknown connection. EV: empty vector RNAi.</p

Relazione del Consiglio all'Assemblea per l'approvazione del bilancio al 31.12.2003

Consiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome / CNR - Consiglio Nazionale delle RichercheSIGLEITItal