Figure 1.
Specific downregulation of host immune effector gene expression following P. aeruginosa infection requires gacA, lasR and rhlR.
(A–B) Expression of the host defense effector genes thn-2, lys-7, spp-1, abf-2, F08G5.6, and lys-2 in wildtype worms exposed to P. aeruginosa PA14, S. typhimurium SL1344, and E. faecalis V583. (C) Expression of downregulated host defense effectors genes in wildtype worms exposed to the PA14 mutants gacA, lasR, and rhlR. Mean transcript levels are plotted relative to matched controls exposed to OP50-1. Error bars indicated SEM. At least 3 replicates of each condition were examined. * t-test, p<0.05 comparison to N2 on OP50-1.
Figure 2.
The downregulation of host defense genes is specifically attenuated in daf-2 insulin-like signaling mutants.
(A–B) The transcriptional response to PA14 infection in wildtype N2 versus (A) daf-2(e1370) and (B) sma-6(wk7) worms is plotted based on whole-genome microarray analysis. Values are log2 scale transformations of the response to infection. The subset of genes that were differentially regulated in N2 worms is shown (p<0.05 and absolute difference >2 fold). Linear regressions are plotted for induced (red) and repressed (green) genes independently. (A) In daf-2(e1370), the response to induced genes to PA14 infection was largely intact (r2 = 0.4280, p<0.0001), but the response to repressed genes was strongly attenuated (r2 = 0.0009, p = 0.8306). (B) In sma-6(wk7), the response of both induced and repressed genes was largely intact (p<0.0001). (C–E) The transcriptional response to PA14 infection in wildtype N2 and (A) daf-2(e1370), (B) sek-1(km4), and (C) sma-6(wk7) animals is plotted for a set of 146 candidate immune and stress response genes based on qRT-PCR measurement. Linear regressions were calculated for induced (red) and repressed (green) genes independently. The associated correlation coefficients (r2) and p-values are indicated. (C) In daf-2(e1370), the response to induced genes to PA14 infection was largely intact (r2 = 0.62, p<0.0001), but the response to repressed genes was strongly attenuated (r2 = 0.061, p = 0.1103). (D) In sek-1(km4) the response of both induced and repressed genes was largely intact (p<0.0001 and p = 0.0026, respectively). (E) In sma-6(wk7) the response of both induced and repressed genes was largely intact (p<0.0001 each).
Figure 3.
DAF-2/DAF-16 insulin-like signaling mediates the downregulation of host defense effector gene expression by P. aeruginosa.
Expression of the host defense effectors (A) thn-2, (B) lys-7, (C) spp-1, (D) abf-2, (E) F08G5.6, and (F) lys-2 were measured in N2, daf-2(e1370), daf-16(mu86), and daf-16(mu86);daf-2(e1370) exposed to OP50-1 and PA14. Mean transcript levels from at least 3 independent experiments were plotted relative to N2 exposed to OP50-1. Error bars represent SEM. t-test * p<0.05 comparison to N2 OP50-1, ˆ p<0.05 comparison to N2 PA14, + p<0.05 comparing OP50-1 to PA14.
Figure 4.
P. aeruginosa infection causes delocalization of nuclear DAF-16 in the intestine.
(A–D) Representative fluorescence micrographs of DAF-16::GFP expressing adult worms with nuclear localized DAF-16::GFP fusion protein after exposure to (A) OP50-1 (B) PA14 gacA, and (C) wildtype PA14, and also (D) nuclear delocalized DAF-16::GFP in worms after exposure to wildtype PA14. (E) Mean number of nuclei with localized DAF-16::GFP after 18 hr exposure to either OP50-1 or PA14 following acute heat shock. ** t-test, p<0.001 (F–H) Proportion of worms in a population categorized as either showing predominately nuclear localized (“nuclear”) or predominately “delocalized” intestinal DAF-16::GFP. Nuclear localization was induced by either heat shock (70–90 minutes at 37°C) (F) or loss of germline proliferation (G–H) and then worms were exposed to (F–H) OP50-1, PA14, PA14 gacA, (G) SL1344, V583, (H) PA14 lasR, or PA14 rhlR. *** Fisher's exact test, p<0.0001.
Figure 5.
Insulin-like signaling is required for the delocalization of nuclear DAF-16 during P. aeruginosa infection.
(A) Proportion of daf-2(e1370); DAF-16::GFP worms exposed to OP50-1 or PA14 categorized as showing either predominately nuclear localized (“nuclear”) or predominately “delocalized” DAF-16::GFP. No nuclear delocalization is observed in daf-2(e1370) mutants. (B) Expression of insulin-like genes ins-11 and ins-7 in wildtype worms exposed to OP50-1, PA14, PA14 gacA, V583, and SL1344. (C) Proportion of worms in a population categorized as either showing predominately nuclear localized (“nuclear”) or predominately “delocalized” DAF-16::GFP. RNAi knockdown of ins-11, ins-7 or vector control RNAi shown. (D) Proportion of DAF-16::GFP and DAF-16::GFP;ins-7(tm1907) worms in a population categorized as either showing predominately nuclear localized (“nuclear”) or predominately “delocalized” DAF-16::GFP. *** Fisher's exact test, p<0.0001.
Figure 6.
Intestinal DAF-16 is specifically required for resistance to P. aeruginosa.
(A–B) Survival of worms cultured on PA14 lawns was monitored over time at 25°C. (A) In wildtype N2 worms, the effects on survival of RNAi knockdown of daf-16 or control RNAi were indistinguishable (logrank, p = 0.85). (B) In VP303 worms, intestinally restricted RNAi knockdown of daf-16 caused enhanced sensitivity to PA14 compared to control RNAi (logrank, p = 0.0002).
Figure 7.
Model of P. aeruginosa activation of insulin-like signaling.
PA14 infection causes an insulin agonist-mediated activation of DAF-2, which likely involves expression of ins-7. Upon DAF-2 activation, several serine-threonine kinases phorphorylate DAF-16, causing the transcription factor to be retained in the cytoplasm and inhibiting its transcriptional-regulatory activity. Consequently, DAF-2–regulated gene expression is affected, causing the downregulation of immune defense genes such as lys-7 and thn-2. Germline signaling and heat shock regulate DAF-16 in parallel to the effect of PA14 and DAF-2 activity. DAF-2 and DAF-16 may function in a network with other signaling components to affect the expression of DAF-2-regulated genes including spp-1.