Intracellular Debugging of the NLR Signaling Pathways

<p>A schematic overview of the major NLR signaling pathways in innate immunity is depicted. Upon detection of their agonists, NLR likely oligomerize through the NOD domain and recruit at least three specific adaptors, including RIP2, CARD9, and ASC. Several modulators of NLR signaling have been recently identified, such as Erbin, Bcl2, and Bcl-xl. The maturation of IL-1β by the inflammasome illustrates the interplay between NLR (i.e., NALP1–3, NAIP5, and IPAF) and other PRM, such as TLR. Better understanding of the spatio-temporal engagement and/or repression of specific NLR might open new avenues for therapeutical intervention.</p

Specific Reduction of <i>PGRP-SC1/2</i> mRNA Using RNA Interference In Vivo

<div><p>Each histogram corresponds to the mean value of four independent experiments (± standard deviation).</p><p>(A) mRNA-level quantification for different PGRPs <i>(PGRP/RpL32)</i> shows that <i>PGRP-SC1</i> and <i>PGRP-SC2</i> mRNA levels are severely reduced in <i>DaGal4;UAS iPGRP-SC</i> flies as compared to <i>UAS iPGRP-SC</i> control flies. <i>PGRP-SA</i> and <i>PGRP-SD</i> mRNA levels are not affected. One hundred percent corresponds to the wild-type value for each transcript. Asterisks indicate that the difference between <i>UAS iPGRP-SC</i> and <i>DaGal4;UAS iPGRP-SC</i> values is statistically significant (<i>p</i> < 0.05).</p><p>(B) Primer specificity in RT-PCR experiments shown by quantification of <i>PGRP-SC1b</i> and <i>PGRP-SC2</i> transcripts. <i>PGRP-SC1b</i> over-expression in <i>HspGal4;UAS PGRP-SC1b</i> flies 1 h after a 30-min heat-shock (37 °C) treatment (HS) is well detected with <i>PGRP-SC1</i> primers but not with those for <i>PGRP-SC2.</i> We can infer that the <i>PGRP-SC2</i> primers used in this study are able to discriminate between <i>PGRP-SC2</i> and <i>PGRP-SC1b</i> transcripts.</p></div

Toll-Pathway Activation Is Wild-Type in <i>DaGal4;UAS iPGRP-SC</i> Flies

<p>Kinetics of <i>drosomycin</i> mrna induction <i>(Drs/RpL32)</i> after infection by gram-positive (upper panel) and gram-negative (lower panel) bacteria. Each histogram corresponds to the mean value of six independent experiments (± standard deviation). Asterisk indicates that the difference between <i>DaGal4;UAS iPGRP-SC</i> and control <i>UAS iPGRP-SC</i> values is statistically significant (<i>p <</i> 0.05). One hundred percent corresponds to the level of activation 24 h after infection in control flies. In the lower panel, <i>drosomycin</i> induction after gram-negative bacterial infections is compared to that of S. aureus infection which is set to 100%. <i>RpL32</i> is used as an internal control.</p

Reduction of PGRP-SC1/2 Levels in the Larval Gut Increases IMD-Pathway Activation after Natural Infection

<div><p>(A) <i>PGRP-SC1</i> and <i>PGRP-SC2</i> mRNAs <i>(PGRP-SC/RpL32)</i> are mainly expressed in the larval gut and are severely reduced in <i>DaGal4;UAS iPGRP-SC</i> and <i>CadGal4;UAS iPGRP-SC</i> larvae. Each histogram corresponds to the mean value of four independent experiments (± standard deviation).</p><p>(B) <i>Diptericin</i> mrna induction levels <i>(Dipt/RpL32),</i> measured 6 h and 24 h after natural infection. Each histogram corresponds to the mean value of variable numbers (shown in parentheses) of independent experiments (± standard deviation). Asterisks indicate that the difference between <i>DaGal4;UAS iPGRP-SC</i> or <i>CadGal4;UAS iPGRP-SC</i> and control <i>UAS iPGRP-SC</i> values is statistically significant (<i>p <</i> 0.05).</p><p>(C) <i>Drosomycin</i> mrna induction levels <i>(Drs/RpL32),</i> measured 24 h after natural infection. Each histogram corresponds to the mean value of four independent experiments (± standard deviation).</p><p>(D) Three hours after natural infection with E. coli GFP, bacteria were found to be highly concentrated in the anterior half of the larval gut. In larvae with reduced gut <i>PGRP-SC</i> levels <i>(CadGal4;UAS iPGRP-SC),</i> feeding on <i>E.coli</i> is sufficient to trigger IMD-pathway activation in the fat body after 24 h (visualized here by the use of a <i>diptericin-LacZ</i> transgene).</p><p>(E) Percentage of larvae showing β-galactosidase activity in the fat body 24 h after natural infection. For each genotype, ten larvae were dissected and stained. Each histogram corresponds to the mean value of five independent experiments (± standard deviation). Asterisks indicate that the difference between <i>diptLacZ;UAS iPGRP-SC</i> and <i>diptLacZ;DaGal4;UAS iPGRP-SC</i> or <i>diptLacZ;CadGal4;UAS iPGRP-SC</i> values and between <i>diptLacZ,UAS iPGRP-SC</i> and <i>diptLacZ;CadGal4;UAS iPGRP-SC</i> values is statistically significant (<i>p</i> < 0.05).</p></div

Reduction of PGRP-SC1/2 Levels Sensitizes Larvae to Bacterial Infection

<div><p>(A) The percentage of dead larvae is measured 24 h after natural infection. Numbers in parentheses correspond to the total number of infected larvae. Each histogram corresponds to the mean value of six independent experiments (± standard deviation) for E. coli and five for <i>E. carotovora carotovora.</i> Asterisks indicate that the difference between <i>UAS iPGRP-SC</i> and <i>DaGal4;UAS iPGRP-SC</i> values is statistically significant (<i>p</i> < 0.05).</p><p>(B) The percentage of adults showing wing notching is measured 7 d after natural infection. Numbers in parentheses correspond to the total number of infected larvae.</p><p>(C–F) Natural infection with <i>E. carotovora carotorova</i> and E. coli triggers increased cell death and developmental defects in <i>DaGal4;UAS iPGRP-SC</i> flies. Wing imaginal discs dissected from <i>DaGal4;UAS iPGRP-SC</i> larvae (F) show higher levels of cell death after natural infection than discs from infected <i>UAS iPGRP-SC</i> control larvae (E). Consistently, some <i>DaGal4;UAS iPGRP-SC</i> adults derived from infected larvae exhibit wing notching (indicated by arrowheads) (D), which was never observed in infected controls (C).</p></div

IMD-Pathway Activation Is Downregulated by PGRP-SC1/2

<div><p>(A) Kinetics of <i>diptericin</i> mrna induction <i>(Dipt/RpL32)</i> after infection by various bacteria. Each histogram corresponds to the mean value of five independent experiments (± standard deviation). Asterisks indicate that the difference between <i>DaGal4;UAS iPGRP-SC</i> and control <i>UAS iPGRP-SC</i> values is statistically significant (<i>p <</i> 0.05). One hundred percent corresponds to the level of activation at 6 h in control flies. In the lower panel, <i>diptericin</i> induction after gram-positive bacterial infections were compared to that obtained after infection by E. cloacae (100%). <i>RpL32</i> is used as an internal control. ci, clean injury; ni, noninfected.</p><p>(B) Quantification of <i>diptericin</i> mRNA levels in <i>UAS iPGRP-SA</i>, <i>DaGal4;UAS iPGRP-SA</i> and <i>PGRP-SA^seml</i> flies shows that reduction of <i>PGRP-SA</i> mRNA levels does not influence IMD-pathway induction 6 h after infection by <i>E. coli.</i> Quantification of <i>drosomycin</i> mRNA levels 24 h after M. luteus infection indicates that PGRP-SA is efficiently knocked down by dsRNA interference. <i>PGRP-SA^seml</i> is a complete loss-of-function mutant for PGRP-SA. Each histogram corresponds to the mean value of five independent experiments (± standard deviation). Asterisk indicates that the difference between <i>DaGal4;UAS iPGRP-SA</i> and control <i>UAS iPGRP-SA</i> values is statistically significant (<i>p</i> < 0.05).</p><p>(C) <i>DaGal4;UAS iPGRP-SC</i> flies are as susceptible to infection by E. cloacae as control flies<i>.</i></p><p>(D) E. cloacae and <i>E. coli AmpR</i> growth in various genetic backgrounds 24 h after infection. Flies with reduced levels of PGRP-SC1/2, unlike <i>IKK</i>γ^key1 mutants, are able to clear bacteria from their hemolymph. Each histogram corresponds to the mean value of four independent experiments (± standard deviation).</p></div

MOESM1 of High carriage of adherent invasive E. coli in wildlife and healthy individuals

Additional file 1: Table S1. Primers used for E. coli virulence genes detection