The Evolutionarily Conserved Mediator Subunit MDT-15/MED15 Links Protective Innate Immune Responses and Xenobiotic Detoxification

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses

Aberrant Activation of p38 MAP Kinase-Dependent Innate Immune Responses Is Toxic to Caenorhabditis elegans

Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the nematode Caenorhabditis elegans. We previously identified an immunostimulatory xenobiotic that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the deleterious effects of the compound, and identified five toxicity suppressors. These strains contained hypomorphic mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1, and pmk-1), demonstrating that hyperstimulation of the p38 MAPK pathway is toxic to animals. To explore mechanisms of immune pathway regulation in C. elegans, we conducted another genetic screen for dominant activators of the p38 MAPK pathway, and identified a single allele that had a gain-of-function (gf) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK pmk-1. The nsy-1(gf) allele caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, had greater levels of phosphorylated p38 MAPK, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. In addition, the nsy-1(gf) mutation was toxic to developing animals. Together, these data suggest that the activity of the MAPKKK NSY-1 is tightly regulated as part of a physiological mechanism to control p38 MAPK-mediated innate immune hyperactivation, and ensure cellular homeostasis in C. elegans

Stimulation of Host Immune Defenses by a Small Molecule Protects C. elegans from Bacterial Infection

The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans–based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens

Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals

Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

Background: Many pathogens secrete toxins that target key host processes resulting in the activation of immune pathways. The secreted Pseudomonas aeruginosa toxin Exotoxin A (ToxA) disrupts intestinal protein synthesis, which triggers the induction of a subset of P. aeruginosa-response genes in the nematode Caenorhabditis elegans. Results: We show here that one ToxA-induced C. elegans gene, the Tribbles pseudokinase ortholog nipi-3, is essential for host survival following exposure to P. aeruginosa or ToxA. We find that NIPI-3 mediates the post-developmental expression of intestinal immune genes and proteins and primarily functions in parallel to known immune pathways, including p38 MAPK signaling. Through mutagenesis screening, we identify mutants of the bZIP C/EBP transcription factor cebp-1 that suppress the hypersusceptibility defects of nipi-3 mutants. Conclusions: NIPI-3 is a negative regulator of CEBP-1, which in turn negatively regulates protective immune mechanisms. This pathway represents a previously unknown innate immune signaling pathway in intestinal epithelial cells that is involved in the surveillance of cellular homeostasis. Because NIPI-3 and CEBP-1 are also essential for C. elegans development, NIPI-3 is analogous to other key innate immune signaling molecules such as the Toll receptors in Drosophila that have an independent role during development. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0334-6) contains supplementary material, which is available to authorized users

Protection from the toxic effects of the xenobiotic RPW-24 requires MDT-15, but not PMK-1.

<p>(A) The thirteen xenobiotic detoxification genes that were induced 4-fold or greater by RPW-24 in the NanoString nCounter gene expression analysis are presented. The top panel compares the RPW-24-mediated induction of these genes in vector control (L4440) and <i>mdt-15(RNAi)</i> animals, and the bottom panel shows these data for wild-type N2 versus <i>pmk-1(km25)</i> animals, as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat-1004143-g002" target="_blank">Figure 2</a>. * <i>p</i><0.05 for the comparison of the RPW-24-induced conditions. (B) Vector control (L4440), <i>mdt-15(RNAi)</i> and <i>pmk-1(RNAi)</i> animals were exposed to 70 µM RPW-24 or the solvent control DMSO from the L1 stage and photographed after 70 hours of development at 20°C. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s004" target="_blank">Figure S4</a> for the quantification data from this experiment.</p

MDT-15 is required for defense against <i>P. aeruginosa</i> infection.

<p>A <i>P. aeruginosa</i> pathogenesis assay with wild-type N2, <i>mdt-15(tm2182)</i> mutant worms and <i>mdt-15(tm2182)</i> animals carrying <i>pmdt-15::mdt-15</i> (three independent lines <i>agEx116</i>, <i>agEx117</i> and <i>agEx118</i>) is shown. The difference in <i>P. aeruginosa</i> susceptibility between <i>mdt-15(tm2182)</i> animals and each of the three transgenic lines carrying <i>pmdt-15::mdt-15</i> is significant, as is the survival difference between N2 and <i>mdt-15(tm2182)</i> animals (<i>p</i><0.001). For sample sizes, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004143#ppat.1004143.s009" target="_blank">Table S3</a>.</p

The Mediator subunit MDT-15 acts downstream of the p38 MAP kinase PMK-1 to regulate the induction of <i>F08G5.6</i> and <i>F35E12.5</i>.

<p>(A) Wild-type or <i>mdt-15(tm2182)</i> mutant synchronized L1 animals containing the <i>pF08G5.6::GFP</i> immune reporter were grown on vector control (L4440), <i>vhp-1</i>(RNAi) or a combination of <i>vhp-1(RNAi)</i> and <i>pmk-1(RNAi)</i> bacteria and then transferred as L4 animals to PA14 for 18 hours. Animals were photographed under the same imaging conditions. (B) qRT-PCR was used to examine the expression levels of <i>F08G5.6</i>, <i>F35E12.5</i> and <i>C32H11.1</i> in wild-type N2 and <i>mdt-15(tm2182)</i> mutant animals exposed to <i>vhp-1(RNAi)</i> or the vector control (L4440) under basal conditions (as described above) and 8 hours after exposure to <i>P. aeruginosa</i>. Knockdown of <i>vhp-1</i> caused significant induction of <i>F08G5.6</i> and <i>F35E12.5</i> in wild-type N2 animals (<i>p</i><0.001), but not in <i>mdt-15(tm2182)</i> animals (<i>p</i>>0.05), under baseline (<i>E. coli</i>) and pathogen-induced conditions. The expression of <i>C32H11.1</i> was significantly induced by <i>vhp-1(RNAi)</i> (<i>p</i><0.001) in an <i>mdt-15</i>-dependent manner under baseline conditions (<i>p</i><0.001), but not following exposure to <i>P. aeruginosa</i>. Data are the average of two biological replicates each normalized to a control gene with error bars representing SEM and are presented as the value relative to the average expression of the indicated gene in the baseline condition (L4440 animals exposed to <i>E. coli</i>).</p

The Mediator subunit MDT-15 regulates the induction of some, but not all, immune genes during <i>P. aeruginosa</i> infection.

<p>The expression of putative <i>C. elegans</i> immune effectors was analyzed by qRT-PCR in vector control (L4440) and <i>mdt-15(RNAi)</i> animals exposed to <i>P. aeruginosa</i> and the negative control <i>E. coli</i> OP50 for 8 hours. Data are the average of three replicates each normalized to a control gene with error bars representing SEM and are presented as the value relative to the average expression from all three replicates of the indicated gene in the baseline condition (L4440 animals exposed to <i>E. coli</i>). <i>mdt-15</i> expression was significantly knocked down by <i>mdt-15(RNAi)</i> in these experiments (<i>p</i><0.001).</p