Ce-Duox1/BLI-3 Generated Reactive Oxygen Species Trigger Protective SKN-1 Activity via p38 MAPK Signaling during Infection in C. elegans

Infected animals will produce reactive oxygen species (ROS) and other inflammatory molecules that help fight pathogens, but can inadvertently damage host tissue. Therefore specific responses, which protect and repair against the collateral damage caused by the immune response, are critical for successfully surviving pathogen attack. We previously demonstrated that ROS are generated during infection in the model host Caenorhabditis elegans by the dual oxidase Ce-Duox1/BLI-3. Herein, an important connection between ROS generation by Ce-Duox1/BLI-3 and upregulation of a protective transcriptional response by SKN-1 is established in the context of infection. SKN-1 is an ortholog of the mammalian Nrf transcription factors and has previously been documented to promote survival, following oxidative stress, by upregulating genes involved in the detoxification of ROS and other reactive compounds. Using qRT-PCR, transcriptional reporter fusions, and a translational fusion, SKN-1 is shown to become highly active in the C. elegans intestine upon exposure to the human bacterial pathogens, Enterococcus faecalis and Pseudomonas aeruginosa. Activation is dependent on the overall pathogenicity of the bacterium, demonstrated by a weakened response observed in attenuated mutants of these pathogens. Previous work demonstrated a role for p38 MAPK signaling both in pathogen resistance and in activating SKN-1 upon exposure to chemically induced oxidative stress. We show that NSY-1, SEK-1 and PMK-1 are also required for SKN-1 activity during infection. Evidence is also presented that the ROS produced by Ce-Duox1/BLI-3 is the source of SKN-1 activation via p38 MAPK signaling during infection. Finally, for the first time, SKN-1 activity is shown to be protective during infection; loss of skn-1 decreases resistance, whereas increasing SKN-1 activity augments resistance to pathogen. Overall, a model is presented in which ROS generation by Ce-Duox1/BLI-3 activates a protective SKN-1 response via p38 MAPK signaling

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points

OpnS, an Outer Membrane Porin of Xenorhabdus nematophila, Confers a Competitive Advantage for Growth in the Insect Host▿ †

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment

Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans.

Dual oxidases (DUOX) are enzymes that contain an NADPH oxidase domain that produces hydrogen peroxide (H2O2) and a peroxidase domain that can utilize H2O2 to carry out a variety of reactions. The model organism Caenorhabditis elegans produces the DUOX, BLI-3, which has roles in both cuticle development and in protection against infection. In previous work, we demonstrated that while certain peroxidases were protective against the human bacterial pathogen Enterococcus faecalis, the peroxidase domain of BLI-3 was not, leading to the postulate that the NADPH oxidase domain is the basis for BLI-3's protective effects. In this work, we show that a strain carrying a mutation in the NADPH oxidase domain of BLI-3, bli-3(im10), is more susceptible to E. faecalis and the human fungal pathogen Candida albicans. Additionally, less H2O2 is produced in response to pathogen using both an established Amplex Red assay and a strain of C. albicans, WT-OXYellow, which acts as a biosensor of reactive oxygen species (ROS). Finally, a C. elegans line containing a BLI-3::mCherry transgene was generated. Previous work suggested that BLI-3 is produced in the hypodermis and the intestine. Expression of the transgene was observed in both these tissues, and additionally in the pharynx. The amount and pattern of localization of BLI-3 did not change in response to pathogen exposure

BLI-3 protein levels are similar in <i>E</i>. <i>faecalis</i> and <i>E</i>. <i>coli</i> fed worms.

<p><b>(A)</b> A representative Western blot of BLI-3 protein expression in non-infected and infected worms. <b>(B)</b> For quantification of BLI-3, a ratio of BLI-3 protein to tubulin was calculated from independent experiments. The error bars represent the standard error of the mean.</p

Less H₂O₂ production is observed in the <i>bli-3</i>(<i>im10</i>) mutant in comparison to the wild type worms during infection.

<p>H₂O₂ production was measured by an Amplex Red assay. While no differences were observed with uninfected animals, significantly less H₂O₂ production was observed with infected <i>bli-3(im10</i>) mutants compared to infected wild type worms (<i>P</i> = 0.0052). The difference between wild type and <i>bli-3(e767)</i> was not significant (<i>P</i> = 0.7112). The experiment was performed in triplicate and one of two replicates is shown.</p

A mutation in the BLI-3 NADPH oxidase domain increases sensitivity to pathogen and shortens lifespan.

<p><i>bli-3(im10)</i> mutants exhibited increased susceptibility after exposure to <b>(A)</b><i>E</i>. <i>faecalis</i>, <b>(B)</b><i>C</i>. <i>albicans</i> compared to wild type animals (<i>P</i> < 0.0001 and <i>P</i> = 0.0056 respectively). Differences were not significant between <i>bli-3(e767)</i> and wild type. <b>(C)</b><i>bli-3(im10)</i> displayed shortened survival in the standard <i>E</i>. <i>coli</i>-based longevity assay compared to wild type (<i>P</i> < 0.0001). <i>bli-3(e767)</i> also exhibited a defect in comparison to wild type in this assay (<i>P</i> < 0.0001). <b>(D)</b><i>bli-3(im10)</i> also had a shortened lifespan on heat killed <i>E</i>. <i>coli</i> compared to wild type (<i>P</i> < 0.0001). The data are representative of experiments repeated three times with an n = 60–90 worms for each condition. All worms were exposed to <i>cdc-25</i>.<i>1</i> RNAi prior to beginning these assays to render them sterile.</p

Localization of BLI-3 to the hypodermis, pharynx and apical membranes of the worm intestinal cells.

<p><b>(A)</b> One of four transgenic lines showing expression of <i>BLI-3</i>::<i>mCherry</i> in the hypodermis (medium white arrow), pharynx (large white arrow) and the apical membranes of the intestinal cells (inset). <b>(B-C)</b> One of two integrated transgenic <i>BLI-3</i>::<i>mCherry</i> lines demonstrating expression in the <b>(B)</b> pharynx, apical membrane and the <b>(C)</b> hypodermis. Large white arrows indicate the pharynx, medium white arrows, the hypodermis, and the small white arrows, the apical membranes of the intestinal cells. Patterns of expression shown are typical for all animals of all lines.</p

Glycolysis regulates KRAS plasma membrane localization and function through defined glycosphingolipids

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential