Ce-Duox1/BLI-3 Generated Reactive Oxygen Species Trigger Protective SKN-1 Activity via p38 MAPK Signaling during Infection in C. elegans Ransome van der Hoeven, Katie C. McCallum, Melissa R. Cruz, Danielle A. Garsin* Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America Abstract 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. Citation: van der Hoeven R, McCallum KC, Cruz MR, Garsin DA (2011) Ce-Duox1/BLI-3 Generated Reactive Oxygen Species Trigger Protective SKN-1 Activity via p38 MAPK Signaling during Infection in C. elegans. PLoS Pathog 7(12): e1002453. doi:10.1371/journal.ppat.1002453 Editor: Frederick M. Ausubel, Massachusetts General Hospital, Harvard Medical School, United States of America Received May 11, 2011; Accepted November 7, 2011; Published December 22, 2011 Copyright: ß 2011 van der Hoeven et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by R01AI076406 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. They had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Danielle.A.Garsin@uth.tmc.edu Introduction Infection by pathogenic microorganisms requires a coordinated response from the host to cope with the multitude of physiological challenges presented by the attack. In addition to producing compounds that have direct antimicrobial activity and countering pathogen virulence strategies, the host must also initiate stress responses to protect cellular resources and processes from the negative consequences of ‘‘friendly-fire.’’ Damage, disease, and sometimes death of the host can occur if immune responses are not controlled or protected against properly. Septic shock and various autoimmune diseases are examples of immune responses gone awry. In this work we explore the connections between infection, immune response and cellular stress response using the well- studied model host Caenorhabditis elegans. A general response to microbial challenge that most animals possess is the production of reactive oxygen species (ROS). The best-studied example is the production of ROS as an antimicrobial response in the phagolysosome of phagocytic cells by the NADPH oxidase gp91 phox . However, this response is not limited to phagocytes, and NADPH oxidases are present in the skin as well as the mucosal epithelium of the oral cavity, respiratory and gastrointestinal tracts of humans [1,2]. Less complex organisms that lack innate immune cells, such as C. elegans, also encode for NADPH oxidases. For instance, the dual oxidase Ce-Duox1/BLI- 3 is present in the hypodermis and in the intestine of C. elegans [3,4]. Our laboratory and others recently demonstrated that an intestinal infection triggers the release of ROS by Ce-Duox1/BLI- 3 in what appears to be a protective response [3,5]. Presumably due to the production of ROS however, there was also evidence of cellular damage as shown by lipofuscin accumulation and loss of protein homeostasis, which was worsened by the knock-down of certain oxidative stress enzymes [6,7]. The goal of this work was to determine if infection, by triggering ROS release by Ce-Duox1/ BLI-3, induces an oxidative stress response in the host as part of the overall response to the pathogen. SKN-1 is a transcription factor that senses oxidative stress and functionally affects resistance to oxidative stress and lifespan in C. elegans. It is an Nrf ortholog, a protein family found in all eukaryotes that upregulates the Phase 2 genes of the three-phase detoxification system [8]. Phase 2 genes encode enzymes that defend against ROS and other reactive compounds [9]. A large PLoS Pathogens | www.plospathogens.org 1 December 2011 | Volume 7 | Issue 12 | e1002453