RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae Xuan Li 1 , Qian Yang 1 , Kristof Dierckens 1 , Debra L. Milton 2,3 , Tom Defoirdt 1,4 * 1 Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Ghent, Belgium, 2 Southern Research Institute, Birmingham, Alabama, United States of America, 3 Department of Molecular Biology, Umea ˚ University, Umea ˚, Sweden, 4 Laboratory of Microbial Ecology and Technology, Ghent University, Ghent, Belgium Abstract Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50–100 mM) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress. Citation: Li X, Yang Q, Dierckens K, Milton DL, Defoirdt T (2014) RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae. PLoS ONE 9(10): e111801. doi:10.1371/journal.pone.0111801 Editor: Riccardo Manganelli, University of Padova, Medical School, Italy Received August 7, 2014; Accepted October 7, 2014; Published October 31, 2014 Copyright: ß 2014 Li 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. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was financially supported by the Scientific Research Fund of Flanders (FWO-Vlaanderen project n u 1.5.013.12N) and the Special Research Fund of Ghent University (GOA project n u BOF12/GOA/022). XL and QY are doctoral researchers funded by a China Scholarship Council grant and a Special Research Grant (BOF-UGent) of Ghent University. TD is a postdoctoral fellow of FWO-Vlaanderen. The funders 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. * Email: Tom.Defoirdt@UGent.be Introduction Vibrio anguillarum is the causative agent of vibriosis, a fatal haemorrhagic septicaemia affecting many aquatic organisms (fish, crustaceans as well as mollusks) [1]. The bacterium is a major pathogen of aquaculture organisms, causing significant economic losses in the aquaculture industry [2]. Several (putative) virulence factors have been identified, although for many of these factors, the specific role in disease is not yet known. Three factors that have been reported to be essential for pathogenicity include the iron uptake system involving the siderophore anguibactin [3–4], chemotactic motility (which is required for entry into the host) [5– 6] and exopolysaccharide production (which is required for attachment to the host) [7]. The bacterium produces a number of other (putative) virulence factors, including haemolysin, lipase and protease [8–10]. However, whether or not these factors are really essential for pathogenicity is currently not clear. As virulence factors are often costly metabolic products, their expression usually is tightly regulated. Quorum sensing, a type of bacterial cell-to-cell communication that uses small signal mole- cules, is one of the regulatory mechanisms controlling the expression of virulence genes in many bacteria [11]. Vibrio anguillarum has been documented to contain two quorum sensing systems, a ‘classical’ acylhomoserine lactone (AHL) system involving the signal synthase/receptor pair VanI/VanR, and a three-channel system as found in many vibrios [12]. Unlike other vibrios, reports published to date indicate that quorum sensing is not involved in regulating the virulence of V. anguillarum [1,12] and we found that this is also the case in gnotobiotic sea bass larvae (our unpublished results). Indole is another molecule that recently gained attention as a putative quorum-sensing signal molecule [13]. Indole is produced by tryptophanase (encoded by the tnaA gene), which reversibly converts tryptophan into indole, pyruvate and ammonia [14]. Despite the fact that many bacteria (including several vibrios) have been known for a long time to produce substantial amounts of indole, its biological role as a signal molecule has only recently been revealed [13]. Most work in this respect has been done on enteric bacteria, mainly E. coli, in which indole has been reported to control virulence-related phenotypes such as biofilm formation, motility, chemotaxis and adherence to epithelial cells [15–16]. In enteropathogenic E. coli, the indole biosynthase TnaA, has been reported to be required for virulence to nematodes [17]. Finally, indole production in E. coli is regulated by the alternative sigma factor RpoS as RpoS induces the expression of the tryptophanase gene tnaA [18]. Thus far, very PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e111801