Lipopolysaccharide O-Antigen Prevents Phagocytosis of Vibrio anguillarum by Rainbow Trout (Oncorhynchus mykiss ) Skin Epithelial Cells Kristoffer Lindell1, Anna Fahlgren1, Erik Hjerde3, Nils-Peder Willassen3, Maria Fa¨ llman1, Debra L. Milton1,2* 1 Department of Molecular Biology, Umea˚ Centre for Microbial Research, Umea˚ University, Umea˚, Sweden, 2 Southern Research Institute, Birmingham, Alabama, United States of America, 3 Department of Chemistry, Faculty of Science and Technology, University of Tromsø, Tromsø, Norway Abstract Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues. Citation: Lindell K, Fahlgren A, Hjerde E, Willassen N-P, Fa¨llman M, et al. (2012) Lipopolysaccharide O-Antigen Prevents Phagocytosis of Vibrio anguillarum by Rainbow Trout (Oncorhynchus mykiss) Skin Epithelial Cells. PLoS ONE 7(5): e37678. doi:10.1371/journal.pone.0037678 Editor: Stephen V. Gordon, University College Dublin, Ireland Received January 27, 2012; Accepted April 26, 2012; Published May 25, 2012 Copyright: 2012 Lindell 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 performed within the Umea˚ Centre for Microbial Research and was supported by funding from the Swedish Council for Environment, Agricultural Sciences and Spatial Planning (221-2O1 0-577), from the Carl Tryggers Foundation, Sweden (CTS: 10-261), from the Swedish Research Council, and from the Natural Science Faculty of Umea˚ University. 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. * E-mail: miltond@southernresearch.org Introduction Vibrio anguillarum, part of the normal microflora on fish and in the aquatic environment, is an opportunistic pathogen that causes a fatal hemorrhagic septicemia in marine fish [1,2]. This pathogen has had devastating impacts on aquaculture worldwide causing large economical losses. Consequently, V. anguillarum is one of the best studied bacterial fish pathogens. Despite the importance of this and other fish pathogens, very few direct studies have been done to characterize bacterial factors required for pathogens to colonize fish tissues [3]. Attachment to and colonization of host tissues are the first steps that many pathogens must take to cause disease. In a study utilizing rainbow trout, V. anguillarum bound glycosphingolipids on the intestinal mucosa suggesting that the intestinal tract is a portal of entry into fish and a major site of colonization and proliferation [4]. Other studies suggested that the fish skin is the initial site of colonization for V. anguillarum and a main point of entry into the animal [5–7]. Recently, two studies directly investigated V. anguillarum infections in rainbow trout and characterized virulence factors required for colonization of the skin tissues. In the first study, a fluorescence-tagged V. anguillarum formed a biofilm-like structure on the fish skin and exopolysac- charide transport genes were shown to be required for formation of the biofilm [8]. A second study investigated the temporal and spatial spread of a V. anguillarum infection in the whole fish animal during an infection using in vivo bioluminescence imaging [9]. In this study, the skin and intestinal mucosal tissues were the first sites for bacterial colonization and proliferation; while, internal organs, such as the spleen and the kidney, were not colonized until the later stages of infection. The bacteria reached a higher cell density on the skin tissues than on the intestinal tissues and colonization of the skin tissues but not the intestinal tissues required siderophore production, the RNA chaperone Hfq, and exopolysaccharide transport genes. In addition, exopolysaccharide transport was shown to aid resistance of V. anguillarum to lysozyme and antimicrobial peptides, which are components of the fish skin immune defense [9]. Fish live in an aquatic environment that is rich in pathogens and the integrity of the skin epidermal mucosa is vital to form a mechanical barrier that protects fish from microorganisms in the environment [10,11]. Fish skin consists of two basic layers, the outer epidermis and the inner dermis and the entire surface of a fish outer body is covered by the epidermal mucosa. The outer epidermis is covered by a layer of mucus, which consists of PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37678