Figure 3. Lipopolysaccharide and exopolysaccharide analyses. (A) Lipopolysaccharides were fractionated by 15%-SDS-PAGE and detected using the Pro-QH Emerald 300 Lipopolysaccharide Gel Stain Kit (Invitrogen). (B) Exopolysaccharides were extracted from culture supernatants and quantified using an Alcian blue technique described by Plante [56]. The amount of EPS is given as an OD620 reading. For both (A) and (B), strains are designated by their mutation. Mutation designations followed by a C are complemented with the respective wild-type gene. doi:10.1371/journal.pone.0037678.g003 the corresponding wild-type gene lead to a very low number of epithelial cells containing bacteria as was seen for the wild type. Taken together, these data clearly demonstrate that skin epithelial cells selectively phagocytize bacteria and that V. anguillarum uses its O-antigen polysaccharide to evade phagocytosis. Mannose abolishes phagocytosis of the O-antigen mutants by the rainbow trout skin epithelial cells Phagocytosis usually involves pattern-recognition receptors, which mediate internalization of foreign particles by binding to O-Antigen Prevents Phagocytosis by Fish Skin Cells molecules found on the surface of the particle [19]. To confirm further that the epithelial cells do internalize the V. anguillarum mutants via a receptor-mediated mechanism, we wondered if phagocytosis could be abolished by pre-treating the cells with a ligand before infection. C-type lectins are eukaryotic carbohy- drate-binding proteins that mediate the recognition of bacterial pathogens [28]. Recent studies show that fish skin tissues contain mannose-binding lectins [29–32]. In addition, mannose is known to inhibit agglutination activity of V. anguillarum [33]. Thus, we wondered if skin epithelial cells utilize a mannose-binding-type lectin for internalization of the Dwzm, Dwzt, and DwbhA mutants. To reveal this, the epithelial cell culture was pretreated with 1 mM mannose before infection with the three mutant strains and the infections were followed by live-cell microscopy (Video S7). Pretreatment of the epithelial cells with mannose decreased membrane ruffling and phagocytosis of all three mutants in comparison to similar infections that used untreated epithelial cells. A video clip showing the Dwzt mutant as a representative for all three mutants is presented as supporting information and frames from Video S7 are presented in Figure 1. Quantification of phagocytosis after mannose treatment using confocal 3D-imaging microscopy further showed that mannose pretreatment of the epithelial cells prevented phagocytosis (Figure 5). These data suggest that a receptor-mediated mechanism is utilized by the skin epithelial cells to internalize the V. anguillarum mutants. O-antigen polysaccharides are essential for V. anguillarum to colonize rainbow trout skin Previously, V. anguillarum was shown to proliferate rapidly and abundantly on rainbow trout skin forming micro-colonies that resemble biofilms [8,9]. We wondered if the O-antigen polysac- charides play a critical role in preventing the removal of V. anguillarum from the fish skin by the epithelial cells. To test this, in vivo bioluminescent imaging was used to follow the spatial and temporal progression of a V. anguillarum infection in the whole rainbow trout animal. As this method detects the emission of light from host cells or tissues with a minimal background signal [34], visualization of V. anguillarum in the fish requires that the bacteria emit light. Thus, the wild type and the Dwzm, Dwzt, and DwbhA mutants were engineered to produce light constitutively by recombining a lac::luxCDABE promoter-operon fusion into an intergenic region of the chromosome as was done in a previous study [9]. Since the production of light is energetically costly for the bacterium and may affect the virulence of the bacterium [35], growth curves of the non-luminescent and luminescent strains were compared for growth differences and no significant differences were seen (data not shown). In addition, photon emission cfu21 was estimated for each strain indicating an approximate equal light emission from all strains (Figure 6). The V. anguillarum strains carrying the lux operon were used to infect rainbow trout (20 g weight) by immersion into seawater containing 104 bioluminescent bacteria ml21. At 24 h and 48 h post infection, fish were analyzed for bioluminescent bacteria. Analyses after the 48 h time point were not done since virulent V. anguillarum strains cause a septicemia making it difficult to determine tissue colonization as the whole fish emits light [9]. In addition, the skin pigment of the rainbow trout blocked detection of light emission from the internal organs. Thus, one side of the fish was removed to image the internal organs after imaging both sides of the whole animal. The light that penetrated from the host tissue was captured as photons, which were used to determine a relative quantification of bacteria during the infection process. Figure 7 shows examples of the in vivo bioluminescent imaging of the external surfaces and the internal organs at 24 h and 48 h post PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e37678