O-Antigen Prevents Phagocytosis by Fish Skin Cells Figure 5. Quantification of phagocytic activity by rainbow trout skin epithelial cells following infection with various V. anguillarum strains. Epithelial cells were isolated from the skin of rainbow trout and either left untreated (closed bars) or pretreated with 1 mM mannose (open bars) before infection with 105 bacteria ml21. Following a 3 h infection, the epithelial cells were fixed and the epithelial cells and bacteria were fluorescently labeled. Confocal 3D- imaging microscopy was done on at least 100 single epithelial cells from three separate experiments to determine the number of cells that contained bacteria. These data are presented as a percent. The V. anguillarum strains that were tested are designated by their mutation. Mutation designations followed by a C are complemented with the respective wild-type gene. Asterisks indicate a p-value of ,0.05 as determined by the Student’s t-test. doi:10.1371/journal.pone.0037678.g005 from Atlantic Salmon (Salmo salar) have previously been observed to internalize bacteria during wound healing and the uptake mechanism appears to be discriminatory as the epithelial cells do not phagocytize all bacteria [16–18]. Even though epithelial cells have the capacity for phagocytosis as they ingest apoptotic bodies during cellular turnover, they have been described as being incapable of internalizing microorganisms [19]. Using bacterial genetics together with live-cell and confocal 3D-imaging micros- copy, our studies unequivocally show that single, migrating skin epithelial cells from rainbow trout (Oncorhynchus mykiss) efficiently phagocytize bacteria in a selective manner most likely by utilizing a phagocytic pattern-recognition receptor that binds mannose. Phagocytosis is the cellular uptake of large particles, such as microorganisms, via a receptor-mediated process. To counteract this host defense, bacterial pathogens have evolved numerous strategies to evade phagocytosis and to resist phagocyte killing [39]. In the present study, O-antigen polysaccharides aided V. anguillarum in the evasion of phagocytosis by rainbow trout epithelial cells. This is a new role for LPS in the virulence of V. anguillarum. The O-antigen polysaccharide of V. anguillarum was previously shown to be essential for virulence in fish animal models, for resistance to complement-mediated killing found in fish blood serum, and for anguibactin-mediated iron transport [40–43]. Our study suggests that the O-antigen masks putative mannose structures on the surface of the bacteria that are recognized by receptors on the surface of the skin epithelial cells. One possible receptor type is the C-type mannose-specific lectin [28]. Mannose-binding lectins may also bind glucose, L-fucose, N- acetyl-mannosamine and N-acetyl-glucosamine. All of which are Figure 6. Correlation of photon emission with the number of V. anguillarum cells. Vibrio anguillarum strains carrying pNQFlaC4-lac::lux on the chromosome were grown to 56108 cells ml21 and serially diluted in a black 96-well plate. Cfu counts were determined for each culture. A bioluminescent image of the 96-well plate was taken using an IVISH SPECTRUM system (Xenogen, Caliper Life Sciences) and photon units (photons sec21 cm22 steradian21) emitting from each well were determined. Strains are designated by their mutation and mutation designations followed by a C are complemented. doi:10.1371/journal.pone.0037678.g006 sugar residues found on bacterial cell surfaces. Fish skin mucus from various species of fish is described to contain a diverse array of lectins, including C-type lectins [44]. Recent studies show that fish skin tissues, including that of rainbow trout (Oncorhynchus mykiss), express mannose-binding lectins [29–31]. In one study, a mannose-specific lectin is shown to be associated with fish skin epithelial cells [32]. An antiphagocytic role has been described for the O-antigen associated with other pathogens, E. coli, Salmonella enterica serovar Typhimurium, Burkholderia cenocepacia, Neisseria gonorrhoeae, and Haemophilus ducreyi [45–49]. In E. coli, the O-antigen is suggested to mask the core region of the LPS, which binds the unique C-type lectin DC-SIGN (dendritic cell specific intercellular adhesion molecule nonintegrin) associated with phagocytic dendritic cells [28,45]. A genome analysis of C-type lectin domains in the puffer fish Fugu rubripes identified all but two groups of C-type lectin domains identified in mammals and the DC-SIGN subgroup is overrepresented [50]. Although an analysis for DC-SIGN receptors has not been done using rainbow trout, the puffer fish studies may suggest that the rainbow trout skin epithelial cells express a DC-SIGN-like receptor that is involved in the phagocytosis of the V. anguillarum O-antigen transport mutants. Vibrio anguillarum colonizes quickly and efficiently the skin tissues of rainbow trout during the early stages of infection [8,9]. The fish skin mucosal layer is an important source of nutrients enabling the bacterium to survive in an otherwise nutrient poor environment. Colonization of the skin tissues greatly aids disease development to a late stage septicemia. Our study is one of the first to provide direct insight into a mechanism that many bacteria may use to colonize the fish skin epidermal mucosa. The highly motile rainbow trout skin epithelial cells play a vital role in the immune defense associated with the skin tissues by phagocytizing bacteria and keeping the skin clear of potential pathogens. To colonize fish skin tissues, other bacteria, like V. anguillarum, may utilize LPS O- antigen to mask surface-located molecular patterns that are recognized by receptors on the phagocytic skin cells. Understand- PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e37678