glycoproteins and is enriched with antimicrobial factors such as antibodies, complement, lysozyme, C-reactive protein, lectins, proteases, transferrin, and antimicrobial peptides [10,11]. Hence, antimicrobial activities are vital functions associated with the fish skin tissues for disease prevention. The epidermal outer layer of fish skin is composed predomi- nately of epithelial cells, which may also be known by other names such as keratocytes, Malpighian cells, or filamentous cells [10]. In this study, the term epithelial cell will be used. In teleost fish, the epidermal epithelial cells are metabolically active and migrate with speeds of 5–12 mm per minute [12,13]. Due to their fast motility, the migrating epithelial cells are thought to play a role in wound repair and protection against infectious disease [14,15]. Immedi- ately following skin injury, epithelial cells at the edge of the wound migrate as a network maintaining intercellular contacts and close the wound completely within a few hours to days. In addition to wound repair, Atlantic salmon (Salmo salar) skin epithelial cells are suggested to possess discriminatory phagocytic activities as they internalize Carnobacterium piscicola, Pseudomonas fluorescens, Aliivibrio wodanis, and Aeromonas salmonicida subsp. salmonicida but not Staphylococcus intermedius or Moritella viscosa [16–18]. Thus, skin epithelial cells are proposed to play a role in the immune defense of the skin tissues of teleost fish by removing debris and bacteria from the skin epidermis. Nothing is known about the mechanisms used by the fish skin epithelial cells to internalize bacteria selectively or how bacteria such as V. anguillarum that do colonize the skin tissues may evade this host immune response. This study aimed to identify bacterial components of V. anguillarum that are required to evade phagocytosis by the epithelial cells. Using live-cell and confocal 3D-imaging microscopy, single, migrating rainbow trout skin epithelial cells were shown to phagocytize bacteria. Moreover, V. anguillarum utilized lipopolysaccharide (LPS) O-antigen to evade the phagocytosis by the epithelial cells. Using in vivo biolumines- cence imaging, mutants defective in O-antigen polysaccharide transport failed to colonize rainbow trout skin even though they did colonize the intestinal tissues suggesting that the epithelial cells cleared the mutant strains from the skin tissues preventing colonization. The O-antigen transport mutants were also less resistant to antimicrobial components associated with the skin epidermal mucosa compared to the wild type. Thus, these newly identified roles for the O-antigen polysaccharide in V. anguillarum provide protection during the early stages of infection so that the bacteria may colonize and proliferate rapidly on the fish skin. In addition, rainbow trout skin epithelial cells were shown to phagocytize bacterial cells providing the epithelial cells with the capacity to play a major role in the immune defense of the fish. Results Vibrio anguillarum evades internalization by rainbow trout skin epithelial cells Epithelial cells are generally not believed to internalize microbial cells although they do have phagocytic activities to remove apoptotic bodies [19]. However, fish skin epithelial cells at the edge of a wound are proposed to phagocytize bacteria aiding wound repair by preventing infections due to opportunistic pathogens [17,18]. Vibrio anguillarum proficiently colonizes the skin epithelial layer of rainbow trout and forms a biofilm-like structure [8,9]. Colonization of the skin appears to be vital for V. anguillarum to cause disease. Consequently, V. anguillarum may encode mechanisms for evasion of phagocytosis by the fish skin epithelial cells. To test this, skin epithelial cells were isolated from rainbow trout and live-cell microscopy was used to visualize the motility of O-Antigen Prevents Phagocytosis by Fish Skin Cells individual epithelial cells and to determine if the epithelial cells could phagocytize V. anguillarum. The motile epithelial cells showed the classical single, canoe-shaped lamellipodium at the leading edge that extends around most of the cell body, which is located at the back of the cell (Figure 1) [20,21]. To test if the cells are phagocytic, latex beads (2.5 mm in diameter) were added to the epithelial cells. Live-cell microscopy showed that the epithelial cells internalized the latex beads, which were localized to the cell body resulting in cell rounding similar to what has previously been described (data not shown) [16]. To determine if V. anguillarum is internalized by the epithelial cells, bacteria (105 cells ml21) from a fresh sub-inoculation were added to the epithelial cell culture and live-cell microscopy was performed. In the resulting video (Video S1), the epithelial cells displaced the bacteria either from the surface so that they swam away or to the side so that they remained on the glass surface. The epithelial cells were also seen to migrate over a bacterial cell without disturbing it. Although a few percent of the wild-type bacteria attached to and moved along the lamellipodium to the cell body, the majority of the bacteria were not phagocytized by the epithelial cells. Figure 1 shows three frames from Video S1 at various time points showing a migrating epithelial cell as it contacts the wild-type V. anguillarum. Several bacteria are circled that were in contact with the lamellipodium but were not internalized as they either swam away or were pushed aside on the glass surface. These data indicate that V. anguillarum evades phagocytosis and likely encodes gene products utilized in an evasion mechanism. Identification of a DNA locus involved in the evasion of phagocytosis by rainbow trout skin epithelial cells To identify genes involved in evading phagocytosis, a previously described mini-Tn5phoAcm transposon mutant library [8] was screened using live-cell microscopy for a mutant unable to evade phagocytosis by the skin epithelial cells. Each mutant was used to infect an epithelial cell culture. One mutant, KM97, was internalized by a migrating epithelial cell and the data are shown in Video S2 and Figure 1. When the migrating epithelial cell came in contact with the KM97 mutant at the leading edge of the lamellipodium, increased membrane ruffling was observed and most bacteria adhered to the epithelial cell and were transported along the lamellipodium to the cell body and internalized. This is in strong contrast to what was observed in the presence of the wild type. In the KM97 mutant, the transposon was localized to the wzm gene, which is the first gene of a putative three gene operon containing wzm-wzt-wbhA (Figure 2A). The putative operon lies within an O-antigen polysaccharide biosynthesis gene cluster (unpublished genome sequence of the NB10 V. anguillarum strain, E. Hjerde, D.L. Milton, and N.P. Willassen) [22]. Figure 2B shows a cartoon in which the O-antigen polysaccharide is shown as one of the three main components of lipopolysaccharide found on the outer surface of Gram-negative bacteria. The wzm and wzt genes are found in numerous O-polysaccharide biosynthesis gene clusters [23]. In Escherichia coli, Wzm and Wzt make up an ATP- binding cassette (ABC) transporter that shares similarities to the ABC-2 subfamily of ABC transporters that are conserved in many Gram-negative bacteria and that are shown so far to transport only unbranched O-polysaccharides [23,24]. Wzm is an integral membrane protein that contains an average of six membrane- spanning domains. Wzt contains an ATP-binding motif or a Walker box and Wzt proteins associated with O-antigen biosyn- thesis have an extended C-terminal domain that determines substrate specificity [25]. In V. anguillarum, the Wzm protein is predicted to contain six transmembrane domains, which were PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37678