fixation, the epithelial cells were washed 3 times in PBS, permeabilized with 1 ml of PBS containing 0.5% Triton X-100 (Sigma) for 3 minutes, and washed twice in PBS. The cells were then treated with PBS containing 1% bovine serum albumin to block non-specific binding sites prior to staining the cells. To visualize internalized bacteria, a V. anguillarum whole-cell antise- rum was added to the permeabilized epithelial cells and incubated for 30 min at room temperature. After washing twice in PBS, the cells were incubated with a FITC-conjugated Donkey Anti-Rabbit IgG antiserum (Jackson ImmunoResearch Laboratories) for 30 minutes and washed twice in PBS. To visualize the epithelial cells, actin was stained using Alexa Fluor 568 phalloidin (Invitrogen) according to the manufacturer’s instructions. The coverslip with the attached epithelial cells was mounted onto a glass slide using ProLongH Gold antifade mounting reagent (Invitrogen). Epithelial cells were imaged using a Nikon D-Eclipse C1 fluorescence confocal microscope equipped with a Plan Apo VC 606/1.40 oil objective. Epithelial cells were detected using lasers with an excitation/emission of 578/600 nm and bacteria were detected using lasers with an excitation/emission of 515/ 530 nm. A 3D rendering of a confocal z-stack covering the entire cell thickness using two colors and image cropping was done with the imaging software NIS-Elements EZ-C1 Ver. 3.90. All images were processed identically in Adobe Photoshop CS2. All confocal imaging analyses were repeated in at least three-independent experiments and the images presented are representative of these experiments. To quantify the number of epithelial cells with internalized bacteria, a minimum of 100 single epithelial cells from three separate experiments were analyzed by doing a 3D image stack of each epithelial cell and cropping this image as described above. Rainbow trout infections Rainbow trout (Oncorhynchus mykiss) with an approximate weight of 20 g were infected with V. anguillarum strains carrying pNQFlaC4-lac::lux at 18uC by bathing in seawater containing 104 bacteria ml21 as described previously [51]. After 30 min, the infected seawater was removed and replaced with fresh seawater and the fish were held at 18uC. At 24 h and 48 h, the fish were euthanized by an overdose of anesthetic before visualization in the in vivo imaging system. Two independent infections were done using 10 fish for each infection. In-vivo bioluminescent imaging of V. anguillarum during an infection of rainbow trout To visualize V. anguillarum during an infection of rainbow trout using in vivo bioluminescent imaging, the bacterial strains used were engineered to produce light as describe previously [9]. The R6K-origin-based suicide plasmid pNQFlaC4-lac::lux that carries the Photorhabdus luminescens luxCDABE operon, which is constitu- tively expressed in V. anguillarum, was recombined into an intergenic region of the chromosome of each strain. Photon emission was correlated with bacterial cell numbers by growing each strain containing pNQFlaC4-lac::lux to an OD600 of 0.7 (56108 cells ml21) in TSB-1%, serially diluting the cells in 100 ml of the same medium in a black 96-well plate, and taking an image of the 96-well plate using an IVISH SPECTRUM system (Xenogen, Caliper Life Sciences). Luminescent signals were collected for 10 sec, images were processed with a binning of 4, and other photographic parameters were identical for each dilution series. Photon units (photons sec21 cm22 steradian21) emitting from each well was determined using the Living ImageH 3.0 software from Xenogen. Cfu counts were determined for each culture. O-Antigen Prevents Phagocytosis by Fish Skin Cells Rainbow trout were infected with V. anguillarum strains carrying pNQFlaC4-lac::lux as described above. At 24 h and 48 h, images of the fish were obtained as described above except that luminescent signals were collected for 5 min. The skin was imaged and then one side of the fish was removed to image the internal organs as the skin pigment blocked the detection of photon emission from internal organs. To enhance detection of bacteria in the intestines, the faecal contents were pushed out the anus. Sensitivity to lysozyme and polymyxin B Overnight cultures of V. anguillarum grown in TSB-1% at 24uC with aeration were diluted in the same medium and incubated to an OD600 of 0.2 (108 cells ml21). The bacterial cells were diluted 10- fold in PBS containing either 10 mg ml21 of hen egg white lysozyme (Fluka) or 10 ng ml21 of freshly prepared polymyxin B sulfate (Sigma) and incubated at room temperature. Bacterial survival was measured by determining the cfu in each sample at 0 min and 20 min for lysozyme and at 0 min and 60 min for polymyxin B. The percent survival given is the average of three experiments. Supporting Information Video S1 Live-cell microscopy video demonstrating that the wild-type V. anguillarum evades phagocytosis by rainbow trout skin epithelial cells. Skin epithelial cells were isolated from rainbow trout and infected with 105 bacteria ml21. Following infection, phagocytic activities of motile epithelial cells were tracked using live-cell microscopy. (MP4) Video S2 Live-cell microscopy video demonstrating that the V. anguillarum transposon mutant KM97 is phago- cytized by rainbow trout skin epithelial cells. Skin epithelial cells were isolated from rainbow trout and infected with 105 bacteria ml21. Following infection, phagocytic activities of motile epithelial cells were tracked using live-cell microscopy. The transposon was localized to the wzm gene, which is the first gene of a three-gene operon involved in LPS O-antigen transport. (MP4) Video S3 Live-cell microscopy video demonstrating that the V. anguillarum Dwzt mutant, which is defective for O-antigen transport, is phagocytized by rainbow trout skin epithelial cells. Skin epithelial cells were isolated from rainbow trout and infected with 105 bacteria ml21. Following infection, phagocytic activities of motile epithelial cells were tracked using live-cell microscopy. (MP4) Video S4 Live-cell microscopy video demonstrating that the V. anguillarum Dwzm mutant, which is defective for O-antigen transport, is phagocytized by rainbow trout skin epithelial cells. Skin epithelial cells were isolated from rainbow trout and infected with 105 bacteria ml21. Following infection, phagocytic activities of motile epithelial cells were tracked using live-cell microscopy. (MP4) Video S5 Live-cell microscopy video demonstrating that the V. anguillarum DwbhA mutant, which is defective for O-antigen transport, is phagocytized by rainbow trout skin epithelial cells. Skin epithelial cells were isolated from rainbow trout and infected with 105 bacteria ml21. Following infection, phagocytic activities of motile epithelial cells were tracked using live-cell microscopy. (MP4) PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37678