O-Antigen Prevents Phagocytosis by Fish Skin Cells Figure 2. Genetic organization of the O-antigen biosynthesis gene cluster containing wzm-wzt-wbhA. (A) The genetic locus was identified from the draft genome sequence of the NB10 V. anguillarum strain (unpublished data, E. Hjerde, D.L. Milton, and N.P. Willassen). Each open reading frame (Orf) is indicated by a box and the direction of transcription is indicated by an arrowhead within each box. Grey boxes indicate the genes under investigation in this study, which are suggested to be involved in the transport of O-antigen polysaccharide. wbhA encodes a putative S- adenosylmethionine methyltransferase and wzt and wzm encode components of an ABC-2 subfamily of polysaccharide transporters. The four rml genes have homology to proteins involved in the biosynthesis of dTDP-L-rhamnose, a possible O-antigen precursor [42]. rmlC encodes a dTDP-6- deoxy-D-glucose-3,5-epimerase; rmlD encodes dTDP-6-deoxy-L-mannose dehydrogenase; rmlA encodes glucose-1-phosphate thymidylyltransferase; rmlB encodes dTDP-glucose 4,6-dehydratase. OrfA and OrfB are both part of two putative operons that encode proteins likely involved LPS biosynthesis. Orfs downstream of wbhA encode two putative transposases and a hypothetical protein. (B) A cartoon image of the structure of Gram- negative lipopolysaccharide. The lipopolysaccharide is a component of the outer membrane in Gram-negative bacteria and is composed of lipid A, core polysaccharide, and the O-antigen, which is a repeating sugar unit. OM, outer cell membrane; P, peptidoglycan; IM, inner membrane. doi:10.1371/journal.pone.0037678.g002 antigen polysaccharides. As group 2 capsular polysaccharides in E. coli are also exported by an ABC transporter similar to Wzm and Wzt [24], each mutation was also tested for the loss of exopolysaccharide. Figure 3B shows that the exopolysaccharide levels were similar to that of the wild type. Thus, Wzm, Wzt, and WbhA are essential for O-antigen polysaccharide biosynthesis but not for production of exopolysaccharides. O-antigen polysaccharides are required for V. anguillarum to evade phagocytosis by the rainbow trout skin epithelial cells The data so far suggest that the O-antigen polysaccharides are required for V. anguillarum to evade phagocytosis by the migrating epithelial cells. To confirm this notion, the Dwzm, Dwzt, and DwbhA mutants were tested for their ability to evade phagocytosis by the epithelial cells. Skin epithelial cell cultures were infected with each mutant (105 bacteria ml21) and migrating epithelial cells were visualized by live-cell microscopy, Video S3, S4, and S5). As for the KM97 transposon mutant, all three (Dwzm, Dwzt, and DwbhA) mutants were phagocytized by the epithelial cells. When bacteria and epithelial cell came into contact at the leading edge of the lamellipodium, the bacteria attached to and migrated along the lamellipodium to the cell body where they were internalized. In addition, an increase in membrane ruffling of the lamellipo- dium occurred in the presence of the mutant strains as compared to contact with the wild type. Figure 1 shows frames at various time points from the video of the Dwzt mutant. Since all three mutants gave similar results with the live-cell microscopy, only one set of video frames is shown as a representative for all mutants. Complementation of each mutation with the respective wild-type gene re-gained the wild-type phenotype of evading phagocytosis by the epithelial cells. Figure 1 and Video S6 (supporting information) are presented as representative images for all three mutants. To confirm that the bacteria were internalized, confocal 3D- imaging microscopy was used to measure internalization and to localize the bacteria inside the cell body of the epithelial cells. Figure 4 gives a three dimensional image of a motile epithelial cell following infection by the wild type and the Dwzm, Dwzt, and DwbhA mutants. In this image, the epithelial cells are fluorescently labeled using phallodin (red), which binds to the actin filaments, and the bacteria are labeled with a FITC-labeled secondary antibody (green), which binds antibodies bound to the cell body of V. anguillarum. To visualize the bacteria in the cell body, a 3D rendering of a confocal microscopy z-stack covering an entire cell was done followed by progressively cropping the phalloidin signal along the Z-axis of the epithelial cell. As seen in Figure 4, the green signal or bacteria are clearly located within the cell body of the epithelial cells that were infected with the Dwzm, Dwzt, and DwbhA mutants but not in the cell infected with the wild-type strain. To quantify the frequency of internalization of the wild type versus the three mutants, confocal 3D-imaging microscopy was used to determine the percentage of epithelial cells containing internalized bacteria (Figure 5). Following infection with the wild type, very few epithelial cells contained bacteria; whereas, the majority of epithelial cells contained bacteria when infected with any one of the mutants. Complementation of each mutation with PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37678