O-Antigen Prevents Phagocytosis by Fish Skin Cells Figure 4. Confocal microscopy 3D-imaging of rainbow trout skin epithelial cells following infections. Skin epithelial cells were infected with the wild type and the Dwzt, Dwzm, and DwbhA mutants. Following fixation, the epithelial cells were visualized by labeling actin with Alexa Fluor 568 phalloidin (red). After permeabilization of the epithelial cell, bacteria were labeled using a whole-cell antiserum and a FITC-conjugated Donkey anti-rabbit IgG (green). Using the NIS-elements AR 3.2 software, a 3D rendering of a confocal microscopy z-stack covering an entire cell was done and is presented as the left most image for each strain. In each image, the phalloidin (red) signal was progressively cropped along the z-axis to reveal internal bacteria. Strains are designated by their mutations and the designations are given to the left of the image. doi:10.1371/journal.pone.0037678.g004 infection for each strain. Table 1 summarizes the frequency of bacterial colonization of the skin, intestines, and spleen of all fish used in the infections at both 24 h and 48 h post infection. Uninfected fish that were treated similarly did not emit light. The wild type colonized the fish skin at random sites on 95% and 100% of the fish at 24 h and 48 h post infection, respectively. However, the Dwzm, Dwzt, and DwbhA mutants were never detected on the fish skin. Complementation of the mutation with the correspond- ing wild type gene regained the wild-type phenotype. The internal organs were also analyzed. To enhance detection of bacteria in the intestines, the faecal content was pushed out towards the anus. The wild type was detected in 85% and 95% of the intestines at 24 h and 48 h post infection, respectively. However, the mutant strains were slower than the wild type to colonize the intestines in the first 24 h (10–35%) but by 48 h, the mutant strains colonized the intestines (80–95%) as well as the wild type. The delay in colonization of the intestines by the mutant strains at 24 h could be complemented with the respective wild-type gene. Bacteria were only detected in the spleen of one fish infected with the wild type at the 48 h time point. These data indicate that the O-antigen polysaccharide is required for colonization of the fish skin and one role for the O-antigen polysaccharide may be to protect V. anguillarum from the phagocytic activities of the skin epithelial cells during an infection. O-antigen polysaccharides protect against antimicrobial peptide and lysozyme activities Studies suggest that the LPS O-antigen polysaccharides provide protection against antimicrobial factors, such as antimicrobial peptides [36]. The mucus layer covering the fish skin contains antimicrobial factors such as lysozyme and antimicrobial peptides that play a role in the innate immune defense of the skin [37]. Thus, the Dwzm, Dwzt, and DwbhA mutants were tested for a decreased resistance to lysozyme and polymyxin B compared to the wild type (Figure 8). Lysozyme hydrolyses the glycosidic bonds found in peptidoglycan; whereas, polymyxin B, an effective, commonly used antimicrobial peptide leads to membrane permeabilization. For lysozyme (10 mg ml21), 78% of the wild type survived a 20 min exposure; whereas, only 40% of all three mutants survived the same treatment. For polymyxin B (10 ng ml21), a 57% survival was seen for the wild type after a 1 h exposure; while, the three mutants showed only 25% survival after the same treatment. Complementation of each mutation with the corresponding wild-type gene resulted in wild-type survival rates. Hence, O-antigen polysaccharides also protect the bacterial cells from antimicrobial agents found in fish skin mucus. Discussion Most reports on bacterial attachment to mucosal tissues of fish are indirect, descriptive in vitro studies that have been done using cell lines [3]. However, two recent in vivo studies give direct evidence that V. anguillarum forms biofilm-like microcolonies within the skin mucosal tissues of rainbow trout where it proliferates rapidly during the early stages of infection [8,9]. During growth on the skin tissues, V. anguillarum is believed to produce extracellular products such as proteases, which may damage skin tissues [38], providing fast access to the blood leading to septicemia and death of the host animal. This study aimed to further our understanding of the immune defenses of the rainbow trout skin tissues and the mechanisms used by V. anguillarum to evade these host defenses enabling it to colonize the fish skin tissues. Fish skin is a formidable barrier for bacterial pathogens to penetrate partly due to the numerous immune defenses associated with the skin tissues [10,37]. The highly motile skin epithelial cells PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37678