O-Antigen Prevents Phagocytosis by Fish Skin Cells Figure 1. Image frames from various time points of live-cell microscopy videos of rainbow trout skin epithelial cells following infection with various V. anguillarum strains. Using live-cell microscopy, a video clip (Videos S1, S2, S3, S6, and S7 supporting information) was recorded of the phagocytic activity of rainbow trout skin epithelial cells after infection with the wild type, the KM97 transposon mutant, the Dwzt mutant, and the Dwzt mutant complemented with the wild-type wzt gene. Three frames from each video progressing in time from left to right are shown indicating the phagocytic activity of an epithelial cell after contact with each strain. Similar videos were made for the Dwzm and DwbhA mutants, and similar results to that of the Dwzt mutant were seen (supporting information Videos S4 and S5). Thus, the Dwzt mutant results are given as a representative for all three mutant strains. In the first frame from each video, three bacterial cells were circled and labeled. These three bacteria were followed in the two additional time frames to indicate movement of the bacteria during the movie. If the cell is no longer labeled in frames 2 and 3, then the bacterial cell was displaced from the glass surface and swam away. In the bottom set of frames, the epithelial cells were pretreated with 1 mM mannose before infection with the Dwzt mutant. The three-image sets are designated by the strain mutation to the left and mutation designations followed by a C are complemented. doi:10.1371/journal.pone.0037678.g001 determined by the TMHMM Server v. 2.0 at the Center for Biological Sequence Analysis at the Technical University of Denmark. Using the Blast search program at NCBI, the Wzt protein of V. anguillarum was shown to contain an extended C- terminus while the N-terminus contains an ATP-binding site, an ABC transporter signature motif, and a Walker box. The wbhA gene [26] encodes a hypothetical protein that contains an S- adenosylmethionine-dependent methyltransferase motif that was determined by a Blast search at NCBI. In E. coli serotype O8 and O9a, a wbdD gene, which encodes a methyltransferase, is found downstream of wzm and wzt homologues within a locus encoding O-antigen polysaccharide assembly systems [24]. The WbdD methyltransferase is involved in chain length determination of the lipopolysaccharide O-antigen, which is coupled to export of the O- antigen polysaccharide using the Wzm-Wzt ABC transporter dependent pathway [23,27]. Consequently, WbhA, together with Wzm and Wzt, may be predicted to function in O-antigen polysaccharide biosynthesis. The Wzm-Wzt ABC transporters and the methyltransferase WbhA are essential for biosynthesis of O-antigen polysaccharide To determine if Wzm, Wzt, and WbhA play a role in O-antigen polysaccharide biosynthesis, in-frame mutations were made that deleted the genes encoding these proteins and each mutant was tested for LPS production. All three (Dwzm, Dwzt, and DwbhA) mutants showed a loss of the O-antigen polysaccharides (Figure 3A). Complementation of each mutation with the respective wild-type gene regained the wild-type level of O- PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37678