anguillarum However, the enzymatic characteristics of Plp in V

anguillarum. However, the enzymatic characteristics of Plp in V. anguillarum were not described. Usually, phospholipases are divided into phospholipases A (A1 and A2), C, and D CP 690550 according to the cleavage position on target phospholipids. Most of lipolytic enzymes AZD0156 mouse contain a putative lipid catalytic motif (GDSL) that was previously demonstrated in other bacterial and eukaryotic phospholipases [30]. However, Molgaard [16] demonstrated that four amino acid residues (SGNH) form a catalytic site, and are conserved in all members of the phospholipase family; therefore, phospholipases were re-named as the SGNH

subgroup of the GDSL family [30]. Multiple alignment analysis of 17 phospholipase homologues (Figure 1) demonstrates that V. anguillarum Plp belongs LY2835219 datasheet to the SGNH hydrolase subgroup, since the GSDL motif was not fully conserved in these proteins (Figure 1). Recently, it was reported that mutation of the serine residue in the SGNH motif resulted in the complete loss of the phospholipase and hemolytic

activities of VHH in V. harveyi[31] demonstrating the importance of this motif on the activity of phospholipase. In contrast to the similarities of their catalytic motifs, the biochemical characteristics of bacterial phospholipases appear to be variable. For example, V. mimicus PhlA has a phospholipase A activity, which cleaves the fatty acid at either sn-1or sn-2 position, but no lysophospholipase activity [28]. Two phospholipases identified from mesophilic Aeromonas sp.

serogroup O:34 show phospholipase A1 and C activity [32]. In addition, TLH of V. parahaemolyticus has PLA2 and lysophospholipase activity, and demonstrates a loss of activity at 55°C for 10 min [23]. In this report, we show that V. anguillarum Plp has PLA2 activity, and is able to maintain activity at 64°C for 1 h (Figures 6 and 7). Therefore, the enzymatic characteristics of specific phospholipases are distinct even when they all belong about to the SGNH hydrolase family (Figure 1). Phospholipases have been implicated in the pathogenic activities of a number of bacteria [33, 34]. It is known that phospholipase activities often lead to cell destruction by degrading the phospholipids of cell membranes [33, 35]. However, the relationships between phospholipases and virulence are not always clear. While the purified rPlp exhibits strong hemolytic activity against Atlantic salmon erythrocytes (Figure 7), Rock and Nelson [8] showed that a knock-out mutation of either the plp gene or the vah1 gene in V. anguillarum did not affect virulence of V. anguillarum during an infection study on juvenile Atlantic salmon. In this report, we show that when groups of rainbow trout are infected with either a plp mutant or a plp vah1 double mutant there is no significant difference in mortalities compared to fish infected with the wild type strain.

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