In this report, we describe the characteristics of the V. anguillarum phospholipase protein (Plp) encoded by plp, and its contribution to the hemolytic activity of V. anguillarum. Specifically, we show that Plp is a secreted phospholipase with A2 activity with specificity for phosphatidylcholine. The enzyme has a broad temperature optimum (37 – 64°C) and a broad pH optimum (pH 5.5 – 8.7). Phospholipases are broadly distributed among the Vibrionaceae and often contribute to the virulence of the pathogenic members of this family. For example, the TLH or LDH of V. parahaemolyticus[23–25] was the first well-studied lecithin-dependent PLA/lysophospholipase . A lecithinase (encoded by lec) was also identified in V. cholerae. Fiore et al. found that a lec mutant strain was unable to degrade lecithin and the culture supernatant exhibited decreased cytotoxicity. However, the mutant did not exhibit decreased fluid accumulation in a rabbit ileal loop assay, suggesting that fluid accumulation in animals is not affected by lecithinase activity. Additionally, the phospholipase A (PhlA) in V. mimicus was found to exhibit hemolytic activity against trout and tilapia erythrocytes and was cytotoxic to the fish cell line CHSE-214 . Recently, the V. harveyi hemolysin (VHH) was shown to be a virulence factor during flounder infection and also had phospholipase activity on egg yolk agar . Rock and Nelson  reported that the putative phospholipase gene (plp) from V. anguillarum exhibits 69% amino acid identity with the V. cholerae lec gene. Both plp and lec are located divergently adjacent to a hemolysin gene (vah1 and hlyA, respectively) [8, 27]. Additionally, Rock and Nelson  demonstrated that functional plp repressed transcription of its adjacent hemolysin gene, vah1, 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 according to the cleavage position on target phospholipids. Most of lipolytic enzymes contain a putative lipid catalytic motif (GDSL) that was previously demonstrated in other bacterial and eukaryotic phospholipases . However, Molgaard  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 . Multiple alignment analysis of 17 phospholipase homologues (Figure 1) demonstrates that V. anguillarum Plp belongs 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 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 . Two phospholipases identified from mesophilic Aeromonas sp. serogroup O:34 show phospholipase A1 and C activity . In addition, TLH of V. parahaemolyticus has PLA2 and lysophospholipase activity, and demonstrates a loss of activity at 55°C for 10 min . 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 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  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. Our data suggest that neither plp nor vah1 are major virulence factors during pathogenesis of salmonids. It was also reported that the deletion of lecithinase (Lec) activity in V. cholerae did not significantly diminish fluid accumulation in the rabbit ileal loop assay, indicating the lecithinase activity does not contribute significantly to enterotoxin activity . Lec is a homologue of Plp . In contrast, the direct IP injection of purified V. harveyi VHH protein caused the death of flounder with an LD50 of about 18.4 μg protein/fish . The rPhlA of V. mimicus also has a direct cytotoxic effect on the fish cell line CHSE-214  suggesting that this phospholipase is a virulence factor during fish infection. In addition, the lecithinase purified from A. hydrophila (serogroup O:34) has been shown to be an important virulence factor to rainbow trout and mouse . We note that infection experiments in both Atlantic salmon and rainbow trout demonstrate that mutation of plp does not attenuate virulence. We propose that V. anguillarum is able to compensate for the loss of Plp-mediated hemolytic activity in vivo by up-regulating the transcription of vah1, as previously described by Rock and Nelson . Additionally, transcription of rtxA is also increased in a plp mutant (Mou and Nelson, unpublished data).
Generally, the hemolytic activity of phospholipases is dependent upon the hydrolysis of the phospholipids that reside in the erythrocyte membrane. Erythrocytes contain various phospholipids including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM). PC makes up 58% of the total erythrocyte phospholipids in the Atlantic salmon , but only 34% and 1% in rabbit and sheep erythrocytes, respectively . Taken together with the high specificity of rPlp for PC (Figure 6), it was not surprising that rPlp was able to lyse the fish erythrocytes, but not sheep erythrocytes (Figure 7), and that the plp mutant had decreased hemolytic activity on LB20-fish blood agar (Figure 2). Our results are consistent were those reported for V. mimicus PhlA  and V. harveyi VHH , in which PhlA and VVH specifically lyse the fish erythrocytes.
We have previously reported that there are two hemolysin gene clusters in V. anguillarum M93Sm, the vah1-plp cluster and rtxACHBDE cluster  and have described their regulation by H-NS and HlyU [17, 37]. Mutation of both vah1 and rtxA results in the loss of all hemolytic activity on TSA-sheep blood agar , which is consistent with the data reported here that Plp has no activity on sheep erythrocytes. We have also previously demonstrated that Plp is a putative repressor of Vah1, since mutation of plp increases vah1 expression by 2–3 fold . In this report, we examined the hemolytic activity of various hemolysin mutants using freshly collected Rainbow trout blood (Table 2) to investigate the relationships among three hemolysins of V. anguillarum. While culture supernatants from two of the three single mutants (JR1 and S123) and one of three double mutants (S183) exhibited ≥94% of the hemolytic activity as supernatants from the wild type strain M93Sm (Table 2), the hemolytic activity of one single mutant (S262, plp) and two double mutants (JR03, plp vah1 and S187, plp rtxA) were reduced to 28%, 14%, and 12% of that in M93Sm, respectively. Our data indicate that only the loss of the plp gene has a significant effect on hemolysis of fish erythrocytes by V. anguillarum culture supernatant, while the loss of rtxA and/or vah1 has little effect. Further, supernatant from the hemolysin triple mutant XM90 (vah1 rtxA plp) exhibits no hemolytic activity on fish blood compared to M93Sm (Table 2), indicating that Vah1, RtxA, and Plp are responsible for all secreted hemolytic activity by V. anguillarum. Finally, complementation of any plp mutant with plp (in trans) restores hemolytic activity to V. anguillarum culture supernatant (Table 2).