BACKGROUND
vibrio anguillarum, a highly motile marine member of the γ-proteobacteria, is one of the causative agents of vibriosis, a fatal hemorrhagic septicemic disease of both wild and cultured fish, crustaceans, and bivalves  <cit> .
fish infected with v. anguillarum display skin discoloration and erythema around the mouth, fins, and vent.
necrotic lesions are also observed in the abdominal muscle  <cit> .
mortality rates in infected fish populations range as high as 30-100%  <cit> .
vibriosis has caused severe economic losses to aquaculture worldwide  <cit>  and affects many farm-raised fish including pacific salmon, atlantic salmon, sea bass, cod, and eel  <cit> .
v. anguillarum enters its fish host through the gastrointestinal tract  and quickly colonizes this nutrient rich environment  <cit> .
garcia et al.
<cit>  have shown that v. anguillarum grows extremely well in salmon intestinal mucus and that mucus-grown cells specifically express a number of different proteins, including several outer membrane proteins  <cit>  and the extracellular metalloprotease empa  <cit> .
several genes have been reported to be correlated with virulence by v. anguillarum, including the vah <dig> hemolysin cluster  <cit> , the rtx hemolysin cluster  <cit> , the siderophore mediated iron transport system  <cit> , the empa metalloprotease  <cit> , and the flaa gene  <cit> .
hemolytic activity of v. anguillarum has been considered to be the virulence factor responsible for hemorrhagic septicemia during infection  <cit> .
we have identified two hemolysin gene clusters in v. anguillarum that contribute to the virulence of this pathogen  <cit> .
one gene cluster, rtxachbde, encodes a martx toxin and its type i secretion system  <cit> .
the second hemolysin gene cluster in v. anguillarum strain m93sm contains the hemolysin gene vah <dig> flanked by two putative lipase-related genes  immediately downstream and upstream by a divergently transcribed hemolysin-like gene  that appears to function as a repressor of vah1-dependent hemolytic activity  <cit> .
the plp-encoded protein has very high sequence similarity to phospholipases found in other pathogenic vibrio species  <cit> .
however, the enzymatic characteristics of plp in v. anguillarum were not described.
generally, phospholipases are divided into several subgroups depending on their specificity for hydrolysis of ester bonds at different locations in the phospholipid molecule.
phospholipases a  cleave long chain fatty acids at sn- <dig>  or sn- <dig>  position from phospholipid to yield lysophospholipid and free fatty acid; phospholipases c  cleave phospholipid into diacylglycerol and a phosphate-containing head group; and phospholipases d  cleave phospholipid into phosphatidic acid and an alcohol.
it is known that some phospholipid products are used as secondary messages, which play a central role in signal transduction  <cit> .
in this study, we determined that plp encodes a phospholipase a <dig> in v. anguillarum, and then purified recombinant plp protein  from e. coli to investigate its biochemical properties.
we also described the contribution and specificity of rplp for hydrolysis of phospholipids, and its ability to lyse fish erythrocytes.
RESULTS
identification of a putative phospholipase gene plp
previously, a putative phospholipase gene, plp, was identified in the vah <dig> hemolysin cluster of v. anguillarum strain m93sm  <cit> .
the 1251-bp plp gene  was predicted to encode a protein of  <dig> amino acids.
a blastx  <cit>  search revealed that the deduced plp amino acid sequence exhibited homology with many lipolytic enzymes including the phospholipase/lecithinase/hemolysin of vibrio vulnificus ; the lecithin-dependent hemolysin / thermolabile hemolysin  of vibrio parahaemolyticus ; the lipolytic enzyme/hemolysin vhh of vibrio harveyi ; and the thermolabile hemolysin of vibrio cholerae .
the phylogenetic tree created by the clustal-w program from  <dig> plp homologous proteins revealed that while the most closely related plp proteins were all from pathogenic members of the genus vibrio, the plp of v. anguillarum was an outlier among the vibrio species, as demonstrated by the neighbor joining analysis .
according to flieger’s classification  <cit> , the alignment of plp with other homologous proteins indicated that plp could be classified into subgroup b of this lipolytic family with its long n-terminal tail  prior to the block i  <cit> .
additionally, close examination of the amino acid sequences of these enzymes revealed that the typical gdsl motif for lypolytic enzymes is not totally conserved in all of these  <dig> proteins, in which leucines  are replaced with isoleucines  in photobacterium, marinomonas, and shewanella .
in this case, v. anguillarum plp should be considered as a member of the sgnh hydrolase family, based on the molgaard’s suggestion that only four amino acids  are completely conserved among the gdsl proteins  <cit> .
plp affects hemolysis of fish erythrocytes
the hemolysin gene vah <dig> is divergently transcribed from plp <cit> .
mutation of plp increased hemolytic activity by 2-3-fold on trypticase soy agar plus 5% sheep blood  plate compared with wild type strain    <cit> .
rock and nelson  <cit>  also demonstrated that the plp mutant had increased vah <dig> transcription , indicating that plp is a putative repressor of vah <dig>  previously, we demonstrated that a double mutant in vah <dig> and rtxa resulted in a hemolysis negative mutant when plated on tsa-sheep blood agar  <cit> .
similar results were observed when using luria-bertani broth plus 2% nacl plus 5% sheep blood  agar .
however, on lb <dig> plus 5% rainbow trout blood  agar, the plp mutant exhibited a smaller zone of hemolysis compared to wild type strain m93sm  ; complementation of plp restored the hemolytic activity of the mutant strain .
similar results were observed when using lb <dig> plus 5% atlantic salmon blood agar , suggesting that the ability of plp to lyse erythrocytes is dependent upon the source of erythrocytes and, therefore, their lipid composition.
plp has phospholipase a <dig> activity
thin layer chromatography  was used to examine the pattern of phospholipid cleavage by plp.
bodipy-labeled phosphatidylcholine  was incubated with various enzyme standards, including phospholipase a <dig> , phospholipase c , or phospholipase d .
tlc analysis revealed distinct cleavage patterns  by these standard enzymes indicating that bpc was an appropriate substrate to examine plp activity.
cell lysate prepared from e. coli strain s <dig>  which contains the shuttle plasmid psup202-plp that was able to complement the plp mutation in v. anguillarum <cit> , cleaved bpc to yield bodipy-lysophosphatidylcholine   plus unlabeled free fatty acid  that is not detectable.
the cleavage products were identical to those generated by pla <dig>  and demonstrate that plp has phospholipase a <dig> activity.
additionally, the culture supernatant from s <dig> had only ~5% of the activity of that in cell lysate, indicating that plp accumulated in the cell lysate instead of being secreted by the e. coli strain.
no phospholipase activity was detected in pbs buffer or in e. coli dh5α containing only the psup <dig> vector .
further, phospholipase a <dig> activity was examined in various subcellular fractions prepared from e. coli strain s <dig>  including cytoplasm, cytoplasmic membrane, and outer membrane fractions.
most plp activity was detected in tween- <dig> soluble membrane fraction, indicating that plp was mainly localized in the cytoplasmic membrane of e. coli s <dig> .
no bodipy-labeled free fatty acid   was detected in the tlc analysis when an apolar solvent was used , and bodipy-labeled lpc was not further degraded by plp in the reaction, indicating that plp had no lysophospholipase or phospholipase b activity.
enzymatic characteristics of rplp protein
to examine the enzymatic characteristics of plp, the entire coding sequence of plp was cloned and inserted into the expression vector pqe <dig>  which adds a his <dig>  tag to the carboxyl end of plp.
the over-expressed recombinant plp  formed inclusion bodies in e. coli.
to recover active rplp, purification of the inclusion bodies followed by solubilization under mild conditions and re-folding was performed as described in the methods.
purity of refolded rplp protein was confirmed by sds-page and silver staining .
the final concentration of purified rplp protein was 8 μg/ml with a recovery of <10%.
subsequently, the enzymatic characteristics of refolded rplp were examined under various chemical and physical conditions.
the enzymatic activity of rplp positively correlated to its concentration from  <dig> μg/ml to  <dig> μg/ml ; therefore,  <dig> μg/ml rplp protein was routinely used in other activity assays.
the enzymatic activity unit of refolded rplp  was about  <dig> -fold higher than standard pla <dig> enzyme extracted from porcine pancreas, which indicated that plp had a high activity against the bpc phospholipid substrate.
plp enzyme activity exhibited a broad temperature optimum from 37°c to 64°c  with 75% activity retained at 27°c and 50% activity at 20°c.
while rplp activity rapidly decreased at temperatures above 70°c, the enzyme retained full activity at 64°c for at least 1 h. the data demonstrate that rplp is a relatively themostable phospholipase.
the effect of ph on enzyme activity was determined for ph values ranging from  <dig> to  <dig>  the data showed that rplp had a broad ph optimum from ph  <dig>  to ph  <dig>  with activity dropping off rapidly at ph values above and below the optimum .
rplp activity was not affected by treatment with the chelating reagents egta  or edta  at concentrations up to 100 mm.
additionally, treatment with divalent metal ions, such as calcium or magnesium had no effect on activity .
plp is a secreted protein in v. anguillarum
subcellular fractions from v. anguillarum strains m93sm and s <dig>  were prepared and phospholipase a <dig> activity examined using bpc and tlc.
initial studies revealed that at 37°c phospholipase a <dig> activity was detected in all cell fractions, including the culture supernatant, periplasm, cytoplasm, cytoplasmic membrane, and outer membrane, from both m93sm and s <dig> .
however, when the assay was performed at 64°c , phospholipase a <dig> activity in s <dig> was significantly decreased in all fractions including the supernatant .
additionally, when the assay was performed at 64°c for m93sm subcellular fractions, only the culture supernatant exhibited phospholipase activity against bpc .
the data demonstrated that plp was secreted into the culture supernatant of v. anguillarum, which corresponds with in silico analysis of the deduced plp amino acid sequence  by signalp that plp has a signal peptide  <cit> .
tlc results also revealed that there was at least one other protein in v. anguillarum m93sm exhibiting phospholipase a <dig> activity besides the secreted, heat stable plp protein.
this was a themolabile pla <dig> activity inactivated at 64°c.
in order to confirm that plp was localized in the supernatant of v. anguillarum, protein samples prepared from various subcellular fractions were separated by sds-page and analyzed by western blot analysis using polyclonal rabbit anti-plp antiserum.
an immuno-reactive band of ~45 kda was detected only in the supernatant of m93sm, but was absent in the supernatant of plp mutant .
taken together with the phospholipase a <dig> activity data, these data indicate that plp is a secreted protein in v. anguillarum.
rplp has a specific activity against phosphatidylcholine
various fluorescently-labeled phospholipid substrates  were used to determine the specificity of the rplp protein.
rplp exhibited high activity against phosphatidylcholine, cleaving bpc to yield blpc and free fatty acid .
however, rplp had almost no activity against both nbd-phosphatidylethanolamine   and nbd-phosphatidylserine  , showing only 2% and 5%, respectively, of the activity of the standard pla <dig> protein against each of the substrates.
the data indicated that the rplp protein does not efficiently cleave either phosphatidylethanolamine or phosphatidylserine.
additionally, unlike the standard sphingomyelinase, rplp was not able to cleave the nbd-sphingomyelin into the nbd-ceramide and phosphocholine , indicating that rplp had no sphingomyelinase activity.
taken together, the data demonstrated that plp is a phosphatidylcholine-specific pla <dig> enzyme.
rplp is able to lyse the fish erythrocytes directly
membrane phospholipid compositions are quite varied among the animal species, especially for phosphatidylcholine.
it is known that phosphatidylcholine makes up 58% of the total phospholipid in fish erythrocytes  <cit> ; however, no phosphatidylcholine is found in sheep erythrocytes  <cit> .
in order to determine whether the differential hemolysis observed for plp mutants of v. anguillarum  is due to the activity of plp against pc, we tested the ability of purified rplp to lyse atlantic salmon erythrocytes.
addition of recombinant plp resulted in the lysis of atlantic salmon erythrocytes, with the amount of lysis directly related to the amount of rplp added to the blood suspension .
in contrast, addition of rplp to a suspension of sheep erythrocytes resulted in no lysis of those cells .
these data show that plp has phosphatidylcholine-specific phospholipase a <dig> activity and can directly lyse fish erythrocytes.
plp is one of the hemolysins of v. anguillarum
previously, we demonstrated that there are two major hemolysin gene clusters in the m93sm, the vah <dig> cluster  <cit>  and the rtxa cluster  <cit> .
mutation of both vah <dig> and rtxa completely eliminated the hemolytic activity of m93sm on tsa-sheep blood agar  <cit> .
mutation of the plp gene resulted in 2-3-fold increased hemolytic activity on tsa-sheep blood agar because vah <dig> expression increased both transcriptionally and translationally in the plp mutant, indicating that plp is a putative repressor of vah <dig> <cit> .
plp also has hemolytic activity against fish erythrocytes due to its phosphatidylcholine-specific activity .
to investigate the relationship of the three hemolysins, culture supernatants obtained from various v. anguillarum strains  were used to examine the hemolytic activity against the fish blood .
wild-type and various
v. anguillarum
mutant strains against rainbow trout blood cells
v. anguillarum
a
ahemolytic activity assays carried out using the tube assay method as described in the methods.
hemolysis by m93sm was given the value of  <dig> .
the data are representative of two independent experiments, each with three replicates, ± one standard deviation .
bstatistically different from hemolytic activity for m93sm .
in contrast to the strong hemolytic activity against 5% rainbow trout blood mixed with culture supernatant from the wild type strain m93sm, hemolytic activity of culture supernatant from strain s <dig>  declined by >70% .
additionally, all mutants containing a knockout of plp exhibited significant declines  in hemolytic activity.
the triple hemolysin mutant, xm <dig>  had no ability to lyse fish erythrocytes .
however, mutations in either vah <dig> or rtxa, but not plp, resulted in little or no decline in hemolytic activity against fish erythrocytes compared to supernatants from wild type cells .
further, complementation of plp restored the hemolytic activity of supernatants from both the plp-complemented strains  .
taken together, these data clearly demonstrate that plp is the major hemolytic enzyme responsible for the lysis of rainbow trout erythrocytes by culture supernatants of v. anguillarum.
plp is not a major virulence factor for v. anguillarum during fish infection
in order to determine whether the plp gene affects virulence in fish, an infection study was performed by inoculating rainbow trout by ip injection with either the wild type strain m93sm or mutant strains s <dig>  or jr <dig> .
the results of this experiment  indicated that there were no statistical differences in mortality between the three strains.
this suggested that mutation of either plp or vah <dig> or both genes did not decrease the virulence of m93sm.
these results are consistent with our previous observations that rtxa is a major virulence factor of m93sm and that mutation of vah <dig> does not affect virulence  <cit> , and demonstrate that plp is not a major virulence factor in the v. anguillarum m93sm.
discussion
in this report, we describe the characteristics of the v. anguillarum phospholipase protein  encoded by plp, and its contribution to the hemolytic activity of v. anguillarum.
specifically, we show that plp is a secreted phospholipase with a <dig> activity with specificity for phosphatidylcholine.
the enzyme has a broad temperature optimum  and a broad ph optimum .
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 <cit>  was the first well-studied lecithin-dependent pla/lysophospholipase  <cit> .
a lecithinase  was also identified in v. cholerae <cit> .
fiore et al.
<cit>  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  in v. mimicus was found to exhibit hemolytic activity against trout and tilapia erythrocytes and was cytotoxic to the fish cell line chse- <dig>  <cit> .
recently, the v. harveyi hemolysin  was shown to be a virulence factor during flounder infection and also had phospholipase activity on egg yolk agar  <cit> .
rock and nelson  <cit>  reported that the putative phospholipase gene  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   <cit> .
additionally, rock and nelson  <cit>  demonstrated that functional plp repressed transcription of its adjacent hemolysin gene, vah <dig>  in v. anguillarum.
however, the enzymatic characteristics of plp in v. anguillarum were not described.
usually, phospholipases are divided into phospholipases a , c, and d according to the cleavage position on target phospholipids.
most of lipolytic enzymes contain a putative lipid catalytic motif  that was previously demonstrated in other bacterial and eukaryotic phospholipases  <cit> .
however, molgaard  <cit>  demonstrated that four amino acid residues  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  <cit> .
multiple alignment analysis of  <dig> phospholipase homologues  demonstrates that v. anguillarum plp belongs to the sgnh hydrolase subgroup, since the gsdl motif was not fully conserved in these proteins .
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 <cit>  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- <dig> position, but no lysophospholipase activity  <cit> .
two phospholipases identified from mesophilic aeromonas sp.
serogroup o: <dig> show phospholipase a <dig> and c activity  <cit> .
in addition, tlh of v. parahaemolyticus has pla <dig> and lysophospholipase activity, and demonstrates a loss of activity at 55°c for 10 min  <cit> .
in this report, we show that v. anguillarum plp has pla <dig> activity, and is able to maintain activity at 64°c for 1 h .
therefore, the enzymatic characteristics of specific phospholipases are distinct even when they all belong to the sgnh hydrolase family .
phospholipases have been implicated in the pathogenic activities of a number of bacteria  <cit> .
it is known that phospholipase activities often lead to cell destruction by degrading the phospholipids of cell membranes  <cit> .
however, the relationships between phospholipases and virulence are not always clear.
while the purified rplp exhibits strong hemolytic activity against atlantic salmon erythrocytes , rock and nelson  <cit>  showed that a knock-out mutation of either the plp gene or the vah <dig> 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 vah <dig> 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 vah <dig> are major virulence factors during pathogenesis of salmonids.
it was also reported that the deletion of lecithinase  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  <cit> .
lec is a homologue of plp  <cit> .
in contrast, the direct ip injection of purified v. harveyi vhh protein caused the death of flounder with an ld <dig> of about  <dig>  μg protein/fish  <cit> .
the rphla of v. mimicus also has a direct cytotoxic effect on the fish cell line chse- <dig>  <cit>  suggesting that this phospholipase is a virulence factor during fish infection.
in addition, the lecithinase purified from a. hydrophila  has been shown to be an important virulence factor to rainbow trout and mouse  <cit> .
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 vah <dig>  as previously described by rock and nelson  <cit> .
additionally, transcription of rtxa is also increased in a plp mutant .
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 , phosphatidylethanolamine , phosphatidylserine , phosphatidylinositol , and sphingomyelin .
pc makes up 58% of the total erythrocyte phospholipids in the atlantic salmon  <cit> , but only 34% and 1% in rabbit and sheep erythrocytes, respectively  <cit> .
taken together with the high specificity of rplp for pc , it was not surprising that rplp was able to lyse the fish erythrocytes, but not sheep erythrocytes , and that the plp mutant had decreased hemolytic activity on lb20-fish blood agar .
our results are consistent were those reported for v. mimicus phla  <cit>  and v. harveyi vhh  <cit> , 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  <cit>  and have described their regulation by h-ns and hlyu  <cit> .
mutation of both vah <dig> and rtxa results in the loss of all hemolytic activity on tsa-sheep blood agar  <cit> , 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 vah <dig>  since mutation of plp increases vah <dig> expression by 2– <dig> fold  <cit> .
in this report, we examined the hemolytic activity of various hemolysin mutants using freshly collected rainbow trout blood  to investigate the relationships among three hemolysins of v. anguillarum.
while culture supernatants from two of the three single mutants  and one of three double mutants  exhibited ≥94% of the hemolytic activity as supernatants from the wild type strain m93sm , the hemolytic activity of one single mutant  and two double mutants  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 vah <dig> has little effect.
further, supernatant from the hemolysin triple mutant xm <dig>  exhibits no hemolytic activity on fish blood compared to m93sm , indicating that vah <dig>  rtxa, and plp are responsible for all secreted hemolytic activity by v. anguillarum.
finally, complementation of any plp mutant with plp  restores hemolytic activity to v. anguillarum culture supernatant .
CONCLUSIONS
v. anguillarum plp is a secreted hemolysin with phosphatidylcholine-specific phospholipase a <dig> activity.
the ability of plp to digest the abundant phosphatidylcholine found in the membrane of fish erythrocytes causes their lysis.
the three hemolysins, plp, vah <dig> and rtxa, account for all hemolytic activity in v. anguillarum culture supernatant under the experiment conditions described in this study.
finally, infection studies in rainbow trout demonstrate that the plp and vah <dig> genes are not required for virulence.

 vibrio anguillarum, a highly motile marine member of the γ-proteobacteria, is one of the causative agents of vibriosis, a fatal hemorrhagic septicemic disease of both wild and cultured fish, crustaceans, and bivalves. fish infected with v. anguillarum display skin discoloration and erythema around the mouth, fins, and vent. necrotic lesions are also observed in the abdominal muscle. mortality rates in infected fish populations range as high as 30-100%. vibriosis has caused severe economic losses to aquaculture worldwide and affects many farm-raised fish including pacific salmon, atlantic salmon, sea bass, cod, and eel. v. anguillarum enters its fish host through the gastrointestinal tract and quickly colonizes this nutrient rich environment. garcia et al. have shown that v. anguillarum grows extremely well in salmon intestinal mucus and that mucus-grown cells specifically express a number of different proteins, including several outer membrane proteins and the extracellular metalloprotease empa. several genes have been reported to be correlated with virulence by v. anguillarum, including the vah hemolysin cluster, the rtx hemolysin cluster, the siderophore mediated iron transport system, the empa metalloprotease, and the flaa gene. hemolytic activity of v. anguillarum has been considered to be the virulence factor responsible for hemorrhagic septicemia during infection. we have identified two hemolysin gene clusters in v. anguillarum that contribute to the virulence of this pathogen. one gene cluster, rtxachbde, encodes a martx toxin and its type i secretion system. the second hemolysin gene cluster in v. anguillarum strain m93sm contains the hemolysin gene vah flanked by two putative lipase-related genes immediately downstream and upstream by a divergently transcribed hemolysin-like gene that appears to function as a repressor of vah1-dependent hemolytic activity. the plp-encoded protein has very high sequence similarity to phospholipases found in other pathogenic vibrio species. however, the enzymatic characteristics of plp in v. anguillarum were not described. generally, phospholipases are divided into several subgroups depending on their specificity for hydrolysis of ester bonds at different locations in the phospholipid molecule. phospholipases a cleave long chain fatty acids at sn- or sn- position from phospholipid to yield lysophospholipid and free fatty acid; phospholipases c cleave phospholipid into diacylglycerol and a phosphate-containing head group; and phospholipases d cleave phospholipid into phosphatidic acid and an alcohol. it is known that some phospholipid products are used as secondary messages, which play a central role in signal transduction. in this study, we determined that plp encodes a phospholipase a in v. anguillarum, and then purified recombinant plp protein from e. coli to investigate its biochemical properties. we also described the contribution and specificity of rplp for hydrolysis of phospholipids, and its ability to lyse fish erythrocytes.  identification of a putative phospholipase gene plp previously, a putative phospholipase gene, plp, was identified in the vah hemolysin cluster of v. anguillarum strain m93sm. the 1251-bp plp gene was predicted to encode a protein of amino acids. a blastx search revealed that the deduced plp amino acid sequence exhibited homology with many lipolytic enzymes including the phospholipase/lecithinase/hemolysin of vibrio vulnificus; the lecithin-dependent hemolysin / thermolabile hemolysin of vibrio parahaemolyticus; the lipolytic enzyme/hemolysin vhh of vibrio harveyi; and the thermolabile hemolysin of vibrio cholerae. the phylogenetic tree created by the clustal-w program from plp homologous proteins revealed that while the most closely related plp proteins were all from pathogenic members of the genus vibrio, the plp of v. anguillarum was an outlier among the vibrio species, as demonstrated by the neighbor joining analysis. according to flieger’s classification, the alignment of plp with other homologous proteins indicated that plp could be classified into subgroup b of this lipolytic family with its long n-terminal tail prior to the block i. additionally, close examination of the amino acid sequences of these enzymes revealed that the typical gdsl motif for lypolytic enzymes is not totally conserved in all of these proteins, in which leucines are replaced with isoleucines in photobacterium, marinomonas, and shewanella. in this case, v. anguillarum plp should be considered as a member of the sgnh hydrolase family, based on the molgaard’s suggestion that only four amino acids are completely conserved among the gdsl proteins. plp affects hemolysis of fish erythrocytes the hemolysin gene vah is divergently transcribed from plp. mutation of plp increased hemolytic activity by 2-3-fold on trypticase soy agar plus 5% sheep blood plate compared with wild type strain. rock and nelson also demonstrated that the plp mutant had increased vah transcription, indicating that plp is a putative repressor of vah previously, we demonstrated that a double mutant in vah and rtxa resulted in a hemolysis negative mutant when plated on tsa-sheep blood agar. similar results were observed when using luria-bertani broth plus 2% nacl plus 5% sheep blood agar. however, on lb plus 5% rainbow trout blood agar, the plp mutant exhibited a smaller zone of hemolysis compared to wild type strain m93sm; complementation of plp restored the hemolytic activity of the mutant strain. similar results were observed when using lb plus 5% atlantic salmon blood agar, suggesting that the ability of plp to lyse erythrocytes is dependent upon the source of erythrocytes and, therefore, their lipid composition. plp has phospholipase a activity thin layer chromatography was used to examine the pattern of phospholipid cleavage by plp. bodipy-labeled phosphatidylcholine was incubated with various enzyme standards, including phospholipase a, phospholipase c, or phospholipase d. tlc analysis revealed distinct cleavage patterns by these standard enzymes indicating that bpc was an appropriate substrate to examine plp activity. cell lysate prepared from e. coli strain s which contains the shuttle plasmid psup202-plp that was able to complement the plp mutation in v. anguillarum, cleaved bpc to yield bodipy-lysophosphatidylcholine plus unlabeled free fatty acid that is not detectable. the cleavage products were identical to those generated by pla and demonstrate that plp has phospholipase a activity. additionally, the culture supernatant from s had only ~5% of the activity of that in cell lysate, indicating that plp accumulated in the cell lysate instead of being secreted by the e. coli strain. no phospholipase activity was detected in pbs buffer or in e. coli dh5α containing only the psup vector. further, phospholipase a activity was examined in various subcellular fractions prepared from e. coli strain s including cytoplasm, cytoplasmic membrane, and outer membrane fractions. most plp activity was detected in tween- soluble membrane fraction, indicating that plp was mainly localized in the cytoplasmic membrane of e. coli s. no bodipy-labeled free fatty acid was detected in the tlc analysis when an apolar solvent was used, and bodipy-labeled lpc was not further degraded by plp in the reaction, indicating that plp had no lysophospholipase or phospholipase b activity. enzymatic characteristics of rplp protein to examine the enzymatic characteristics of plp, the entire coding sequence of plp was cloned and inserted into the expression vector pqe which adds a his tag to the carboxyl end of plp. the over-expressed recombinant plp formed inclusion bodies in e. coli. to recover active rplp, purification of the inclusion bodies followed by solubilization under mild conditions and re-folding was performed as described in the methods. purity of refolded rplp protein was confirmed by sds-page and silver staining. the final concentration of purified rplp protein was 8 μg/ml with a recovery of <10%. subsequently, the enzymatic characteristics of refolded rplp were examined under various chemical and physical conditions. the enzymatic activity of rplp positively correlated to its concentration from μg/ml to μg/ml; therefore, μg/ml rplp protein was routinely used in other activity assays. the enzymatic activity unit of refolded rplp was about -fold higher than standard pla enzyme extracted from porcine pancreas, which indicated that plp had a high activity against the bpc phospholipid substrate. plp enzyme activity exhibited a broad temperature optimum from 37°c to 64°c with 75% activity retained at 27°c and 50% activity at 20°c. while rplp activity rapidly decreased at temperatures above 70°c, the enzyme retained full activity at 64°c for at least 1 h. the data demonstrate that rplp is a relatively themostable phospholipase. the effect of ph on enzyme activity was determined for ph values ranging from to the data showed that rplp had a broad ph optimum from ph  to ph  with activity dropping off rapidly at ph values above and below the optimum. rplp activity was not affected by treatment with the chelating reagents egta or edta at concentrations up to 100 mm. additionally, treatment with divalent metal ions, such as calcium or magnesium had no effect on activity. plp is a secreted protein in v. anguillarum subcellular fractions from v. anguillarum strains m93sm and s were prepared and phospholipase a activity examined using bpc and tlc. initial studies revealed that at 37°c phospholipase a activity was detected in all cell fractions, including the culture supernatant, periplasm, cytoplasm, cytoplasmic membrane, and outer membrane, from both m93sm and s. however, when the assay was performed at 64°c, phospholipase a activity in s was significantly decreased in all fractions including the supernatant. additionally, when the assay was performed at 64°c for m93sm subcellular fractions, only the culture supernatant exhibited phospholipase activity against bpc. the data demonstrated that plp was secreted into the culture supernatant of v. anguillarum, which corresponds with in silico analysis of the deduced plp amino acid sequence by signalp that plp has a signal peptide. tlc results also revealed that there was at least one other protein in v. anguillarum m93sm exhibiting phospholipase a activity besides the secreted, heat stable plp protein. this was a themolabile pla activity inactivated at 64°c. in order to confirm that plp was localized in the supernatant of v. anguillarum, protein samples prepared from various subcellular fractions were separated by sds-page and analyzed by western blot analysis using polyclonal rabbit anti-plp antiserum. an immuno-reactive band of ~45 kda was detected only in the supernatant of m93sm, but was absent in the supernatant of plp mutant. taken together with the phospholipase a activity data, these data indicate that plp is a secreted protein in v. anguillarum. rplp has a specific activity against phosphatidylcholine various fluorescently-labeled phospholipid substrates were used to determine the specificity of the rplp protein. rplp exhibited high activity against phosphatidylcholine, cleaving bpc to yield blpc and free fatty acid. however, rplp had almost no activity against both nbd-phosphatidylethanolamine and nbd-phosphatidylserine, showing only 2% and 5%, respectively, of the activity of the standard pla protein against each of the substrates. the data indicated that the rplp protein does not efficiently cleave either phosphatidylethanolamine or phosphatidylserine. additionally, unlike the standard sphingomyelinase, rplp was not able to cleave the nbd-sphingomyelin into the nbd-ceramide and phosphocholine, indicating that rplp had no sphingomyelinase activity. taken together, the data demonstrated that plp is a phosphatidylcholine-specific pla enzyme. rplp is able to lyse the fish erythrocytes directly membrane phospholipid compositions are quite varied among the animal species, especially for phosphatidylcholine. it is known that phosphatidylcholine makes up 58% of the total phospholipid in fish erythrocytes; however, no phosphatidylcholine is found in sheep erythrocytes. in order to determine whether the differential hemolysis observed for plp mutants of v. anguillarum is due to the activity of plp against pc, we tested the ability of purified rplp to lyse atlantic salmon erythrocytes. addition of recombinant plp resulted in the lysis of atlantic salmon erythrocytes, with the amount of lysis directly related to the amount of rplp added to the blood suspension. in contrast, addition of rplp to a suspension of sheep erythrocytes resulted in no lysis of those cells. these data show that plp has phosphatidylcholine-specific phospholipase a activity and can directly lyse fish erythrocytes. plp is one of the hemolysins of v. anguillarum previously, we demonstrated that there are two major hemolysin gene clusters in the m93sm, the vah cluster and the rtxa cluster. mutation of both vah and rtxa completely eliminated the hemolytic activity of m93sm on tsa-sheep blood agar. mutation of the plp gene resulted in 2-3-fold increased hemolytic activity on tsa-sheep blood agar because vah expression increased both transcriptionally and translationally in the plp mutant, indicating that plp is a putative repressor of vah. plp also has hemolytic activity against fish erythrocytes due to its phosphatidylcholine-specific activity. to investigate the relationship of the three hemolysins, culture supernatants obtained from various v. anguillarum strains were used to examine the hemolytic activity against the fish blood. wild-type and various v. anguillarum mutant strains against rainbow trout blood cells v. anguillarum a ahemolytic activity assays carried out using the tube assay method as described in the methods. hemolysis by m93sm was given the value of. the data are representative of two independent experiments, each with three replicates, ± one standard deviation. bstatistically different from hemolytic activity for m93sm. in contrast to the strong hemolytic activity against 5% rainbow trout blood mixed with culture supernatant from the wild type strain m93sm, hemolytic activity of culture supernatant from strain s declined by >70%. additionally, all mutants containing a knockout of plp exhibited significant declines in hemolytic activity. the triple hemolysin mutant, xm had no ability to lyse fish erythrocytes. however, mutations in either vah or rtxa, but not plp, resulted in little or no decline in hemolytic activity against fish erythrocytes compared to supernatants from wild type cells. further, complementation of plp restored the hemolytic activity of supernatants from both the plp-complemented strains. taken together, these data clearly demonstrate that plp is the major hemolytic enzyme responsible for the lysis of rainbow trout erythrocytes by culture supernatants of v. anguillarum. plp is not a major virulence factor for v. anguillarum during fish infection in order to determine whether the plp gene affects virulence in fish, an infection study was performed by inoculating rainbow trout by ip injection with either the wild type strain m93sm or mutant strains s or jr. the results of this experiment indicated that there were no statistical differences in mortality between the three strains. this suggested that mutation of either plp or vah or both genes did not decrease the virulence of m93sm. these results are consistent with our previous observations that rtxa is a major virulence factor of m93sm and that mutation of vah does not affect virulence, and demonstrate that plp is not a major virulence factor in the v. anguillarum m93sm. discussion in this report, we describe the characteristics of the v. anguillarum phospholipase protein encoded by plp, and its contribution to the hemolytic activity of v. anguillarum. specifically, we show that plp is a secreted phospholipase with a activity with specificity for phosphatidylcholine. the enzyme has a broad temperature optimum and a broad ph optimum. 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 was the first well-studied lecithin-dependent pla/lysophospholipase. a lecithinase 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 in v. mimicus was found to exhibit hemolytic activity against trout and tilapia erythrocytes and was cytotoxic to the fish cell line chse-. recently, the v. harveyi hemolysin 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 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. additionally, rock and nelson demonstrated that functional plp repressed transcription of its adjacent hemolysin gene, vah in v. anguillarum. however, the enzymatic characteristics of plp in v. anguillarum were not described. usually, phospholipases are divided into phospholipases a, c, and d according to the cleavage position on target phospholipids. most of lipolytic enzymes contain a putative lipid catalytic motif that was previously demonstrated in other bacterial and eukaryotic phospholipases. however, molgaard demonstrated that four amino acid residues 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 phospholipase homologues demonstrates that v. anguillarum plp belongs to the sgnh hydrolase subgroup, since the gsdl motif was not fully conserved in these proteins. 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- position, but no lysophospholipase activity. two phospholipases identified from mesophilic aeromonas sp. serogroup o: show phospholipase a and c activity. in addition, tlh of v. parahaemolyticus has pla 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 pla activity, and is able to maintain activity at 64°c for 1 h. therefore, the enzymatic characteristics of specific phospholipases are distinct even when they all belong to the sgnh hydrolase family. phospholipases have been implicated in the pathogenic activities of a number of bacteria. it is known that phospholipase activities often lead to cell destruction by degrading the phospholipids of cell membranes. however, the relationships between phospholipases and virulence are not always clear. while the purified rplp exhibits strong hemolytic activity against atlantic salmon erythrocytes, rock and nelson showed that a knock-out mutation of either the plp gene or the vah 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 vah 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 vah are major virulence factors during pathogenesis of salmonids. it was also reported that the deletion of lecithinase 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 ld of about μg protein/fish. the rphla of v. mimicus also has a direct cytotoxic effect on the fish cell line chse- suggesting that this phospholipase is a virulence factor during fish infection. in addition, the lecithinase purified from a. hydrophila 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 vah as previously described by rock and nelson. additionally, transcription of rtxa is also increased in a plp mutant. 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, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin. 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, it was not surprising that rplp was able to lyse the fish erythrocytes, but not sheep erythrocytes, and that the plp mutant had decreased hemolytic activity on lb20-fish blood agar. 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. mutation of both vah 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 vah since mutation of plp increases vah expression by 2– fold. in this report, we examined the hemolytic activity of various hemolysin mutants using freshly collected rainbow trout blood to investigate the relationships among three hemolysins of v. anguillarum. while culture supernatants from two of the three single mutants and one of three double mutants exhibited ≥94% of the hemolytic activity as supernatants from the wild type strain m93sm, the hemolytic activity of one single mutant and two double mutants 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 vah has little effect. further, supernatant from the hemolysin triple mutant xm exhibits no hemolytic activity on fish blood compared to m93sm, indicating that vah rtxa, and plp are responsible for all secreted hemolytic activity by v. anguillarum. finally, complementation of any plp mutant with plp restores hemolytic activity to v. anguillarum culture supernatant.  v. anguillarum plp is a secreted hemolysin with phosphatidylcholine-specific phospholipase a activity. the ability of plp to digest the abundant phosphatidylcholine found in the membrane of fish erythrocytes causes their lysis. the three hemolysins, plp, vah and rtxa, account for all hemolytic activity in v. anguillarum culture supernatant under the experiment conditions described in this study. finally, infection studies in rainbow trout demonstrate that the plp and vah genes are not required for virulence. 
additionally, rplp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rplp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. the plp gene of v. anguillarum encoding a phospholipase with a activity is specific for phosphatidylcholine and, therefore, able to lyse fish erythrocytes, but not sheep erythrocytes. however, v. anguillarum strains with mutations in plp or in plp and vah exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout. we also demonstrated that plp was secreted using thin layer chromatography and immunoblot analysis. further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxa and/or vah had little effect. several extracellular proteins secreted by v. anguillarum have been shown to contribute to virulence. mutation of plp does not affect the virulence of v. anguillarum in rainbow trout. his6-tagged recombinant plp protein was over-expressed in e. coli. hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that plp has different effects against erythrocytes from different species. while two hemolysin gene clusters, vah1-plp and rtxachbde, have been previously identified and described, the activities of the protein encoded by the plp gene were not known. the plp gene, one of the components in vah cluster, encodes a 416-amino-acid protein, which has homology to lipolytic enzymes containing the catalytic site amino acid signature sgnh. purified and re-folded active rplp exhibited phospholipase a activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. vibrio anguillarum is the causative agent of vibriosis in fish.
 vibrio anguillarum is the causative agent of vibriosis in fish. several extracellular proteins secreted by v. anguillarum have been shown to contribute to virulence. while two hemolysin gene clusters, vah1-plp and rtxachbde, have been previously identified and described, the activities of the protein encoded by the plp gene were not known. here we describe the biochemical activities of the plp-encoded protein and its role in pathogenesis.  the plp gene, one of the components in vah cluster, encodes a 416-amino-acid protein, which has homology to lipolytic enzymes containing the catalytic site amino acid signature sgnh. hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that plp has different effects against erythrocytes from different species. his6-tagged recombinant plp protein was over-expressed in e. coli. purified and re-folded active rplp exhibited phospholipase a activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. characterization of rplp revealed broad optimal activities at ph 5– and at temperatures of 30-64°c. divalent cations and metal chelators did not affect activity of rplp. we also demonstrated that plp was secreted using thin layer chromatography and immunoblot analysis. additionally, rplp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rplp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxa and/or vah had little effect. however, v. anguillarum strains with mutations in plp or in plp and vah exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.  the plp gene of v. anguillarum encoding a phospholipase with a activity is specific for phosphatidylcholine and, therefore, able to lyse fish erythrocytes, but not sheep erythrocytes. mutation of plp does not affect the virulence of v. anguillarum in rainbow trout. vibrio anguillarumvbriosisphospholipasehemolysisvirulence 