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Characterization of Plp, a phosphatidylcholine-specific phospholipase and hemolysin of Vibrio anguillarum.

Li L, Mou X, Nelson DR - BMC Microbiol. (2013)

Bottom Line: Divalent cations and metal chelators did not affect activity of rPlp.However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.Mutation of plp does not affect the virulence of V. anguillarum in rainbow trout.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, University of Rhode Island, 120 Flagg Rd,, Kingston, RI 02881, USA. dnelson@uri.edu.

ABSTRACT

Background: 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.

Results: The plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), 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 (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5-9 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 vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.

Conclusion: The plp gene of V. anguillarum encoding a phospholipase with A2 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.

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Effects of chemical and physical conditions on rPlp activity. (A) Effect of rPlp concentration on enzymatic activity. (B) The effect of temperature on rPlp activity. (C) The effect of pH on rPlp activity. (D) The effect of EGTA rPlp activity.
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Figure 4: Effects of chemical and physical conditions on rPlp activity. (A) Effect of rPlp concentration on enzymatic activity. (B) The effect of temperature on rPlp activity. (C) The effect of pH on rPlp activity. (D) The effect of EGTA rPlp activity.

Mentions: 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 1 μg/ml to 8 μg/ml (Figure 4A); therefore, 4 μg/ml rPlp protein was routinely used in other activity assays. The enzymatic activity unit of refolded rPlp (1 unit = amount of protein that cleaves 1 μmole of BODIPY-PC per minute) was about 2,500-fold higher than standard PLA2 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 (Figure 4B) 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.


Characterization of Plp, a phosphatidylcholine-specific phospholipase and hemolysin of Vibrio anguillarum.

Li L, Mou X, Nelson DR - BMC Microbiol. (2013)

Effects of chemical and physical conditions on rPlp activity. (A) Effect of rPlp concentration on enzymatic activity. (B) The effect of temperature on rPlp activity. (C) The effect of pH on rPlp activity. (D) The effect of EGTA rPlp activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222444&req=5

Figure 4: Effects of chemical and physical conditions on rPlp activity. (A) Effect of rPlp concentration on enzymatic activity. (B) The effect of temperature on rPlp activity. (C) The effect of pH on rPlp activity. (D) The effect of EGTA rPlp activity.
Mentions: 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 1 μg/ml to 8 μg/ml (Figure 4A); therefore, 4 μg/ml rPlp protein was routinely used in other activity assays. The enzymatic activity unit of refolded rPlp (1 unit = amount of protein that cleaves 1 μmole of BODIPY-PC per minute) was about 2,500-fold higher than standard PLA2 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 (Figure 4B) 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.

Bottom Line: Divalent cations and metal chelators did not affect activity of rPlp.However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.Mutation of plp does not affect the virulence of V. anguillarum in rainbow trout.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, University of Rhode Island, 120 Flagg Rd,, Kingston, RI 02881, USA. dnelson@uri.edu.

ABSTRACT

Background: 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.

Results: The plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), 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 (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5-9 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 vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.

Conclusion: The plp gene of V. anguillarum encoding a phospholipase with A2 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.

Show MeSH
Related in: MedlinePlus