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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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Hemolytic activity of M93Sm and S262 (plp) on TSA-sheep blood agar (A) and LB20 + 5% rainbow trout blood agar (B). A single colony of M93Sm and S262 was transferred onto each of the blood agars and incubated at 27°C for 24 h. The zones of hemolysis were measured and the diameters were given in the figure. This is a representative experiment from 3 replicate trials, each performed in triplicate.
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Figure 2: Hemolytic activity of M93Sm and S262 (plp) on TSA-sheep blood agar (A) and LB20 + 5% rainbow trout blood agar (B). A single colony of M93Sm and S262 was transferred onto each of the blood agars and incubated at 27°C for 24 h. The zones of hemolysis were measured and the diameters were given in the figure. This is a representative experiment from 3 replicate trials, each performed in triplicate.

Mentions: The hemolysin gene vah1 is divergently transcribed from plp[17]. Mutation of plp increased hemolytic activity by 2-3-fold on Trypticase soy agar plus 5% sheep blood (TSA-sheep blood) plate compared with wild type strain (M93Sm) (Figure 2A) [8]. Rock and Nelson [8] also demonstrated that the plp mutant had increased vah1 transcription (by 2-4-fold), indicating that Plp is a putative repressor of vah1. Previously, we demonstrated that a double mutant in vah1 and rtxA resulted in a hemolysis negative mutant when plated on TSA-sheep blood agar [9]. Similar results were observed when using Luria-Bertani broth plus 2% NaCl plus 5% sheep blood (LB20-sheep blood) agar (data not shown). However, on LB20 plus 5% rainbow trout blood (LB20-rainbow trout blood) agar, the plp mutant exhibited a smaller zone of hemolysis compared to wild type strain M93Sm (diameter: 9.5 ±0.5 mm vs. 12 ± 0.0 mm, P < 0.05) (Figure 2B); complementation of plp restored the hemolytic activity of the mutant strain (Figure 2B). Similar results were observed when using LB20 plus 5% Atlantic salmon blood agar (data not shown), suggesting that the ability of Plp to lyse erythrocytes is dependent upon the source of erythrocytes and, therefore, their lipid composition.


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

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

Hemolytic activity of M93Sm and S262 (plp) on TSA-sheep blood agar (A) and LB20 + 5% rainbow trout blood agar (B). A single colony of M93Sm and S262 was transferred onto each of the blood agars and incubated at 27°C for 24 h. The zones of hemolysis were measured and the diameters were given in the figure. This is a representative experiment from 3 replicate trials, each performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Hemolytic activity of M93Sm and S262 (plp) on TSA-sheep blood agar (A) and LB20 + 5% rainbow trout blood agar (B). A single colony of M93Sm and S262 was transferred onto each of the blood agars and incubated at 27°C for 24 h. The zones of hemolysis were measured and the diameters were given in the figure. This is a representative experiment from 3 replicate trials, each performed in triplicate.
Mentions: The hemolysin gene vah1 is divergently transcribed from plp[17]. Mutation of plp increased hemolytic activity by 2-3-fold on Trypticase soy agar plus 5% sheep blood (TSA-sheep blood) plate compared with wild type strain (M93Sm) (Figure 2A) [8]. Rock and Nelson [8] also demonstrated that the plp mutant had increased vah1 transcription (by 2-4-fold), indicating that Plp is a putative repressor of vah1. Previously, we demonstrated that a double mutant in vah1 and rtxA resulted in a hemolysis negative mutant when plated on TSA-sheep blood agar [9]. Similar results were observed when using Luria-Bertani broth plus 2% NaCl plus 5% sheep blood (LB20-sheep blood) agar (data not shown). However, on LB20 plus 5% rainbow trout blood (LB20-rainbow trout blood) agar, the plp mutant exhibited a smaller zone of hemolysis compared to wild type strain M93Sm (diameter: 9.5 ±0.5 mm vs. 12 ± 0.0 mm, P < 0.05) (Figure 2B); complementation of plp restored the hemolytic activity of the mutant strain (Figure 2B). Similar results were observed when using LB20 plus 5% Atlantic salmon blood agar (data not shown), suggesting that the ability of Plp to lyse erythrocytes is dependent upon the source of erythrocytes and, therefore, their lipid composition.

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