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Identification and characterization of additional flagellin genes from Vibrio anguillarum.

ABSTRACT: Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made. Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament. However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.

SUBMITTER: McGee K

PROVIDER: S-EPMC178316 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Identification and characterization of additional flagellin genes from Vibrio anguillarum.

McGee K K Hörstedt P P Milton D L DL

Journal of bacteriology 19960901 17

Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between bo ...[more]

PMID: 8752337

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Project description:Vibrio anguillarum is a causative agent of vibriosis in fish. Hemolytic activity has been suggested as a virulence factor by contributing to hemorrhagic septicemia and diarrhea. In order to identify and characterize the hemolysin genes and examine the role of hemolytic activity in virulence, a mini-Tn10Kan mutagenesis clone bank of V. anguillarum was screened. While no hemolysin-negative strains were observed, several mutants with two- to threefold-increased hemolytic activity were found. The region containing the insertion mutation was cloned, sequenced, and found to contain the V. anguillarum hemolysin (vah1) and two other open reading frames, coding for a putative lactonizing lipase (llpA) and a putative phospholipase (plp). The mini-Tn10Kan was inserted into plp. Site-directed mutagenesis of each gene revealed that mutations in vah1 and llpA did not affect hemolytic activity, but insertions into plp caused a two- to threefold increase in hemolysis. Double mutations in plp and either vah1 or llpA resulted in wild-type hemolytic activity. Complementation of plp restored hemolytic activity to wild-type levels. Spectrophotometric determination of hemolysin specific activity revealed that activity on a per cell basis peaked during the first 2 h of growth in LB20. Real-time quantitative reverse transcriptase PCR used to quantitate transcription of the hemolysin genes plp and vah1 in V. anguillarum wild-type strains M93Sm and NB10 revealed that transcription of plp and vah1 peaked at 2 h of growth in LB20. Additionally, expression of vah1 measured in the plp mutant strain, JL01, during the first 2 h of growth was >8 times higher than that in M93Sm. Mutations in plp and llpA did not affect virulence of V. anguillarum. The mutation in vah1 attenuated V. anguillarum virulence in fish. These data show that several genes are responsible for hemolytic activity in V. anguillarum. At least three genes (plp, llpA, and vah1) are responsible for one hemolytic activity. The data also suggest that plp acts as a negative regulator of vah1 and llpA.

Project description:BackgroundVibrio 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.ResultsThe 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.ConclusionThe 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.

Dataset Information

Identification and characterization of additional flagellin genes from Vibrio anguillarum.

Publications

Identification and characterization of additional flagellin genes from Vibrio anguillarum.

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