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Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Sirikharin R, Taengchaiyaphum S, Sanguanrut P, Chi TD, Mavichak R, Proespraiwong P, Nuangsaeng B, Thitamadee S, Flegel TW, Sritunyalucksana K - PLoS ONE (2015)

Bottom Line: In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B.A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium).The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

View Article: PubMed Central - PubMed

Affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.

ABSTRACT
Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE analysis of 60% AS fractions from broth of non-AHPND isolate S02 (Lane 2), and VPAHPND isolates 5HP (Lane 3) and CN (Lane 4).Lane 1: 60% AS from culture broth without bacteria. There are 2 major bands (ToxA at ~16kDa and ToxB at ~50 kDa) present in lanes for the VPAHPND isolates 5HP and CN, but absent or at low level in non-AHPND isolate S02.
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pone.0126987.g002: SDS-PAGE analysis of 60% AS fractions from broth of non-AHPND isolate S02 (Lane 2), and VPAHPND isolates 5HP (Lane 3) and CN (Lane 4).Lane 1: 60% AS from culture broth without bacteria. There are 2 major bands (ToxA at ~16kDa and ToxB at ~50 kDa) present in lanes for the VPAHPND isolates 5HP and CN, but absent or at low level in non-AHPND isolate S02.

Mentions: When the 60% AS fractions were analyzed by SDS-PAGE (Fig 2), two prominent protein bands with molecular masses (according to the gel marker) of approximately 16 kDa (later named ToxA) and 50 kDa (later named ToxB) were found to be prominent in the fractions from VPAHPND isolates 5HP and CN, but not in the fraction from the non-AHPND isolate VP S02. When these two bands were excised from the gel and subjected to LC-MS/MS analysis, the peptide profiles for the two bands from 5HP matched those from CN. Comparison with known protein records using the MASCOT program revealed that the highest similarities for ToxA and ToxB were for two hypothetical proteins in the draft genome of V. parahaemolyticus M0605 contig034 (GenBank JALL01000066.1) that originated from AHPND bacteria in Mexico [7]. ToxA matched GenBank protein accession number ETZ12074.1 (MASCOT score 189 with 33% sequence coverage) while ToxB matched GenBank protein accession number ETZ12073 (MASCOT score of 386 with 26% sequence coverage). In this Mexican sequence, it was found that the ToxA and ToxB genes were closely linked (i.e., separated by 12 nucleotides). From this sequence, primers were designed to amplify the full length ToxA and ToxB genes from CN and 5HP for cloning and sequencing. The results revealed that the sequences from CN and 5HP were identical to those in JALL01000066.1 and that the two Tox genes were also separated by the same 12 nucleotides. After completion of this work, draft sequences of 6 VPAHPND and 4 non-AHPND VP from Thailand were published [11, 12], including the sequence for our 5HP and CN isolates [11]. In all 7 of the VPAHPND isolates listed at GenBank (6 from Thailand and one from Mexico), ToxA and ToxB were present with identical nucleic acid and deduced amino acid sequences, with identical genome arrangements and separated by 12 identical nucleotides, except that the 5’–3’ orientation of the two protein genes in the Mexican record (JALL01000066.1) was opposite to that in all the other records. Neither of these toxin genes were found in the chromosomal DNA of these AHPND isolates or in the total DNA of the non-AHPND isolates examined [7, 11–13].


Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Sirikharin R, Taengchaiyaphum S, Sanguanrut P, Chi TD, Mavichak R, Proespraiwong P, Nuangsaeng B, Thitamadee S, Flegel TW, Sritunyalucksana K - PLoS ONE (2015)

SDS-PAGE analysis of 60% AS fractions from broth of non-AHPND isolate S02 (Lane 2), and VPAHPND isolates 5HP (Lane 3) and CN (Lane 4).Lane 1: 60% AS from culture broth without bacteria. There are 2 major bands (ToxA at ~16kDa and ToxB at ~50 kDa) present in lanes for the VPAHPND isolates 5HP and CN, but absent or at low level in non-AHPND isolate S02.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4446338&req=5

pone.0126987.g002: SDS-PAGE analysis of 60% AS fractions from broth of non-AHPND isolate S02 (Lane 2), and VPAHPND isolates 5HP (Lane 3) and CN (Lane 4).Lane 1: 60% AS from culture broth without bacteria. There are 2 major bands (ToxA at ~16kDa and ToxB at ~50 kDa) present in lanes for the VPAHPND isolates 5HP and CN, but absent or at low level in non-AHPND isolate S02.
Mentions: When the 60% AS fractions were analyzed by SDS-PAGE (Fig 2), two prominent protein bands with molecular masses (according to the gel marker) of approximately 16 kDa (later named ToxA) and 50 kDa (later named ToxB) were found to be prominent in the fractions from VPAHPND isolates 5HP and CN, but not in the fraction from the non-AHPND isolate VP S02. When these two bands were excised from the gel and subjected to LC-MS/MS analysis, the peptide profiles for the two bands from 5HP matched those from CN. Comparison with known protein records using the MASCOT program revealed that the highest similarities for ToxA and ToxB were for two hypothetical proteins in the draft genome of V. parahaemolyticus M0605 contig034 (GenBank JALL01000066.1) that originated from AHPND bacteria in Mexico [7]. ToxA matched GenBank protein accession number ETZ12074.1 (MASCOT score 189 with 33% sequence coverage) while ToxB matched GenBank protein accession number ETZ12073 (MASCOT score of 386 with 26% sequence coverage). In this Mexican sequence, it was found that the ToxA and ToxB genes were closely linked (i.e., separated by 12 nucleotides). From this sequence, primers were designed to amplify the full length ToxA and ToxB genes from CN and 5HP for cloning and sequencing. The results revealed that the sequences from CN and 5HP were identical to those in JALL01000066.1 and that the two Tox genes were also separated by the same 12 nucleotides. After completion of this work, draft sequences of 6 VPAHPND and 4 non-AHPND VP from Thailand were published [11, 12], including the sequence for our 5HP and CN isolates [11]. In all 7 of the VPAHPND isolates listed at GenBank (6 from Thailand and one from Mexico), ToxA and ToxB were present with identical nucleic acid and deduced amino acid sequences, with identical genome arrangements and separated by 12 identical nucleotides, except that the 5’–3’ orientation of the two protein genes in the Mexican record (JALL01000066.1) was opposite to that in all the other records. Neither of these toxin genes were found in the chromosomal DNA of these AHPND isolates or in the total DNA of the non-AHPND isolates examined [7, 11–13].

Bottom Line: In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B.A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium).The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

View Article: PubMed Central - PubMed

Affiliation: Shrimp-virus interaction laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.

ABSTRACT
Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

No MeSH data available.


Related in: MedlinePlus