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The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

ABSTRACT: Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

SUBMITTER: Lee CT

PROVIDER: S-EPMC4553777 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Lee Chung-Te CT Chen I-Tung IT Yang Yi-Ting YT Ko Tzu-Ping TP Huang Yun-Tzu YT Huang Jiun-Yan JY Huang Ming-Fen MF Lin Shin-Jen SJ Chen Chien-Yu CY Lin Shih-Shun SS Lightner Donald V DV Wang Han-Ching HC Wang Andrew H-J AH Wang Hao-Ching HC Hor Lien-I LI Lo Chu-Fang CF

Proceedings of the National Academy of Sciences of the United States of America 20150810 34

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental ...[more]

PMID: 26261348

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Project description:Translucent post-larvae disease (TPD), caused by Vibrio parahaemolyticus (Vp TPD), has become an emerging shrimp disease, affecting more than 70%-80% of coastal shrimp nurseries in China in spring 2020. Here, we investigated the key virulence factors of Vp TPD by analyzing protein fragments, related genomic information, as well as experimental challenge tests. After investigating the toxic effects of purified protein fragments with different molecular weights (MWs) from Vp TPD, we found that only the protein fragments with MW >100 kDa showed similar lethality to live Vp TPD in the experimental challenge test using post-larvae shrimp. Meanwhile, similar histopathological changes exhibiting in the hepatopancreas and midgut of the diseased individuals were observed in the post-larvae shrimp challenged with either bacterial protein fragments (MW >100 kDa) or live Vp TPD. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, two novel proteins, Vibrio high virulent protein (VHVP)-1 and VHVP-2, were identified as the candidates of key virulence factors to cause TPD. Moreover, VHVP-1 and VHVP-2 were found to be encoded by two genes (vhvp-1 and vhvp-2) tandemly located on a 187,791-bp plasmid and were predicted to depend on the same promoter following a comparative genomic analysis. Further epidemiological investigation and challenge test indicated that the V. parahaemolyticus isolate carrying only the vhvp-1 gene and lacking vhvp-2 gene could not cause mortality of experimental Penaeus vannamei post-larvae. The mutant (Δvhvp-2) by deleting vhvp-2 gene could only cause 4.92% of accumulative mortality of post-larvae that is similar to the non-Vp TPD Vibrio strain. Additionally, the complemented strains, Δvhvp-2/pBT3-vhvp-2 and Δvhvp-2/pwtCas9-vhvp-2, showed similar virulence to the wild-type Vp TPD. The results demonstrated that V. parahaemolyticus becomes lethal to post-larval shrimp by acquiring the VHVP-2 virulence factor. This study sheds light on further investigations of the pathogenic mechanism of Vp TPD and the development of strategies for early diagnosis of TPD in shrimp hatcheries. As a severe emerging shrimp disease, TPD has heavily impacted the shrimp aquaculture industry and resulted in serious economic losses in China since spring 2020. This study aimed to identify the key virulent factors and related genes of the Vp TPD, for a better understanding of its pathogenicity of the novel highly lethal infectious pathogen, as well as its molecular epidemiological characteristics in China. The present study revealed that a novel protein, Vibrio high virulent protein-2 (MW >100 kDa), is responsible to the lethal virulence of V. parahaemolyticus to shrimp post-larvae. The results are essential for effectively diagnosing and monitoring novel pathogenic bacteria, like Vp TPD, in aquaculture shrimps and would be beneficial to the fisheries department in early warning of Vp TPD emergence and developing prevention strategies to reduce economic losses due to severe outbreaks of TPD. Elucidation of the key virulence genes and genomics of Vp TPD could also provide valuable information on the evolution and ecology of this emerging pathogen in aquaculture environments.

Project description:ObjectivesThe purpose of this study was to determine the structural features and transferability of the multidrug-resistance (MDR) plasmid, and resistance phenotypes for the tested antimicrobials in foodborne Vibrio parahaemolyticus.MethodsPlasmids were isolated from a V. parahaemolyticus strain of seafood origin, then sequenced using the Illumina NovaSeq 6000 and PacBio Sequel II sequencing platforms to obtain the complete genome data. Characterization of the MDR plasmid pVP52-1, including determination of antimicrobial resistance genes (ARGs), plasmid incompatibility groups, and transferability, was carried out.ResultsV. parahaemolyticus strain NJIFDCVp52 contained two circular chromosomes and two circular plasmids (pVP52-1 and pVP52-2). Plasmid typing indicated that pVP52-1 belonged to the incompatibility group IncA/C2 and the sequence type pST3. pVP52-1 carried 12 different ARGs, an IS110-composite transposon consisting of aac(6')-Ib-cr, qnrVC1, aac(6')-Ib, dfrA14, and the IS26-mphA-IS6100 unit flanked by inverted sequences of IS5075 and IS4321. pVP52-2 carried no ARGs. A plasmid elimination assay showed that only pVP52-1 and its ARGs were lost, the loss of resistance to several antimicrobials, causing a change from the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-trimethoprim/sulfamethoxazole resistance pattern to the ampicillin resistance pattern. In accordance, a conjugation transfer assay showed that only pVP52-1 and its ARGs were horizontally transferred, leading to increased antimicrobial resistance in Escherichia coli strain EC600, causing a change from the ampicillin-nalidixic acid resistance pattern to the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-nalidixic acid-chloramphenicol-tetracycline-trimethoprim/sulfamethoxazole-azithromycin resistance pattern. Further transferability experiments revealed that pVP52-1 could be transferred to other enterobacterial strains of E. coli and Salmonella.DiscussionThis study emphasizes the urgent need for continued surveillance of resistance plasmids and changes in antimicrobial resistance profiles among the V. parahaemolyticus population.

Project description:Our ability to predict evolutionary trajectories of pathogens is one of the promising leverages to fight against the pandemic disease, yet few studies have addressed this question in situ, due to the difficulty in monitoring the milestone evolutionary events for a given pathogen and in understanding the evolutionary strategies. In this study, we monitored the real-time evolution of Vibrio parahaemolyticus in response to successive antibiotic treatment in three shrimp farms in North China from 2011 to 2018 by whole-genome sequencing. Results showed that the stepwise emergence of resistance was associated with the antibiotic usage. Genomic analysis of resistant isolates showed that the acquisition of the resistant mobile genetic elements flanked by an insertion sequence (ISVal1) closely mirrored the antibiotics used in shrimp farms since 2014. Next, we also identified 50 insertion sites of ISVal1 in the chromosome, which facilitated the formation of pathogenicity islands (PAIs) and fitness islands in the following years. Further, horizontal transfers of a virulent trh-nik-ure genomic island (GI) and two GIs improving the fitness have been observed in two farms since 2016. In this case study, we proposed that the insertion sequence triggered four major evolutionary events during the outbreaks of shrimp disease in three farms, including horizontal transfer of transposon (HTT) (stage 1), the formation of resistance islands (stage 2) and the PAIs (stage 3), and horizontal transfer of the PAIs (stage 4). This study presented the first in vivo evolutionary trajectories for a given bacterial pathogen, which helps us to understand the emergence mechanisms of new genotypes.IMPORTANCE Most human infectious diseases originate from animals. Thus, how to reduce or prevent pandemic zoonoses before they emerge in people is becoming a critical issue. Continuous genomic surveillance of the evolutionary trajectories of potential human pathogens on farms is a promising strategy to realize early warning. Here, we conducted an 8-year surveillance of Vibrio parahaemolyticus in three shrimp farms. The results showed that the use of antibiotics and horizontal transfer of transposons (HTT) drove the evolution of V. parahaemolyticus, which could be divided into four stages: HTT, formation of resistance islands, formation of pathogenicity islands (PAIs), and horizontal transfer of PAIs. This study presented the first in vivo monitoring of evolutionary trajectories for a given bacterial pathogen, providing valuable information for the prevention of pandemic zoonoses.

Dataset Information

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Publications

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

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