Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

Kyung Ku Jang; Zee Won Lee; Bityeoul Kim; Young Hyun Jung; Ho Jae Han; Myung Hee Kim; Byoung Sik Kim; Sang Ho Choi

doi:10.1074/jbc.M117.791657

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A₂ essential for pathogenesis

Kyung Ku Jang, Zee Won Lee, Bityeoul Kim, Young Hyun Jung, Ho Jae Han, Myung Hee Kim, Byoung Sik Kim, Sang Ho Choi

Food Science and Biotechnology

Research output: Contribution to journal › Article › peer-review

32 Scopus citations

Abstract

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A₂ secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase– dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA. Molecular biological analyses revealed that plpA expression is controlled via the promoter, P_plpA, and that HlyU and CRP directly bind to P_plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A₂ regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

Original language	English
Pages (from-to)	17129-17143
Number of pages	15
Journal	Journal of Biological Chemistry
Volume	292
Issue number	41
DOIs	https://doi.org/10.1074/jbc.M117.791657
State	Published - 13 Oct 2017

Bibliographical note

Funding Information:
This work was supported by National Research Foundation Mid-career Researcher Program Grant 2015R1A2A1A13001654 from the Ministry of Science, ICT, and Future Planning and the R&D Convergence Center Sup-port Program of the Ministry of Agriculture, Food, and Rural Affairs, Repub-lic of Korea (to S. H. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Tables S1 and S2. † Deceased November 2, 2015. 1To whom correspondence may be addressed. Tel.: 82-42-879-8212; E-mail: bskim@kribb.re.kr. 2To whom correspondence may be addressed. Tel.: 82-2-880-4857; E-mail: choish@snu.ac.kr.

Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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10.1074/jbc.M117.791657

Cite this

@article{67491e7908c544999e750b7e5775d984,

title = "Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis",

abstract = "The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase– dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA. Molecular biological analyses revealed that plpA expression is controlled via the promoter, PplpA, and that HlyU and CRP directly bind to PplpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.",

author = "Jang, {Kyung Ku} and Lee, {Zee Won} and Bityeoul Kim and Jung, {Young Hyun} and Han, {Ho Jae} and Kim, {Myung Hee} and Kim, {Byoung Sik} and Choi, {Sang Ho}",

note = "Funding Information: This work was supported by National Research Foundation Mid-career Researcher Program Grant 2015R1A2A1A13001654 from the Ministry of Science, ICT, and Future Planning and the R&D Convergence Center Sup-port Program of the Ministry of Agriculture, Food, and Rural Affairs, Repub-lic of Korea (to S. H. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Tables S1 and S2. † Deceased November 2, 2015. 1To whom correspondence may be addressed. Tel.: 82-42-879-8212; E-mail: bskim@kribb.re.kr. 2To whom correspondence may be addressed. Tel.: 82-2-880-4857; E-mail: choish@snu.ac.kr. Publisher Copyright: {\textcopyright} 2017 by The American Society for Biochemistry and Molecular Biology, Inc.",

year = "2017",

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T1 - Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

AU - Jang, Kyung Ku

AU - Lee, Zee Won

AU - Kim, Bityeoul

AU - Jung, Young Hyun

AU - Han, Ho Jae

AU - Kim, Myung Hee

AU - Kim, Byoung Sik

AU - Choi, Sang Ho

N1 - Funding Information: This work was supported by National Research Foundation Mid-career Researcher Program Grant 2015R1A2A1A13001654 from the Ministry of Science, ICT, and Future Planning and the R&D Convergence Center Sup-port Program of the Ministry of Agriculture, Food, and Rural Affairs, Repub-lic of Korea (to S. H. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Tables S1 and S2. † Deceased November 2, 2015. 1To whom correspondence may be addressed. Tel.: 82-42-879-8212; E-mail: bskim@kribb.re.kr. 2To whom correspondence may be addressed. Tel.: 82-2-880-4857; E-mail: choish@snu.ac.kr. Publisher Copyright: © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2017/10/13

Y1 - 2017/10/13

N2 - The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase– dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA. Molecular biological analyses revealed that plpA expression is controlled via the promoter, PplpA, and that HlyU and CRP directly bind to PplpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

AB - The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase– dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA. Molecular biological analyses revealed that plpA expression is controlled via the promoter, PplpA, and that HlyU and CRP directly bind to PplpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

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