TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages
Abstract Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secret...
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| Format: | Article |
| Language: | English |
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BMC
2023-07-01
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| Series: | Veterinary Research |
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| Online Access: | https://doi.org/10.1186/s13567-023-01194-6 |
| _version_ | 1827894954061463552 |
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| author | Yaofang Hu Changsheng Jiang Yueqiao Zhao Hua Cao Jingping Ren Wei Zeng Mengjia Zhang Yongtao Li Qigai He Wentao Li |
| author_facet | Yaofang Hu Changsheng Jiang Yueqiao Zhao Hua Cao Jingping Ren Wei Zeng Mengjia Zhang Yongtao Li Qigai He Wentao Li |
| author_sort | Yaofang Hu |
| collection | DOAJ |
| description | Abstract Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP. |
| first_indexed | 2024-03-12T22:14:46Z |
| format | Article |
| id | doaj.art-41e9189903d04ae4b05cafc364c17abb |
| institution | Directory Open Access Journal |
| issn | 1297-9716 |
| language | English |
| last_indexed | 2024-03-12T22:14:46Z |
| publishDate | 2023-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Veterinary Research |
| spelling | doaj.art-41e9189903d04ae4b05cafc364c17abb2023-07-23T11:22:32ZengBMCVeterinary Research1297-97162023-07-0154111410.1186/s13567-023-01194-6TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophagesYaofang Hu0Changsheng Jiang1Yueqiao Zhao2Hua Cao3Jingping Ren4Wei Zeng5Mengjia Zhang6Yongtao Li7Qigai He8Wentao Li9National Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityNational Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural UniversityAbstract Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.https://doi.org/10.1186/s13567-023-01194-6Actinobacillus pleuropneumoniaeApxI exotoxinTurboID enzyme-catalyzed proximity labeling methodinteracting proteomes |
| spellingShingle | Yaofang Hu Changsheng Jiang Yueqiao Zhao Hua Cao Jingping Ren Wei Zeng Mengjia Zhang Yongtao Li Qigai He Wentao Li TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages Veterinary Research Actinobacillus pleuropneumoniae ApxI exotoxin TurboID enzyme-catalyzed proximity labeling method interacting proteomes |
| title | TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages |
| title_full | TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages |
| title_fullStr | TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages |
| title_full_unstemmed | TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages |
| title_short | TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages |
| title_sort | turboid screening of apxi toxin interactants identifies host proteins involved in actinobacillus pleuropneumoniae induced apoptosis of immortalized porcine alveolar macrophages |
| topic | Actinobacillus pleuropneumoniae ApxI exotoxin TurboID enzyme-catalyzed proximity labeling method interacting proteomes |
| url | https://doi.org/10.1186/s13567-023-01194-6 |
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