TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells
ABSTRACT Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser’s disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fus...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-10-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02307-22 |
| _version_ | 1811329689028919296 |
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| author | Changsheng Jiang Ningning Ma Hua Cao Wei Zeng Jingping Ren Yaofang Hu Jiaru Zhou Mengjia Zhang Chang Li Yifei Lang Wentao Li Qigai He |
| author_facet | Changsheng Jiang Ningning Ma Hua Cao Wei Zeng Jingping Ren Yaofang Hu Jiaru Zhou Mengjia Zhang Chang Li Yifei Lang Wentao Li Qigai He |
| author_sort | Changsheng Jiang |
| collection | DOAJ |
| description | ABSTRACT Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser’s disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser’s disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser’s disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms. |
| first_indexed | 2024-04-13T15:48:21Z |
| format | Article |
| id | doaj.art-24ae98602615483d93a8abf7276055bf |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-13T15:48:21Z |
| publishDate | 2022-10-01 |
| publisher | American Society for Microbiology |
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| series | Microbiology Spectrum |
| spelling | doaj.art-24ae98602615483d93a8abf7276055bf2022-12-22T02:40:54ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-10-0110510.1128/spectrum.02307-22TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM CellsChangsheng Jiang0Ningning Ma1Hua Cao2Wei Zeng3Jingping Ren4Yaofang Hu5Jiaru Zhou6Mengjia Zhang7Chang Li8Yifei Lang9Wentao Li10Qigai He11State Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaKey Laboratory of Prevention and Control Agents for Animal Bacteriosis, Ministry of Agriculture and Rural Affairs, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, ChinaSwine Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaState Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Veterinary Medicine, Huazhong Agricultural University, Wuhan, ChinaABSTRACT Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser’s disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser’s disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser’s disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms.https://journals.asm.org/doi/10.1128/spectrum.02307-22Glaesserella parasuisouter membrane protein P2 (OmpP2)proximity labelingTurboIDinteracting proteomes |
| spellingShingle | Changsheng Jiang Ningning Ma Hua Cao Wei Zeng Jingping Ren Yaofang Hu Jiaru Zhou Mengjia Zhang Chang Li Yifei Lang Wentao Li Qigai He TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells Microbiology Spectrum Glaesserella parasuis outer membrane protein P2 (OmpP2) proximity labeling TurboID interacting proteomes |
| title | TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells |
| title_full | TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells |
| title_fullStr | TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells |
| title_full_unstemmed | TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells |
| title_short | TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells |
| title_sort | turboid screening of the ompp2 protein reveals host proteins involved in recognition and phagocytosis of glaesserella parasuis by ipam cells |
| topic | Glaesserella parasuis outer membrane protein P2 (OmpP2) proximity labeling TurboID interacting proteomes |
| url | https://journals.asm.org/doi/10.1128/spectrum.02307-22 |
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