Frontiers in cellular and infection microbiology · 2026
TM9SF4 acts as a receptor mediating Glaesserella parasuis cytolethal distending toxin-induced cytotoxicity in PK15 cells.
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Lei Li, Xu Shiyu, Yang Zhen, Pan Xingyu, Xu Jiali, Du Senyan, Zhao Qin, Huang Xiaobo, Cao Sanjie, Wu Rui, Wang Yiping, Yan Qigui, Wen Yiping
Аннотация
BACKGROUND: Cytolethal Distending Toxin (CDT) is the only exotoxin that Glaesserella parasuis (G. parasuis) can secrete. G. parasuis CDT (GpCDT) triggers DNA damage responses, leading to irreversible cell cycle arrest and apoptosis, playing an important role in the pathogenic process of G. parasuis. Currently, research on the host cell receptors of GpCDT remains limited. Screening and identification of host cell receptors that interact with GpCDT are crucial for systematically elucidating the cytotoxic mechanisms induced by this toxin.
METHODS: This study employed Co-immunoprecipitation (Co-IP) combined with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to identify potential host proteins interacting with GpCDT in PK15 cells. Nine Proteins were selected for further evaluation based on subcellular localization and Gene Ontology classification. Eukaryotic expression and Co-IP validated four interacting proteins. Subsequently, heterozygous knockout PK15 cell lines for these genes were generated via CRISPR/Cas9, and CCK-8 assays identified TM9SF4 as having the most significant impact on GpCDT virulence. Therefore, a homozygous TM9SF4 knockout PK15 cell line (KO) was generated via limited dilution, and a stable TM9SF4-overexpressing PK15 cell line (OE) was established through lentiviral packaging. Western blotting and qRT-PCR confirmed protein and gene expression, and CCK-8 assays combined with cytopathic effect (CPE) observation determined the role of TM9SF4 in GpCDT-induced cytotoxicity. Finally, indirect immunofluorescence was performed to assess co-localization of TM9SF4 with GpCDT.
RESULTS: We identified 287 proteins in PK15 cells that potentially interact with GpCDT, among which 58 were localized to the plasma membrane or extracellular. Nine proteins were selected for further investigation. Among them, EPHB4, LITAF, TM9SF4, SLC12A4 interacted with GpCDT, but only the deficiency of TM9SF4 significantly inhibited the virulence of the GpCDT. Results from CCK-8 and CPE showed that KO cells exhibited significantly higher survival rates and suppressed GpCDT-induced cellular distention and cell death, whereas OE cells showed decreased survival rates and typical cytopathic change. Finally, indirect immunofluorescence confirmed strong co-localization between TM9SF4 and GpCDT.
CONCLUSION: We initially proposed TM9SF4 as a receptor for GpCDT in PK15 cells, essential for GpCDT binding and cytotoxicity. This study may provide a new theoretical basis for targeted prevention and treatment of swine Glässer's disease.
METHODS: This study employed Co-immunoprecipitation (Co-IP) combined with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to identify potential host proteins interacting with GpCDT in PK15 cells. Nine Proteins were selected for further evaluation based on subcellular localization and Gene Ontology classification. Eukaryotic expression and Co-IP validated four interacting proteins. Subsequently, heterozygous knockout PK15 cell lines for these genes were generated via CRISPR/Cas9, and CCK-8 assays identified TM9SF4 as having the most significant impact on GpCDT virulence. Therefore, a homozygous TM9SF4 knockout PK15 cell line (KO) was generated via limited dilution, and a stable TM9SF4-overexpressing PK15 cell line (OE) was established through lentiviral packaging. Western blotting and qRT-PCR confirmed protein and gene expression, and CCK-8 assays combined with cytopathic effect (CPE) observation determined the role of TM9SF4 in GpCDT-induced cytotoxicity. Finally, indirect immunofluorescence was performed to assess co-localization of TM9SF4 with GpCDT.
RESULTS: We identified 287 proteins in PK15 cells that potentially interact with GpCDT, among which 58 were localized to the plasma membrane or extracellular. Nine proteins were selected for further investigation. Among them, EPHB4, LITAF, TM9SF4, SLC12A4 interacted with GpCDT, but only the deficiency of TM9SF4 significantly inhibited the virulence of the GpCDT. Results from CCK-8 and CPE showed that KO cells exhibited significantly higher survival rates and suppressed GpCDT-induced cellular distention and cell death, whereas OE cells showed decreased survival rates and typical cytopathic change. Finally, indirect immunofluorescence confirmed strong co-localization between TM9SF4 and GpCDT.
CONCLUSION: We initially proposed TM9SF4 as a receptor for GpCDT in PK15 cells, essential for GpCDT binding and cytotoxicity. This study may provide a new theoretical basis for targeted prevention and treatment of swine Glässer's disease.