Abstract
Background: The cytolethal distending toxin (CDT), a significant exotoxin, is closely linked to the pathogenicity of Glaesserella parasuis (GPS), but its pathogenic not yet fully elucidated. Previously, we identified Rab4b as a potential host factor contributing to the cytotoxicity of GpCDT through a whole-genome CRISPR/Cas9 screen technology, and subsequently confirmed its association with GpCDT cytotoxicity in PK-15 cells. Aims: In this study, our data first indicated that Rab4b could interact with the active subunit of the Glaesserella parasuis cytolethal distending toxin. Methods: Investigating the relationship between Rab4b and GpCDT subunits as confirmed by coimmunoprecipitation assay. Next, the porcine alveolar macrophage cell line 3D4/21 was used to establish an infected cell model. Using CRISPR/Cas9 gene editing, we established Rab4b and EEA1-expression-deficient 3D4/21 cell lines. 3D4/21 cells, Rab4b-KO cells and EEA1-KO cells were treated with GpCDT. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Western blotting and qRT-PCR were used to measure the expression of related proteins and genes, and cell morphology observation and indirect immunofluorescence were performed to evaluate the GpCDT-mediated cytotoxicity. Then utilise transcriptome sequencing analysis to investigate its specific mechanisms. Result: In this study, our data first indicated that Rab4b could interact with the active subunit of the GpCDT. Next, we demonstrated that Rab4b also influences GpCDT-induced cytotoxicity and vesicle trafficking in 3D4/21 cells. To investigate the Rab4b-mediated cytotoxicity of GpCDT in 3D4/21 cells, we screened for EEA1, a gene critical in this process, by transcriptome sequencing analysis. 3D4/21 cells exposed to GpCDT exhibit upregulated EEA1 expression, an event that is lost in the absence of Rab4b. Using CRISPR/Cas9 gene editing, we established EEA1 expression-deficient 3D4/21 cell lines that fail to internalize GpCdtB, resulting in resistance to GpCDT-induced toxic effects. Conclusions: We suggest that Rab4b facilitates the cellular uptake of GpCDTby upregulating EEA1 protein expression, thereby facilitating the vesicular transport of GpCDT in 3D4/21 cells. Our findings may provide new insights into the pathogenicity of GpCDT and lay the experimental foundation for a deeper understanding of the role of Rab4b proteins.