Host factor Rab4b mediates Internalization and Intoxication of 3D4/21 cells by the Active Subunit of the Glaesserella parasuis Cytolethal Distending Toxin via influencing EEA1 expression

Yiwen Zhang¹Zhen Yang^1,2Senyan Du¹QIN ZHAO¹Xiaobo Huang¹Rui Wu¹Yiping Wang¹Qigui Yan¹Sanjie Cao¹Yiping Wen^1*

• ¹Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
• ²ChongQing Academy of Animal Sciences, Chongqing, China

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. In this study, our data first indicated that Rab4b could interact with the active subunit of the Glaesserella parasuis cytolethal distending toxin. Next, the porcine alveolar macrophage cell line 3D4/21 was used to establish an infected cell model, demonstrating that Rab4b also influences GpCDT from Glaesserella parasuis-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. We suggest that Rab4b facilitates the cellular uptake of GpCDT by 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.