Cytotoxicity Modelling of Pasteurella multocida Toxin and Its Histological Study

Abstract

In this study, we investigated the molecular mechanism by which the recombinant multicidal Bartonella toxin rPMT damages PK15 cells. We successfully constructed the prokaryotic expression vector pCold I-toxA and identified suitable expression and purification conditions for rPMT. Using the CCK8 assay, we established a cellular damage model and found that PK15 cells were significantly affected by rPMT infection at a concentration of 20 ug/mL for 24 h. Flow cytometry experiments revealed that rPMT induced apoptosis in PK15 cells. To further understand the underlying mechanism, we prepared a potent murine anti-polyclonal antibody against rPMT and evaluated its effectiveness (potency of 1:1000). In mouse experiments, the LD50 of rPMT was determined to be 0.460 ng/g. Transcriptome sequencing data indicated that rPMT injury to PK15 cells led to elevated expression of inflammation-related pathways and genes. Additionally, QPCR experiments confirmed that rPMT injury significantly upregulated the expression of inflammation-related factors, including NLRP3, IL-1 β , IL-6, IL-8, and TNF- α , compared to normal PK15 cells. In conclusion, the recombinant PMT toxin (rPMT) used in this study exhibited high biological activity and caused significant damage to PK15 cells, possibly through an inflammatory validation effect. These findings shed light on the molecular mechanisms underlying rPMT-induced cellular damage and its potential role in inflammation-related pathways.