Development of a GFP-labeled Pasteurella multocida strain for in vivo organ infection tracing using a stable RCR-based genetic manipulation plasmid

Advances in microscopy have enabled the widespread application of in vivo pathogen tracking approaches to bacterial pathogenesis. Pasteurella multocida ( P. multocida ) is a major zoonotic pathogen causing severe respiratory diseases in livestock. However, genetic manipulation of clinical swine-derived isolates remains hindered by the lack of stable genetic tools. As a result, no genetically engineered in vivo tracking strain limits research in P. multocida infection dynamics. In this study, we evaluated diverse replication mechanisms and identified that rolling circle replication (RCR)-type replicons exhibit superior transformation robustness in swine isolates. Leveraging the temperature-sensitive pSET4s backbone and an optimized mpheS counter-selection marker, we developed a high-efficiency, markerless genetic manipulation system. The system exhibited high copy numbers and significantly enhanced transformation yields across diverse clinical swine isolates. To validate gene knockout efficiency, we performed markerless deletions of the capsule biosynthesis genes hyaD and hyaE . Phenotypic characterization revealed that hyaluronic acid (HA) deficiency led to complete attenuation of virulence in a murine model. Furthermore, we engineered a 15-Pm::GFP reporter strain via site-specific gene knock-in without detectable effects on bacterial growth or virulence. This strain enables direct visualization of bacterial colonization in the lung and liver by fluorescence imaging and allows real-time, in situ monitoring of pathogen and Kupffer cell interactions. Collectively, our results established an effective genetic toolkit for swine-derived P. multocida and developed fluorescently labeled strains for in vivo tracking. This plasmid provides a useful tool for investigating pathogenic mechanisms and in vivo infection dynamics of P. multocida .

IMPORTANCE

P. multocida is a critical threat to global animal health, while the difficulty of genetic modification in swine isolates has interrupted the link of the genomic and pathogenic phenotypes. This study provides a highly efficient, RCR-based markerless manipulation system optimized with cross-serogroup applicability for swine isolates. Validated across 15 clinical swine isolates, this plasmid offers a robust tool for genetic analysis and the rational design of next-generation vaccines. By defining the essential roles of hyaD and hyaE , we demonstrate that the hyaluronic acid capsule is required for virulence and pulmonary colonization. Moreover, the development of a biologically fluorescent reporter strain enables high-resolution, in situ tracking of infection dynamics, providing a powerful tool for real-time visualization during P. multocida infection.