PLO genomic diversity underpins differential immunogenicity of Trueperella pyogenes strains from deer and swine

Trueperella pyogenes (T. pyogenes), an opportunistic pathogen, colonizes animal mucosal membranes (respiratory, genitourinary, gastrointestinal) and globally causes systemic infections including endometritis and pneumonia. Its primary virulence factor, pyolysin (PLO), has been extensively studied to elucidate the pathogen's mechanisms and to develop vaccines, yet effective prevention strategies have not been achieved. This study characterized two T. pyogenes strains isolated from sika deer (D-T. pyogenes) and swine (S-T. pyogenes) through comparative genomics and immunological analyses. Whole-genome sequencing revealed significant genetic divergence in the plo gene, with 85 nucleotide differences (94.7% identity) and 19 amino acid substitutions (96.4% identity) between the strains. The S-T. pyogenes strain possessed unique virulence factors, including nutritional toxicity and specialized secretion systems, which may explain its enhanced virulence in murine models. Immunization with prokaryotically expressed recombinant PLO proteins (D-rPLO and S-rPLO) elicited robust humoral and cellular immune responses in mice. While D-rPLO induced faster antibody production and higher survival rates post-challenge, S-rPLO triggered stronger pro-inflammatory cytokine responses (IL-2, TNF-α) but conferred inferior protection, likely due to immune evasion associated with its virulence-related epitopes. Flow cytometry analysis revealed a predominant increase in the CD4⁺/CD8⁺ T cell ratio, highlighting Th1-mediated immunity as critical for pathogen clearance. Histopathological examination correlated D-rPLO's superior efficacy with reduced tissue damage, suggesting that host-specific variations in the plo gene influence antigenic epitope recognition. These findings highlight host-driven adaptations shaping bacterial immunogenicity and PLO's functional diversity, advancing T. pyogenes pathogenesis understanding and guiding multi-antigen vaccine design targeting conserved epitopes for balanced efficacy.