Unraveling the Molecular and Physiological Roles of Signal Peptide Peptidase A in Flavobacterium columnare

Columnaris disease, caused by Flavobacterium columnare , represents one of the most economically devastating bacterial infections in global freshwater aquaculture. Despite its significant impact, the molecular mechanisms underlying F. columnare pathogenesis remain largely unexplored due to the challenge in targeted genomic manipulation. Signal peptide peptidase A (SppA) plays a crucial role in bacterial protein secretion by degrading residual signal peptides after protein translocation, yet its function in F. columnare physiology and virulence has not been characterized. Here, we employed a targeted gene deletion approach to investigate the role of sppA in F. columnare . The Δ sppA mutant exhibited pleiotropic phenotypes including increased outer membrane vesicle (OMV) production (3.8-fold higher compared to the wild type), reduced biofilm formation, and loss of gliding motility. Transcriptomic analysis of the Δ sppA mutant revealed significant upregulation of genes involved in membrane stress response and efflux pump system, including algU , osmC and the genes in the MacAB-TolC efflux system, compared to the wild-type state. Importantly, the artificial infection experiment demonstrated the mutant’s significantly attenuated virulence in freshwater Medaka ( Oryzias latipes ), with a 20% higher survival rate of fish compared to the wild type. Our findings reveal that SppA is essential for maintaining membrane homeostasis in F. columnare and serves as one of the virulence factors during columnaris infection. These results provide important insights into the biological function of the sppA gene in F. columnare and highlight the complex relationship between bacterial protein secretion, membrane integrity, and pathogenesis.