Pleiotropic Effects of Signal Peptide Peptidase A Gene Deletion on Membrane Homeostasis, Gliding Motility, and Virulence 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. 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 in-frame gene knockout approach to investigate the role of sppA in F. columnare. The ΔsppA mutant exhibited pleiotropic phenotypes including increased outer membrane vesicle (OMV) production (3.8-fold higher compared to the wild type), loss of gliding motility, enhanced efflux pump activity at the transcriptomic level, and reduced virulence. Transcriptomic profiling of the ΔsppA mutant revealed significant upregulation of genes involved in membrane stress response, including the genes in the MacAB-TolC efflux system, algU, and osmC, compared to the wild-type state. Importantly, survival assays demonstrated its virulence was significantly attenuated 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 and normal cellular physiology 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 its role in bacterial protein secretion, membrane homeostasis, and pathogenesis.