Mutations in the clpS Gene of Riemerella anatipestifer Affect Stress Response and Bacterial Virulence.

The Clp protease regulates antibiotic resistance, biofilm formation, stress response, and virulence in microorganisms through a post-transcriptional mechanism. Substrate recognition by the ClpS subunit is essential for the target protein degradation. However, the function of ClpS in Riemerella anatipestifer (R. anatipestifer) remains unclear. In this study, a clpS gene (B739_1609) in-frame deletion strain of R. anatipestifer was constructed using homologous recombination. The ΔclpS gene mutant exhibited no influence on the growth rate and shape of R. anatipestifer. However, the survival rate was significantly lower than that of the parent strain under different conditions, including 42 °C, hydrogen peroxide (10 mM), hydrochloric acid (20 mM), and iron deficiency (10 mM 2'2-dipyridyl). Moreover, R. anatipestifer can activate transcription of the clpS under heat and oxidative stress. The ΔclpS mutant strain exhibited defects in adhesion and invasion of RAW264.7 cells, as well as reduced pathogenicity in ducklings. Additionally, RNA-seq analysis revealed significant changes in the expression of 77 genes in the ΔclpS mutant strain, with 63 genes upregulated and 14 genes downregulated. The differentially expressed genes were primarily clustered in the following metabolic pathways: Global and Overview Maps, Energy Metabolism, Carbohydrate Metabolism, Metabolism of Cofactors and Vitamins, Glycan Biosynthesis and Metabolism, and Nucleotide Metabolism. These results demonstrate that clpS is involved in the stress response and virulence of R. anatipestifer. Moreover, these results can serve as a reference for understanding the molecular pathogenic mechanisms of R. anatipestifer.