Roles of SPI-2 T3SS effectors in virulence of Salmonella Choleraesuis and Construction of a triple-gene mutant vaccine strain

Effector protein functions of Type III secretion system (T3SS) encoded by Salmonella pathogenicity islands 2 (SPI-2) have not been fully characterized in Salmonella enterica serovar Choleraesuis. This study characterized 21 effectors of SPI-2 T3SS of S. Choleraesuis in terms of macrophage survival and virulence in mice via construction of various gene mutant strains. Eight effector genes including sseF, sseJ, sifB, sseK, sifA, sopD 2 , steC , and steD contributed to bacterial survival in macrophage cell line RAW264.7; whereas only sopD 2 also promoted bacterial virulence in mice like other three effector genes sseL, steA , and spiC . The mutant strain, Δ sopD 2 , Δ sseL , Δ steA , or Δ spiC , led to higher mouse survival compared to the wild-type strain post-oral infection, while their bacterial loads in spleen and liver were not reduced except the Δ spiC that was undetectable in mouse tissues. Then, the triple-gene mutant strain Δ sseL Δ sopD 2 Δ steA was constructed and found to be virulence attenuated with a compromised colonization ability. Finally, immunization of this mutant orally induced robust serum IgG responses and provided 40% protection against lethal S. Choleraesuis challenge. Our study highlights the critical role of four SPI-2 T3SS effectors in S. Choleraesuis pathogenesis.