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 <i>Salmonella</i> pathogenicity islands 2 (SPI-2) have not been fully characterized in <i>Salmonella enterica</i> serovar Choleraesuis. This study characterized 21 effectors of SPI-2 T3SS of <i>S. Choleraesuis</i> in terms of macrophage survival and virulence in mice via construction of various gene mutant strains. Eight effector genes including <i>sseF, sseJ, sifB, sseK, sifA, sopD</i> ₂, <i>steC</i>, and <i>steD</i> contributed to bacterial survival in macrophage cell line RAW264.7; whereas only <i>sopD</i> ₂ also promoted bacterial virulence in mice like other three effector genes <i>sseL, steA</i>, and <i>spiC</i>. The mutant strain, Δ<i>sopD</i> ₂, Δ<i>sseL</i>, Δ<i>steA</i>, or Δ<i>spiC</i>, 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<i>spiC</i> that was undetectable in mouse tissues. Then, the triple-gene mutant strain Δ<i>sseL</i>Δ<i>sopD</i> ₂Δ<i>steA</i> 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 <i>S. Choleraesuis</i> challenge. Our study highlights the critical role of four SPI-2 T3SS effectors in <i>S. Choleraesuis</i> pathogenesis.