Roles of SPI-2 T3SS Effectors in virulence of Salmonella Choleraesuis and Construction of a triplegene mutant vaccine strain

Rui Xu^1,2,3,4Xiangfei Ji^1,2,3,4Junqi Lian⁵Dekang Zhu^1,2,3,4Mafeng Liu^1,2,3,4Mingshu Wang^1,2,3,4Renyong Jia^1,2,3,4Shun Chen^1,2,3,4Qiao Yang^1,2,3,4Ying Wu^1,2,3,4Shaqiu Zhang^1,2,3,4Juan Huang^1,2,3,4Xumin Ou^1,2,3,4Di Sun^1,2,3,4Tian Bin^1,2,3,4Yu He^1,2,3,4Zhen Wu^1,2,3,4Anchun Cheng^1,2,3,4*Xinxin Zhao^6*

• ¹Research Center of Avian Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, China
• ²Institute of Veterinary Medicine and Immunology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, China
• ³Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China
• ⁴Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China, Chengdu, Sichuan, China
• ⁵Hulunbuir Agricultural and Livestock Product Quality and Safety Center, Hulunbuir, Inner Mongolia, China
• ⁶Sichuan Agricultural University, Ya'an, China

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, sopD2, steC, and steD contributed to bacterial survival in macrophage cell line RAW264.7; whereas only sopD2 also promoted bacterial virulence in mice like other three effector genes sseL, steA and spiC. The mutant strain, ΔsopD2, ΔsseL, ΔsteA, or ΔspiC, 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 ΔspiC that was undetectable in mouse tissues. Then, the triple-gene mutant strain ΔsseLΔsopD2ΔsteA 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.