Deletion of Salmonella pathogenicity islands SPI-1, 2 and 3 induces substantial morphological and metabolic alternation and protective immune potential

The Salmonella pathogenicity islands (SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs (SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants (SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant (SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates (17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover, the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3 mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3⁺CD4⁺ and CD3⁺CD8⁺ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups. In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic (including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.