| Salmonella is a facultative intracellular gram-negative pathogen. Up to date, over 2,600 serovars of Salmonella have been identified. Among them, Salmonella enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) infects specifical host which are mainly young chickens within 20 days old, inducing white diarrhea, causing high morbidity and mortality. Adult chickens can be infected with no clinical symptoms, and become carriers of Salmonella Pullorum. The more serious problem is that Salmonella Pullorum can transmit vertically by ovary and eggs, in addition with horizontal transmission. Although Salmonella Pullorum have basically been eliminated in some developed countries, it is still popular in developing countries such as China and other countries, which causes serious economic losses to poultry husbandry.Salmonella pathogenic capacities of invasion to the cells and its intracellular survival are mainly related to Salmonella pathogenicity island (SPI), which encoded type III secretion system (T3SS) and its effectors. Previous studies on T3SS were mainly on the model of Salmonella Enteritidis or Salmonella Typhimurium infecting mammals but little on the other models. Because of the specificity of Salmonella Pullorum on host adaption, non-flagella, and so on, the study of genes encoding T3SS effectors in Salmonella Pullorum is promising and also very limited. In this study, the model of Salmonella Pullorum infecting avian HD-11 cells is used to research T3SS effectors. Salmonella Pullorum strain S06004 sseL gene-deletion strain, its complemented strain and sseL eukaryotic expressed plasmids were successfully constructed. T3SS effctor SseL of S06004 on the function of regulating bacterial virulence and inhibiting celluar inflammatory response have been preliminarily studied.1 Construction and virulent evaluation of sseL gene-deletion strain of Salmonella PullorumIn this study, the sseL gene-deletion strain of Salmonella Pullorum strain S06004 was constructed using λ-Red recombination system, and named as S06004ΔsseL. And its complementary strain S06004ΔsseL::sseL was developed as well. The sseL gene was successfully knocked out in S06004Δsse identified by the PCR test and gene sequencing. There was no significant difference between S06004ΔsseL, S06004ΔsseL::sseL and their parent strain S06004 in their growth and biochemistry characteristics.The result of virulence test of S06004ΔsseL and its parent strain demonstrated that the LD50 of S06004AsseL was 7.6×108 CFU, which rose about 60 folds compared to the LD50 of its parent strain (1.9×107 CFU), in the model of chicken being infected by intramuscular injection. The avian macrophages HD-11 were used as the cell model to investigate the characterization of S06004ΔsseL. The results showed that the adhesion and the invasion rate of S06004ΔsseL had no significant difference compared to those of S06004. However, its proliferation was significantly declined (about 2 folds) at 5 and 10 hours post infection compared to that of S06004 (P<0.05) in HD-11. Live cells rate at 10 h after infected by S06004ΔsseL was 69.3%, higher than that of HD-11 infected by S06004 (P<0.05). The results of competitive index (C.I.) in vitro and in vivo showed that the C.I. of S06004ΔsseL were weaker than those of the parent strain S06004.2 Preliminary study on cellular NF-κB imflammatory pathway inhibited by Salmonella Pullorum effector SseLAfter HD-11 cells infected by S06004ΔsseL and the parent strain S06004, total RNA of the HD-11 cells was extracted at 8 hours post infection, the expression levels of relative cytokines IL-1β, IL-6, IL-4, IFN-γ, CXCLi2, CCLi2 NKAP, TANK MUL1, NKIRA were detected by RT-PCR. The results showed that, HD-11 cells infected by S06004ΔsseL had higher expression levels of pro-inflammatory factors, such as IL-4, IFN-γ, CXCLi2, CCLi2, and NF-κB related cytokines, such as NKAP, TANK. In addition, the eukaryotic expression plasmid pcDNA3.1-SseL-flag was constructed and transfected to HEK293 cells. Western-blot assay showed that pcDNA3.1-SseL-flag had been expressed in HEK293, which size was about 37 KDa. After transfected with plasmid pcDNA3.1-SseL-flag, pCMV-TRL4, pCMV-MD2 and NF-κB luciferase reportor, HEK293 cells were then stimulated with LPS. The expression of S06004 SPI-2 effector SseL could suppress the activation of cellular NF-κB inflammatory pathway about 2-3 folds, comparing with negative control. The function of SseL proteins from Salmonella Pullorum and Salmonella Typhimurium on NF-κB signaling pathway is almost no difference.In conclusion, Salomella Pullorum T3SS effector SseL coding gene sseL was a very important virulence gene. Besides, SseL as a deubiquitin enzyme could suppress the activation of cellular NF-κB inflammatory pathway and then suppress inflammatory response. |
PDF Full Text Request