Construction And Biological Characteristic Of Salmonella Enteritidis Mutants With Deletion Of Genes Encoding T3SS Effectors

Salmonella enteritidis are a common zoonosis pathogens, which can infect both human and animals. Type Three Secretion System(T3SS) encoded by SPI-1and SPI-2is an important virulence factor for Salmonella’s pathogenicity. Different T3SS effectors play different roles in pathogenic process. Currently, the role of T3SS effectors is investigated based on the model of S. typhimurium, however, their role may be different in different serotype of Salmonella. In this study, we selected6T3SS effectors of S. Enteritidis, constructed six deletion mutants by using the method of homologous recombination, and investigated the relationship between these effectors and pathogenicity. This study lays a foundation for further studying the molecular mechanism of these effectors in the process of S. Enteritidis infection.1. Prokaryotic expression and preparation of polyclonal antibody against T3SS effectors of S. Enteritidis.SspH2, SlrP and SseL genes were amplified by PCR, and cloned into two kinds of prokaryotic expression vetors pET-32a(+) and pGEX-6p-1, respectively. The recombinant plasmids pET-32a-SspH2, pET-32a-SlrP, pET-32a-SseL, pGEX-6p-1-SspH2, pGEX-6p-1-SlrP and pGEX-6p-1-SseL were transformed into E.Coli BL21and the expressed recombinant proteins were induced. SDS-PAGE analysis showed that the six recombinant proteins were expressed in predicted sizes. Most of the recombinant proteins were found in the supernatant of bacterial lysates, indicating they were soluble proteins. The purified fusion proteins with GST tag were injected into rabbits subcutaneously to produce polyclonal antibodies.The titers of the antisera were determined by indirect enzyme-linked immunosorbent assay using the fusion proteins with His tag as detecting antigen, The results showed that the antisera with high titer were acquired two weeks after the third vaccination.2. Construction of S. Enteritidis mutants with deletion of genes encoding T3SS effectorsA S. Enteritidis strain C50041was selected as parental strain, and six mutants with deficiency of genes including SspH2, PipB, SseL, SopE2, SlrP and SipB were constructed by the Red recombination system. Six deletion mutants were confirmed by PCR amplification and sequence analysis. The expression of the proteins SspH2, SseL, and SlrP in the related deletion strains were further comfirmed by Western-blot. The result showed that the corresponding proteins of ΔSspH2, ΔSseL and ΔSlrP were not detectable, but theses proteins were dectectd in the other gene deletion mutants. Growth assay and bactericidal assay of sera indicated that deficiency of the six effectors had no significant effect on the growth of the mutants and the surface structures and permeability.3. The role of S. Enteritidis T3SS effectors on pathogenicityThe adhesion and invasion in HeLa cells, and proliferation in macrophages of the SspH2, PipB, SseL, SopE2, SlrP and SipB mutants were determined. The results showed that percentage of adhesion and invasion of HeLa cells by ΔSipB mutant strain was significantly decreased compared with the wild-type strain, and the ΔSseL mutant showed higher adherence ratio and invasion ratio in HeLa cells than the wild-type strain. Nevertheless, there was no significant difference in proliferation ration between the mutants and wild strain in macrophage cell line HD11. The expression levels of cytokine in HeLa cells after the infection of bacterials at different times were determined by q-PCR. The results revealed that there was no significant difference in the expression levels of IFN-γ, TNF-α and IL-18, but IL-8expression induced by ΔSspH2mutant was significantly higher than that induced by wild-type strain, and the IL-1β expression induced by ΔPipB mutant was significantly increased at6h after infection. To determine the LD50,1-day-old SPF chickens were challenged intraperitoneally with wild-type strain and six mutants. The LD50of ΔPipB, ΔSopE2and ΔSspH2were lower than that of wild-type strain, especially in ΔSspH2group, while the LD50of ΔSipB, ΔSseL and ΔSlrP were higher than wild-type strain, especially in ΔSipB group.In summary, the S. Enteritidis mutants with deletion of genes encoding6T3SS effectors were constructed successfully. Defeciency of effectors SipB and SlrP resulted in the attenuation of virulence in S. Enteritidis, defeciency of effectors SspH2and PipB resulted in the enhance of virulence in S. Enteritidis, and defeciency of effectors SseL and SopE2had no significant effect on virulence in S. Enteritidis. The roles of effectors PipB, SspH2, SseL and SopE2in S. Enteritidis were different from that in S. typhimurium.