Function Analysis Of Fimbriae Gene SefA And FimA In Salmonella Enteritidis

Salmonella enteritidis(S.enteritidis) is a non-host-adapted, gram-negative facultative intracellular pathogen of animals and human beings with the ability to colonize various niches in diverse hosts. Most of them have flagella and can be motile. In recent years, with the development of the poultry industry, S. enteritidis infections are becoming more and more serious. They cause serious economic loss and have important significance in public health. During its pathogenic progress, various S. enteritidis fimbriae are among the major virulence factors which can be expressed both in vivo and in vitro. SEF14fimbriae are only expressed in S. enteritidis and closely related S. enterica serovars, SEF21fimbriae are expressed in all salmonella. The objective of this study is aimed at working out the specific roles of S. enteritidis fimbriae SEF14and SEF21during its pathogenic process using wild-type50336and its SEF14and SEF21deletion mutants, as well as their corresponding complementary strains by means of in vitro cell adhesion, expression, and infection experiments conducted on susceptible animals, to provide scientific and technical basis for further study.1Expression condition Analysis of S. enteritidis fimbriae in different culture methodsTo further explore the optimized expression condition of SEF14and the effect of the major subunit gene of SEF14and SEF21on the expression of other fimbriae or flagella. In this work, we detected the expression of SEF14and SEF21fimbriae in wild-type Salmonella enteritidis50336by qRT-PCR as well as the other fimbriae and flagella of mutants50336ΔSefA,50336ΔfimA,50336ΔsefAΔfimA. Based on the different culture methods, static culture at37℃or42℃and fimbriae induce condition(37℃, CFA culture,60h), we found that S. enteritidis wild-type strain expressed13.8-fold more sufficient sefA, the major subunit gene of SEF14fimbriae, in the induce condition(37℃, CFA culture,60h) than the control condition. The expression of the fimA, the major subunit gene of SEF21fimbriae, was the same as the control condition. In the static culture at42℃, the expression of sefA and fimA are5.8and6.1-fold more than the control condition, respectively. There were also have different between the single and double deletion mutants for the other fimbriae and flagella subunit gene expression. In the fimbriae induce condition, the expression of sefA,fimA and fliC in mutant strain50336ΔsefA are2.7,1.5,9.5-fold more than the wild type50336, the expression of sefA, afgA and fliC in mutant strain50336ΔfimA are4.7,0.6,1.6-fold more than the wild type50336, the expression of afgA, fliCA in mutant sarain50336ΔsefAΔfimA are1.04,6.2-fold more than the wild type50336, respectively. In the static culture at42℃, the expression of sefA, fimA and fliC in mutant strain50336ΔsefA are0.2,2.2,1.3-fold more than the wild type50336, the expression of sefA, afgA and fliC in mutant strain50336ΔfimA are1.7,1.9,1.4-fold more than the wild type50336, the expression of afgA, fliCA in mutant sarain50336ΔsefAΔfimA are3.6,2.2-fold more than the wild type50336, respectively. The data showed that SEF14, SEF21fimbriaes single or double deletion mutants promote the other fimbriae or flagella gene expression. We found that the expression of different adhension factors(including flagella) is complementary in different culture methods, and the deletion of adhesion factor will enhance others compensatory expression. It has been emphasized that much research work needs to be done in the field of the mechanism.2Studies on adhesion of SEF14and SEF21fimbriae of S. enteritidis with intestinal epithelial cells IPEC-J2and Caco-2In this part, about the role of SEF14and SEF21fimbriae in the pathogenesis of S. enteritidis, the adhesion ability between the ntestinal epithelial cell models (IPEC-J2, Caco-2) and S.enteritidis wild-strain50336, mutant strains50336AsefA,50336ΔfimA,50336ΔsefΔAfimA, and the complemented strains50336ΔsefA(pBRA),50336ΔfimA(pACYCF),50336ΔsefAΔfimA (pBRA+pACYCF) were tested. The result showed adhesion ability for IPEC-J2cells was2-4folds more than Caco-2when incubated1h and4h. The cell model tests with two different cell lines show that the number of binding cells foe the double mutant50336ΔsefAΔfimA was less than the wild-type50336significantly. Compared to the wild strain, the number of double mutant strain50336ΔsefAΔfimA for binding cells of IPEC-J2at1h or Caco-2at4h incubation reached a significant level. However, the single deletion strains50336ΔsefA and50336ΔfimA had no significant difference compared to the wild type. The results showed that there exist synergistic interactions among adhesion factors and fimbriae, albeit, there are a single factor (i.e.SEF14or SEF21fimbriae) effect on the adhesion between S. enteritidis strain and the intestinal epithelial cell. Caco-2and IPEC-J2cells are two important intestinal epithelial cell lines from different resources, both of them are cultivated as monolayers on plastic surfaces and used for the research of the pathogenesis in S. enteritidis. But in vitro experiment, the cell models cannot be completely same the function and features of the intestinal epithelium. The adhesion ability of S. enteritidis with IPEC-J2was strong than Caco-2, this conclusion show that there exsit some differences between cells and intestinal epithelium. The interaction between S. enteritidis and intestinal epithelial cells is much more complicated and needs more research work to be done.3The assessment of RNA expression levels of Toll-like receptor4, Toll-like receptor15and NRAMP mRNAs in blood of chicken infected with S. enteritidis by Quantitative Real-time PCRToll like receptor4(TLR4), Toll like receptor15(TLR15) and natural resistance-associated macrophage protein(NRAMP) play an important role in the inital process of the pathogen recognition and resistance. To investigate whether SEF14and SEF21fimbriae of S. enteritidis play a role in the chicken infection, in this study, we build chicken infection model to assess the mRNAs levels of TLR4, TLR15and NRAMP in the blood of chickens infected with wild-type S. enteritidis50336and fimbriae gene mutants by qRT-PCR. The results showed that the mRNAs levels of TLR4, TLR15, NRAMP fluctuated at different times following the wild type and the fimbriae gene deletion strains infection, which indicated that the RNA expression of TLR4, TLR15and NRAMP is closely related to S. enteritidis infection. The mRNA levels of TLR4and TLR15in chickens infected with SEF21fimbriae mutant were2.46and2.5-fold respectively than that in wild-type50336infecting chickens at1day post-infection. However, no significant change was measured in chickens infected with single mutant50336ΔsefA and double mutant50336ΔsefAΔfimA compared with the wild type group. It indicated that SEF21fimbriae can affect the expression of TLR4and TLR15in blood of the chickens infected with S. enteritidis. The mRNA level of NRAMP in chickens infected with SEF21fimbriae mutant was11.55folds than wild type group at3days post-infection, which indicated that SEF21fimbriae may affect the expression of NRAMP. No significant changes were measured in chickens infected with other mutant strains compared with the wild type. However, more research work needs to be done about the mechanism of SEF14and SEF21fimbriae in this process.