Study On The Function Of SEF14 Fimbriae From S. Enteritidis

Salmonellae are gram-negative facultative rod-shaped bacteria and a member of the family of Enterobacteriaceae. Salmonellae are important enteric pathogens and they cause one of the most common foodborne diseases. Many types of Salmonella bacteria cause Salmonellosis in kinds of animal and people, and they live in the intestinal tracts of warm and cold blooded animals. The elderly, infants, and those with impaired immune systems may have a more severe illness, and the adults infected with Salmonella usually remain symptomless and unnoticed. Foods of animal origin, such as meat, dairy products, and eggs, have been implicated in outbreaks of human Salmonellosis. The prevalence of S. enteritidis has dramatically increased worldwide, and infection caused by S. enteritidis are similar or exceed the number of S. typhimurium, and it has been reported as the most common serotype in the United States and other developed countries, and many people die of the disease every year.Expression of SEF14 fimbriae is limited to S. enteritidis, S. dublin, all belonging to Salmonella group D. And there is few S. dublin infection cases clinically reported. SEF14 fimbriae may affect serovar-specific virulence traits, and by now the role of SEF14 fimbriae in virulence remains to be further elucidated. This study is focus on SEF14 fimbriae, we analyze difference and variation of the sef14 operon gene clusters in DNA level in S. enteritidis and related serogroup-D Salmonella to probe the clue why SEF14 fimbriae specificly expressed in S. enteritidis and S. dublin; there is few method reported for quikly and specially detecting of S. enteritidis infection in clinincal and SEF14 fimbriae as antigen protein is specially expressed on S. entritidis, we clone and express of the major subunit of SEF14 fimbriae and analyze its immune activity, develop the rSEFA-mediated indirect ELISA using the rSEFA for monitoring serum antibodies against S. enteritidis infection; S. enteritidis mutants from different source of sef14 gene cluster include sefA and sefD subgenes are constructed to study the molecular pathogenesis mechanism of interaction between the SEF14 fimbraie and the susceptible swine and mice cells. The result in this paper will provide some information for treating and controlling of this disease and we try to explore the relationship between Chinese indigenous chicken lines and the susceptibility of S. enteritidis by infection with the S. enteritidis mutants constructed in this test.1 Difference and variation of the sef14 operon gene clusters inS.enteritidis and serogroup-D SalmonellaIn this study, we analyze the difference and variation of the sef14 poeron gene clusters in S. enteritidis and related serogroup-D Salmonella, and we found that prevalence of sefA, sefD and sefR genes in S. enteritidis and S. dublin was 100%. In 18 isolates of S. pullorum, the prevalence of sefA gene was 100%, while the prevalence of sefD and sefR genes was 38.9% (7/18), and 11 strains isolated after 1980s did not contain any gene sefD or sefR. Interestingly, among the 7 strains of S. pullorum before 1980s, the sefD sequence has a missing base pair at position 196 and caused open reading frame (ORF) shift, resulting in a stop codon (TAG) at position 71 amino acid residual (Leu of TTA at position 214-216 shift into stop codon of TAG at position 215-217). Unlike S. pullorum, all S. enteritidis and S. dublin tested could express SEF14 fimbriae in vitro. Based on the data of the difference and variation of sef14 operon gene clusters between S. enteritidis and S. pullorum, we may explain why SEF14 fimbriae in S. pullorum could not be expressed.2 Cloning, expression and immune activity analysis of subunit SEFA of SEF14 fimbriae from S. enteritidisThe major subunit sefA gene of SEF14 fimbriae from S. enteritidis was amplified by PCR using a pair of primers designed and the template from national reference strain 50336 genomic DNA. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the expression vector pET22b+ to construct recombinant plasmid pETsefA, which was confirmed by the means of combination with restriction endonuclease analysis and sequencing, and then pETsefA was transformed to E. coli BL21 (DE3). After IPTG induce, the recombinant BL21 (DE3) with pETsefA expressed the soluble SEFA protein (rSEFA) with size of 15.2kDa by SDS-PAGE and Western-blotting analysis. Both the mouse sera with high titer of anti- rSEFA or against reference strain 50336 infection were raised and detected after being immunized with the purified rSEFA protein or infected with the reference strain, which could recognize the purified SEF14 fimbriae from reference strain 50336 and rSEFA, respectively.3 Development of an indirect ELISA for detection antibodies against S. enteritidisAn indirect ELISA (enzyme-linked immunosorbent assay) was developed for detecting antibodies against S. enteritidis using a recombinant fusion antigen rSEFA. This assay was optimized for antigen coating concentration of 7.5μg/ml and a serum dilutin of 1:100, with a standard incubation time 60 min. A reading of OD>0.346 was scored as positive. The assay was highly specific and has no cross reaction with serum of other relative disease, such as S. pullorum. There was 80.6% agreement in comparison with the agglutination test using S. enteritidis whole-cell antigen when they both were used to test on 100 clinical samples. The application of indirect ELISA would provide a simple and rapid method for monitoring serum antibodies against S. enteritidis infection.4 Constructing S. enteritidis mutants using Red recombination system and suicide vector systemBoth the Red recombination system and suicide vector system can be use to modify gram-negative bacteria chromosome. In this study S. enteritidis (X) mutants from different source of sef14 gene cluster include sefA and sefD subgenes were constructed by both Red recombination and suicide vector systems. Based on the original sequence of sefD gene and the antibiotic resistance cassette in the GenBank, we generated PCR products using primers with the homologies extension of sefD gene to be deleted and template pKD3 carrying selectable antibiotic chloramphenicol (cat) gene that is flanked by FRT sites. The XΔsefD::Cat deletion mutants were contracted from PCR products. After selection, the resistance gene located in the XΔsefD::Cat were eliminated in the second recombination by a temperature-sensitive helper plasmid pCP20 that encoding and expressing the FLP recombinase, which acts on the direct repeated FRT sites flanking the resistance gene. The XΔsefD obtained were further confirmed by PCR amplification and sequencing. The suicide vector system was used to delete sefA gene, we constructed the recombinant suicide plasmid flanking by homologous arms about 1000bp, then transformed into S. enteritidis stains. These recombinant strains were lost their plasmid in plates containing sucrose. The mutants were confirmed by PCR and sequence. Four S. enteritidis mutants XΔsefD and XΔsefA were successfully constructed. Thus, both of the two systems can be used to delete S. entertidis chromosome without any resistant gene. Compared with the suicide vector system, the Red recombination system need high quality PCR product and high quality competent cells, the first recombination reaction is very low, whereas in the second recombination the FRT sites will be stained in the chromosome. However, the protocol of the suicide vector is complex, because is needs to construct the recombination plasmid. The mutants obtained in this study will be the base to study the molecular pathogenesis mechanism of interaction between the SEF14 fimbraie and the susceptible host cells, prevention and control of the S. entertidis - caused disease.5 The effect of LD50 to Balb/c mice and infection of chickens caused by S. enteritidis and its SEF14 mutantsIn this experiment, the LD50 of the national reference strain 50336 and mutant strains 50336ΔsefA,50336ΔsefD were tested in 6-week-old Balb/c mice by intraperitoneal injection (IP). The results showed that the LDso of the three strains were 19.95,7.94,7.94cfu, respectively. It suggested the virulence of S. enteritidis in 6-week-old Balb/c mice increased when injected with mutant strains 50336ΔsefA or 50336ΔsefD. The observation of infection of chickens with the standard strain 50336 and mutant strains 50336ΔsefA,50336AsefD and 50336AsefAΔsefD indicated that they are highly heterogeneous in their ability to cause death in 1-day-chickens. The results of infection of chicken also showed that injection with 50336ΔsefA or 50336ΔsefAΔsefD reduced the gaining in weight in 7-day-old. This suggested that the virulence of 50336ΔsefA or 50336ΔsefAΔsefD were increased in chicken. Test of isolation and identification of bacteria showed that S. enteritidis and mutant strains are widely distributed in the organs after injection. There are kinds of Chinese indigenous chicken lines with diverse genetic backgroud. According to the infection with 5. enteritidis and its SEF14 fimbriae mutants, we test the susceptibility of QINGYUAN chicken line against S. enteritidis and provide some information for breeding program.6 Studies into the role of the SEF14 fimbriae in the pathogenesis of S. enteritidisAdhesion of pathogen to host cells is an important prerequisite for successful colonization and establishment of the pathogenesis. Microphages are so important and involved in number of reaction to encounter S. enteritidis infection in animals, and may be caused dissemination of S. enteritidis through the animals. To investigate the role of the SEF14 fimbriae in pathogenesis, the mutants XΔsefA, XΔsefD, XΔsefAΔsefD of SEF14 fimbriae from S. enteritidis (X) strains which were from different source were constructed and tested in porcine intestinal epithelial cell lines (IPEC-J2) and mouse peritoneal microphages in an in vitro infection model. There was no significant difference between the wild-type strains and the isogenic mutants in their abilities to adhere to IPEC-J2 cells. The mouse peritoneal macrophages were able to internalize high numbers of S. enteritidis. However, the number of entered and surviaval of isogenic XΔsefA, XΔsefD, XΔsefAΔsefD mutant in mouse peritoneal macrophages was significantly different from wild strains. These results indicate that possession of SEF14 fimbriae alone do play a significant role in the pathogenesis of S. enteritidis through the reaction with microphages.