Construction And Characterization Of Salmonella Enteritidis △lpfC And △stdB Mutant Strains

Salmonella Enteritidis (SE) is a gram-negative and facultatively intracellular bacterium, parasitic in human and animal intestines. As an important zoonotic foodborne pathogen, it can cause acute gastroenteritis and even severe systemic infection. SE has flagella and fimbria as motile elements. Fimbria can mediate interactions between bacteria and host, which is involved in the pathogenicity of bacteria, formation of biofilms, motility, invasion and colonization of cells. Fimbria is considered as one of the major virulence factors for infection of Salmonella. In the previous studies of our lab, the SCOTS (selective captured of transcribed sequences) technology was used to determine and screen genes expressed by SE-infected murine and chicken macrophages.Wefound lpfC and stdB genes were up-regulated significantly during the infection process compared with cultivation in vitro. But there are few reports about their roles in virulence and infection of Salmonella, it is speculated that they play an important role in SE infection progress. SE mutants defected in IpfC and stdB were successfully constructed by homologous recombination technology in this study, respectively. Afterwards their virulence to BALB/c mice, adhesion to cells in vitro, gene expression and persistence of bacteria in CBA mice (the genetically resistant) were evaluated. These results will help us to understand the function of IpfC and stdB genes in the further.1. Construction and identification of the mutant strain C50041ΔlpfC and C50041ΔstdBThe IpfC and stdB mutant of SE strain C50041 via suicide plasmid pGMB151 were constructed, and named C50041ΔlpfC, C50041ΔstdB, of Salmonella Enteritidis, respectively, and its corresponding complementary strain C50041ΔlpfCR and C50041ΔstdBR were also developed in this study. Then the biological characteristics and virulence were determined. Among the five strains, there are no obviously differences in growth property and biochemical properties, while pass-generation test showed that the hereditary property of mutant strains and complementary strains was stable. However, the BALB/c mouse lethal test showed that the LD50 of the C50041 ΔlpfC was 2 times than that of the parent strain C50041, the LD50 of the mutant strain C50041 ΔstdB was one third of C50041 LD50. This study provided basic data for further study on the function of lpfC and stdB genes, contributed to reveal the function of Lpf and Std fimbriae from SE.2. Preliminary function analysis of stdB gene in Salmonella Enteritidis strain C50041In this study, the function of stdB gene was studied by measurement of the adhesion and invasion ability to different cells in vitro, the level of gene expression and persistence in CBA mice. Our results showed that there was no difference in the motility between C50041 ΔstdB and the parental strain. However, the adhesion and invasion ability to IPEC-J2 cell was extremely significantly decreased than that of the parental strain (p<0.01). When the pathogen was cultivated in vitro or interacted with RAW264.7, the expression level of stdA-. stdC and stdF from C50041 ΔstdB was lower than that of C50041. When the RAW264.7 cells were infected by C50041, C50041ΔstdB and C50041ΔstdBR,respectively, the expression of IL-12 from C50041ΔstdB infected cells were extremely dramatically lower than the others (p<0.01). Then the CBA mice were used as the animal model to evaluate the persistence of SE and its mutant in different organs, it was shown that the tendency of the bacterial amounts present in Peyer’s patches, mesenteric lymph nodes, spleens, livers, and caecum were similar to those of WT and mutant strain at each time point. However, we found that loss of stdB gene weakened the ability of SE to persist and colonize in the caecum of CBA mice. These results are helpful to understand the function of Std fimbriae in SE.3. Preliminary function analysis of lpfC gene in Salmonella Enteritidis strain C50041In this study, the role of lpfC gene was studied by measurement of the adhesion and invasion ability to different cells in vitro, the level of gene expression, detection of fimbriae by electron microscope and number of the bacteria in CBA mice. The results showed that the C50041 AlpfC strain did not produce fimbriae in transmission electron micrography. The adhesion ability to IPEC-J2 cell of C50041 ΔlpfC was extremely significantly decreased than WT strain (p<0.01), the invasion ability to RAW264.7 and HD-11 cells of the mutant was more decreased, too. When the pathogen was cultivated in vitro or interacted with RAW264.7, the expression level of lpfA, lpfB and lpfD from C50041ΔlpfC was lower than that of C50041. When the RAW264.7 cells were infected by C50041, C50041 ΔlpfC and C50041 ΔlpfCR, respectively, it was shown that the expression of IL-12 from C50041AΔlpfC infected cells were extremely dramatically lower than the others (p<0.01). Then the CBA mice were used as the animal model to evaluate the persistence of SE and its mutant in different organs, the results demonstrated that loss of lpfC gene weakened the ability of SE to persist and colonize in the caecum and the Peyer’s patches of CBA mice. These results are helpful to understand the function of Lpf fimbriae in SE.