Preliminarv Investigation Of The Function Of Salmonella Fimbriae Adhesin FimH

Salmonella is an important zoonotic pathogen with a worldwide concern.It can infect a broad range of different animal hosts and can cause intestinal or systemic infections in both humans and animals.In humans,Salmonella infection is responsible for 16 million cases of typhoid fever and 1.3 billion cases of gastroenteritis,leading to 3 million deaths annually over the world.It was well recognized as one of the main causes of food poisoning in numerous countries and regions.Salmonella type I fimbriae is an adhesion and colonization factor expressed on the surface of the bacteria,which mediating the adhesion of bacteria to host cells,and plays a key role in the process of Salmonella colonization and invasion.On the top of type I fimbria,expressing the FimH,which has a great influence on the adhesion of host cells,receptors and biofilm formation.It resists immune cell phagocytosis and promotes the secretion of inflammatory cytokines during innate immunity.FimH also plays an important role to inhibit cell apoptosis and other aspects.The FimH derived from different Salmonella has a 99%sequence homology generally,and previous studies demonstrated that the slight amino acid sequence differences in FimH adhesin can dramatically affect the binding capabilities to mannose receptors and certain host cells significantly.Although numerous studies have been studied on Salmonella FimH,with a focus on Salmonella enterica subspecies enterica.However,few studies were worked on other that subspecies enterica,further studies regarding to FimH from a broad serovars is warranted.Further studies about diverse functions are needed on other subspecies and serotypes of Salmonella.The main purpose of this study were to:(1)explore the best method for soluble expression of FimH recombinant protein;(2)explore the effect of fimH gene on the biological function and pathogenicity of Salmonella via SL1344 fimH-deleted strain;(3)explore the relationship between FimH amino acid sequence difference and fimbria biological function via complement strains of homologous FimH from S.enterica subspecies ?,?,?b,?,? and other serotypes,including Gallinarum,Enteritidis,Pullorum,Dublin and Typhi.1.Exploration of soluble expression of FimH recombinant proteinBy using three different types of expressing vector,pET30a,pGex-4t-1 and pCold,combined with different types of fimH genes and other gene coding fimbrial structure and chaperone protein of type ? fimbriae,eight different recombinant expression vectors were constructed,and the corresponding FimH recombinant protein was expressed with various conditions.Western blot method was used to detect the expression of recombinant FimH protein.Together,the pCold expression vector that co-expressed FimH and FimC was the most appropriate method approach to obtain soluble recombinant protein of FimH.By immunization,the FimH polyclonal antibody was successfully obtained from the rabbit.The titer of the antibody detected by ELISA was about 1:640,000,which can be used for subsequent FimH protein detection.2.Exploration of the effect of fimH gene on the biological function of SalmonellaSL1344 fimH-deleted strain was constructed through the ?-Red homologous recombination system,and tested by adhesion assay,biofilm assay and Caenorhabditis elegans killing assay.The mannose adhesion assay and the IPEC-J2 cell adhesion assay showed the SL1344 wild type strain had strong adhesion ability,but lost adhesion ability in the ?fimH mutant.Biofilm experiments showed that the ability of Salmonella to form biofilms on polystyrene 96-well plates was enhanced in the ?fimH mutant.And the ability of Salmonella to form biofilms on polystyrene 96-well plates increases with the decreasing temperature,room temperature condition was showed the best for Salmonella to form biofilms.The C.elegans killing experiment results showed that the fimH-deleted strain had a significantly higher killing rate than the SL1344 wild type strain,indicating that the ?fimH was more virulent than the wild type strain.This result may be due to the up regulation of the expression of other virulence factors such as biofilm after the fimH gene deletion.3.Exploration of relationship between amino acid sequence differences of FimH and the biological function of SalmonellaThe low copy plasmid pACYC184 was used to construct the FimH complement plasmid pACYC184-fimHFZ,which could incorporate fimH alleles from S.enterica subspecies ?,?,?b,?,? and different Salmonella serotypes Gallinarum,Enteritidis,Pullorum,Dublin and Typhi.And all fimH alleles complement plasmids pACYC184-fimHFZ was transfered into ?fimH strain to construct complement strains with FimH homologous protein of each subspecies and serotypes.The results of adhesion assay,biofilm assay and C.elegans killing assay suggested that S.enterica subsp.? FimH had a weaker binding properties to mannose,and the FimH of S.Dublin had strong adhesion.All of complement strains did not produce the biofilms under the culture conditions of 37? and 22?.Each complement strains had similar lethality to C.elegans,and there is no significant difference compared with SL1344 wild type strain.Together,this study proved that FimH,as a type ? fimbriae adhesin protein,has a decisive effect on the adhesion function of Salmonella.The difference in FimH amino acid sequence mainly affects the adhesion function of Salmonella and has no effect on other biological characteristics.Deletion of fimH gene can enhance Salmonella virulence,it might be caused by the up-regulating of other Salmonella virulence factors such as biofilm.This study provided additive new information for the functional diversity of Salmonella type ? fimbriae FimH,and improve the knowledge between FimH sequence types and various types of pathogenic functions.