PilA Expression Is Required For Colonic Epithelium Adhesion And Intestinal Colonization Of Enterococcus Faecium

Background: Pilus-like surface protein PilA was widely expressed in nosocomial infections of Enterococcus faecium.Although there is evidence that Gram-positive bacteria are associated with adhesion and colonization,but whether or not the PilA makes the enterococcus faecium easy to survive in the hospital environment and promote enterococcus faecium to colonize in the intestine is still unknown.In addition,the use of enterococcus faecium protein products-Immunoglobulin antibody or vaccine prompting passive/active immunotherapy unite with the current antimicrobial agents is a viable alternative to the treatment of enterococcus faecium,therefore,we need to further explore the immunogenicity of PilA in the human body.Objectives:We investigate the immunogenicity of PilA in human body,and to provide the basis for the preparation of PilA vaccine.And,To construct the pil A gene insertion-deletion mutants and complementary derivatives,the biofilm formation,adhesion experiment in vitro and intestinal colonization ability in vivo were used to evaluate the adhesion and colonization ability of PilA.Methods:The PilA protein expression vector pQE-pil A was constructed and transformed into the competent cells DH5? by recombinant plasmid pQE-30.The recombinant fusion PilA protein was induced by IPTG,then purified.The immunoreactivity of purified PilA protein were analysis by Western Blot.The expression of anti-PilA antibody IgG and IgM in serum was analyzed by Western Blot to determine the immunogenicity of the Pil A protein in the human body.Construction of pil A gene mutant E1162?pilA and complementary derivative E1162?pilA(p AT392::pil A)using insert-deletion and complementation,construct growth curve in vitro to assess the effect of pil A gene deletion/complementation on the expression of PilA,and the expression of PilA on the surface of E1162 was observed by immunoelectron microscopy,and the growth conditions of PilA were studied,then verified by whole cell ELISA,flow cytometry and immunoblotting.The effect of PilA on the adhesion of E1162 was evaluated by biofilm formation experiments,polystyrene adhesion experiments and cell adhesion assays / antibody binding inhibition assays.The effect of PilA on the colonization of E1162 in mammals was assessed by constructing a mouse intestinal colonization model.Results: PilA protein is immunogenic in patients with bacteremia.We found that PilA protein of enterococcus faecium E1162 only expressed in the solid culture(non-broth culture);Moreover,The expression of PilA on the surface of Enterococcus faecium was inhibited by the deletion of pilA gene,while the complementary pilA gene could restore the expression of Pil A protein.Although the deletion of pilA gene had no significant effect on biofilm formation and polystyrene adhesion,the deletion of pilA gene and the use of anti-PilA antibody could significantly reduce the adhesion of E1162 to colon epithelial cells.In vivo experiments,the deletion of the pil A gene significantly reduced the count of enterococcus faecium in feces and reduced the colonization of Enterococcus faecium in the ileum,cecum and distal colon.Conclusions: We successfully expressed the recombinant fusion protein PilA,and found that the immunogenicity of PilA in the human body,that can guide the synthesis of active or passive immunization agents;We successfully constructed pilA gene deletion mutant E1162?pilA and the complementary derivative E1162?pil A(p AT392::pilA)of the mutant.The expression of PilA protein plays an important role in the adhesion and colonization of enterococcus faecium,and this founding suggested that the PilA protein may be a new target of the treatment of enterococcus faecium infection.The findings above provide a new insight for the prevention and treatment of infection of enterococcus faecium.