The Roles Of CsgA, BcsA, And RpoS Genes In Biofilm Formation And Virulence In Salmonella Pullorum And Typhimurium

Salmonella biofilm formation is important to environmental stress resistance and virulence.However,limited information is available regarding the roles of the csgA,bcsA and rpoS genes involved in biofilm formation which affect curli protein;cellulose production and virulence for Salmonella enterica serovar Pullorum and Typhimurium.Furthermore,Salmonella can form a biofilm,which can lead bacteria to increase bacterial resistance to adverse conditions;disinfectants;antibiotics and host immune system,consequently promoting bacterial dispersal and survival and enhancing its virulence.Thus,biofilm formation is associated with the outbreak of salmonellosis and persistent infections in patients.In order to better control biofilm formation bacteria which is caused salmonellosis in humans and animals,it is necessary to clarify the respective roles of csgA;bcsA and rpoS genes in different serotypes of Salmoella such as S.Pullorum(S6702;S11923-3)and S.Typhimurium(S016;S025)strains in biofilm formation and pathogenicity.1.Contribution of the csgA and bcsA genes to Salmonella enterica Serovar Pullorum Biofilm Formation and Virulence.In this study,the mutant strains of csgA gene,bcsA gene or csgA/bcsA genes in S.Pullorum strain S6702 were constructed by using the lambda Red recombination system,so as to evaluate several aspects of biofilm formation and virulence.The biofilm-forming ability of Salmonella strains was detected by crystal violet assay;Congo red/Brilliant blue plate and calcofluor plate were used to detect the components of biofilm.Moreover,phenotype of biofilm formation was detected by the Field emission scanning electron microscope(FESEM)analysis of bacteria grown on coverslips at 28 °C for 24 h.Also the Real-time PCR amplification method was established to compare the expression of genes during the period of biofilm formation,each deletion mutants were combined with gene complementation assay.Growth curve revealed that the growth rates of all mutants were similar as wild-type strains.A quantitative microtiter assay performed by crystal violet staining in 96-well plate and test 'tube showed that the mutants AcsgA and ?csgAAbcsA lost biofilm-formation ability completely.Therefore,?csgA showed decreased production of curli fimbriae,while AbcsA had reduced cellulose production.?csgA was reduced in adhesion and invasion to HeLa cells and exhibited decreased intracellular proliferation in HD 11 macrophages.?bcsA exhibited increased proliferation in HD 11 cells and replicated better in chicken spleens,as compared to the wild-type strain.In addition,mutant ?csgA?bcsA was significantly reduced in adhesion and invasion of epithelial cells and intracellular proliferation in macrophages compared with the wild-type strains.The virulence of mutants ?csgA and ?csgA?bcsA was attenuated significantly in assays involving oral challenge of one-day-old chickens.2.The role of csgA and bcsA genes on Biofilm Formation and Virulence in Salmonella enterica Serovar Typhimurium.In the present study,knockout mutants in the csgA and bcsA genes of S.Typhimurium strain S016 and S025 were constructed,and their biofilm formation ability and virulence were assessed.The AcsgA strains did not produce curli fimbriae,and AbcsA mutants had decreased cellulose production,only the AcsgA strains had no biofilm-forming ability.The AcsgA strains showed decreased adhesion and invasion to HeLa cells and reduced intracellular proliferation in HD 11 macrophages.Whereas,AbcsA mutants presented similar adhesion,invasion and proliferation as compared to the wild-type strains.The AcsgA strains were significantly attenuated in the virulence in assays involving oral challenge of one-day-old chickens.3.Identification and characteristics of an rpoS dependent Salmonella enterica serovar Pullorum strain in biofilm formation.In this study,a S.Pullorum strain S119233 was selected to construct the knockout mutant of rpoS,and catalase test confirmed its successful construction.Determination of biofilm formation ability of the ArpoS strain showed that the mutant strain had similar curli expression,decreased cellulose expression and reduced biofilm formation when compared with the wild-type strain,indicating that S.Pullorum S119233 was an rpoS-dependent strain for biofilm formation.Further,its knockout mutants of csgA and bcsA were constructed,and both AcsgA and AbcsA mutants had reduced biofilm formation ability.Especially,the phenotype of the ArpoS mutant was similar as that of the AbcsA mutant,indicating that the rpoS-dependent strain may regulate the biofilm formation through the expression of cellulose.In addition,the AcsgA mutant showed reduced adhesion and invasion of epithelial cells,decreased intracellular proliferation in macrophages,and attenuated virulence when compared with the wild-type strain.Both ArpoS and ?bcsA strains showed similar adhesion,invasion,proliferation in vitro,and virulence in vivo as the wild-type strain.The finding of an rpoS-dependent strain for biofilm formation will help us to understand the different mechanism for regulation of biofilm formation.In summary,we found that the csgA gene played a major role in biofilm formation and pathogenicity.However,the bcsA gene played different role in different serotype strains.Also,we found an rpoS-dependent strain for biofilm formation,which may help us to understand the mechanism for regulation of biofilm formation.