Construction Of The BolA Gene Mutant Strain Of Salmonella Typhimurium And Study On Its Biological Function

Salmonella Typhimurium(S.Typhimurium)is an important zoonotic pathogen,which is pathogenic to humans,livestock,poultry and other animals,causing great harm and economic losses.Meanwhile,due to the widespread use or abuse of antimicrobial agents in animal husbandry and human clinical,the resistance of S.Typhimurium has been very serious.It is of great significance to find target factors related to drug resistance and pathogenicity of S.Typhimurium for prevention and control of the disease.Transcription factor BolA is found in most gram-negative bacteria and is also an important receptor molecule in the c-di-GMP system.The BolA is pleiotropic and plays an important role in bacterial biofilm formation,outer membrane permeability and drug resistance.In this study,we will study the effect of BolA gene on the biological characteristics of S.Typhimurium to explore the role of BolA gene on the biofilm formation,drug resistance and pathogenicity of S.Typhimurium,providing relevant data for the in-depth study of the drug resistance and pathogenic mechanism of S.Typhimurium.In this study,the multi-drug resistant S.Typhimurium SM26 isolated clinical patient was used as the research object.Red homologous recombination technology was used to successfully construct the BolA gene mutant strain of S.Typhimurium SM26.Meanwhile,the prokaryotic expression vector p BAD-His A was used to construct the restorer SM26△BolAR.The BolA gene mutant strain was cultured on the LB agar plate for 28 consecutive generations.The PCR identification showed that the mutant gene did not undergo reversion mutations,indicating that the BolA gene mutant strain could be stably inherited.This study analyzed the changes in biological characteristics after the deletion of the BolA gene.The growth curve results show that the deletion of BolA gene did not affect the growth rate of S.Typhimurium.The environmental stress resistance of the tested strains was determined and the results showed that the BolA gene mutant strain had significantly reduced resistance to acidic and hydrogen peroxide stress.Crystal violet staining and XTT colorimetry method were used to detect biofilm formation and biofilm activity of the tested strains,respectively.The results showed that the BolA gene mutant strain caused a reduction of biofilm formation by about 30%,a decrease in biofilm activity by about 29%.Congo red and Coomassie bright blue G-plate method was used to determine the biofilm components of the tested strains.The results showed that the production of frizzled pili protein of the BolA gene mutant strain was reduced.The laser confocal was used to observe the structure of the biofilms of the tested strains.The laser confocal image intuitively showed that the biofilms of the wild-type and restorer strains were aggregated into clusters and the structure was dense,while the BolA gene mutant strain is relatively loose and could not be clustered.Analysis of the expression levels of the c-di-GMP pathway-related genes csg D,adr A,stm4511 and yhj H,the results showed that compared with wild-type strain,the expression levels of csg D,adr A,stm4511 in the BolA gene mutant strain were down-regulated by 2 to 10 times,but there was no difference in yhj H.Our results show that the deletion of BolA gene can reduce the biofilm formation ability and biofilm activity of S.Typhimurium.This study determined the antimicrobial susceptibility of the tested strains to 15 antimicrobial agents.The results showed that the susceptibility of the BolA gene mutant strain to chloramphenicol,nalidixic acid,ciprofloxacin,kanamycin,streptomycin,ofloxacin,gentamicin and cefotaxime was increased by 2～8 times and the strain changed from resistance to 6 kinds of antimicrobial agents to resistance to 2 kinds,indicating that the BolA gene has an effect on the resistance of S.Typhimurium.The experiment determined the effect of sub-inhibitory concentrations of antibiotics on the biofilm formation ability and biofilm activity of the tested strains.The results showed that the sub-inhibitory concentrations of cefotaxime increase the biofilm formation ability and biofilm activity of the tested strains,while ciprofloxacin decrease the biofilm formation ability and biofilm activity of the tested strains.Moreover,the sub-inhibitory concentrations of kanamycin increased the biofilm formation ability and biofilm activity of wild-type and restorer strains,but reduced the biofilm formation ability and biofilm activity of BolA gene mutant strain.The results showed that the BolA gene has the effect of affecting the biofilm formation ability and biofilm activity of S.Typhimurium under the condition of sub-inhibitory concentrations of kanamycin.The motility of the tested strains was determined by 0.3% LB agar cultured conditions.The results showed that the ability of motility of the BolA gene mutant strain is significantly lower than that of the wild-type strain and the diameter of BolA gene mutant strain was reduced by about 40%.Analysis of the expression levels of the motility-related genes fim D and flg G,the results showed that compared with wild-type strain,the expression levels of fim D and flg G in the BolA gene mutant strain were significantly reduced.The cell adhesion and invasion experiment was used to determine the effect of BolA on the virulence of S.Typhimurium.The results showed that the ability of adhesion and invasion to Caco-2 cells and He La cells was significantly decreased.Analysis of the expression levels of the virulence-related genes csg G,cpx R,pho P,inv H and fim A.The results showed that the expression levels of inv H,csg G and fim A in the BolA gene mutant strain were significantly reduced.This indicates that the BolA gene plays an important role in the pathogenicity of S.Typhimurium.In conclusion,a multi-drug resistant S.Typhimurium BolA gene mutant and restorer strains were successfully constructed in this study.The study found that the BolA gene has an important role in the biofilm formation,motility,environmental stress resistance,drug resistance and pathogenicity of S.Typhimurium.These research results will provide relevant data for further study of the drug resistance and pathogenic mechanism of S.Typhimurium.