The Roles Of Fructose-specific Phosphotransferase System In The Pathogensis Of Klebsiella Pneumoniae

Objective: Klebsiella pneumoniae,a Gram-negative enterobacter,has become one of the most important pathogens of community-acquired and nosocomial infections in recent years.Phosphotransferase system is an important sugar transport system in bacterial cells and may be related to bacterial pathogenicity.Genome-wide analysis revealed that there is a putative PTS system in KP---the Frw PTS system,the frwC gene encodes the ElI C.The cAMP receptor protein is an important global regulator of pathogens that regulates the metabolism and virulence of bacteria.However,in Klebsiella pneumoniae,whether CRP affects the pathogenicity of bacteria through the regulation of fructose transport system has not been studied.To explore the regulatory relationship between CRP and fructose PTS system,study the function of fructose PTS and improve the regulatory network of CRP,which laid the foundation for the study on virulence of Klebsiella pneumoniae strains.Methods: The Bioinformatics analyzes the binding motif of the global regulatory factor CRP and looks for genes that may have CRP binding sites.The qRT-PCR and lacZ fusion assays revealed that CRP positively regulates frwC gene expression.The CRP protein expression vector was constructed and transformed into BL21 for extracting purified CRP protein.Whether or not CRP has a direct binding to the frwC promoter region was verified by gel blocking experiments.Based on the homologous recombination technology,a deletion fragment of the frwC gene was constructed by using the suicide vector pKO3-Km plasmid.The gene fragment encoding the promoter region and the transcription termination region of the frwC gene was amplified and cloned into the pGEM-T-easy plasmid and transferred to the deletion strain Construction of frwC gene back-up strain.By plotting the growth curve of frwC knockout strain in M9 basal medium plus different concentrations of different carbohydrates,whether the putative fructose PTS system is involved in the fructose utilization of Klebsiella pneumoniae was analyzed.By plotting the growth curve in LB medium,The effect of frwC gene on bacterial growth was analyzed.The biofilm formation ability of wild strain,crp mutant and frwC mutant was compared by crystal violet quantitative assay.The capsular related gene magA was determined by wire drawing,high speed centrifugation and RT-PCR.The effect of frwC gene on capsular formation was analyzed.The effect of frwC gene on the virulence of Klebsiella pneumoniae was analyzed by virulence test in mice.Results: The qRT-PCR,lacZ fusion assays,and EMSA experiments showed that CRP binds directly to the promoter region of frwC and regulates frwC expression.The frwC gene knockout strain Δ frwC and the complementation strain C-frwC were constructed successfully.The frwC gene was not expressed in Δ frwC by RT-PCR.Growth curve experiments showed that the frwC knockout strain had no effect on the utilization of glucose and maltose,but had an impact on the utilization of fructose at high concentrations(0.05% and 0.2%),indicating that the fructose PTS system was effective in fructose utilization of Klebsiella pneumoniae The process works.In LB medium,the growth rates of frwC and crp knockout strains were all slowed down,while the backfilling strains were able to replenish the corresponding growth rate,indicating that frwC affected the growth rate of bacteria.Quantitative detection of biofilm formation by crystal violet staining showed that biofilm formation of frwC knockout was more than that of wild-type and crp mutants.Quantitative experiments using high-speed centrifugation and capsule extraction found that the amount of capsular formation in the frwC knockout strain was more.RT-PCR results showed that the expression of magA in the capsular gene increased after frwC knockout.Drawing experiments showed that the knockout strains were more stick and formed a mass of filaments that could not be formed into filaments.Mouse virulence experiments showed that the survival rate of mice infected with frwC knockout strain was higher than that of wild strain and complement strain.Conclusion:(1)CRP positively regulates the expression of EIIC enzymeFrwC in this PTS system,and CRP directly regulates frwC gene.(2)This fructose PTS system plays a role in fructose utilization of Klebsiella pneumoniae,and frwC affects bacterial growth rate.(3)The negative regulation of frwC gene biofilm and capsule formation.(4)frwC gene affects bacterial virulence.