The Study On The Mechanism Of Fis Mediated Formation Of Persisters In Salmonella Enterica Serovar Typhi

Objective:Persister is one of the important causes for chronic and relapsing infections,which can be detected in many kinds of human pathogens.The mechanism of persisters formation is very complicated,which involves the virulence and energy metabolism of bacteria.This study is aimed to investigate whether Salmonella enterica serovar Typhi exsits persistence,the effect of environmental factors on the formation of persisters and the role of global regulator Fis in the formation of persisters in S.Typhi.Methods:1.Drug sensitivivity test.The bacteria with Mc F of 0.5 was spread uniformly on the M-H plate.The paper of ampicillin,ciprofloxacin and kanamycin were each placed on the surface of the plate to test the antibiotic sensitivity of each drug.2.Minimal inhibitory concentration measurement.Ampicillin and ciprofloxacin with different concentrations were put into 96 wells plate and an equal amount of bacteria was added into each well.The lowest drug concentration which inhibited the growth of bacteria was regarded as minimal inhibitory concentration(MIC).3.Drug survival curve of bacteria.The bacteria strain was incubated to early log phase,with the addition of ampicillin or ciprofloxacin.Aliquots of bacterial cultures(100μL)was taken for counting CFUs once an hour.Drug survial curve of bacteria was drawn with the time of drug treatment as X-axis and the number of colonies at each time point as Y-axis.4.Measurement of Persistence.The bacteria strains were cultured to different OD600,with the addition of ampicillin or ciprofloxacin for 5 h.Bacterial cultures(100 μL)were taken for counting CFUs.The ratio of persisters(persistence)was the number of colonies by antibiotics treatment after 5 h versus the number of colonies before the drug was added.5.Analysis of expression of genes by q RT-PCR.The wild type strain and Δfis were cultured to the specified time points.Total RNAs were extracted from bacteria to perform reverse transcription.q RT-PCR was used to determine the difference in the expression levels of the target genes between WT and Δfis.6.RT-PCR to analyze the construction of glt.To determine whether glt I,glt J,glt K,glt L are located on an operon,we designed two pairs of primers for each gene.The wild type strain was cultured to the early log phase and total RNAs were extracted to perform reverse transcription.c DNA was used for the PCR.7.Construction of glt and fis double deleted mutant and glt complemented mutant of S.Typhi.Homologous glt deleted recombination fragment was ligated with the suicide plasmid p GMB151,the positive plasmid was transferred into the fis deleted mutant as glt and fis double deleted mutant(Δ glt Δ fis).An empty plasmid p BAD/Myc-His A and the identified recombinant plasmid p BAD-glt were transferred into glt and fis double deleted mutant as control strain(Δ glt Δ fis+p BAD)and complemented mutant(ΔgltΔfis+p BAD-glt).Results:1.The drug sensitivity test results showed that the wild type strain and the fis deleted mutant were sensitive to ampicillin,ciprofloxacin and kanamycin.2.The results of MIC test revealed that the wild type strain and the fis deleted mutant showed similar sensitivity to ampicillin and ciprofloxacin.3.The survival curve of S.Typhi under ampicillin or ciprofloxacin treatment was a typical biphasic killing pattern,which was observed with a amount of bacteria alive in the presence of antibiotics.4.The persistence was detected in S.Typhi.The formation of persisters was related to the bacterial cultural condition and growth period.5.q RT-PCR results revealed that fis had the highest expression in the early log phase.Compared with the wild type strain,the expression of rel A and spo T decreased and the expression of gln H,gln P,glt,glt K,glt J and glt S composing the glutamate transporter increased in the fis deleted mutant.6.Compared with the wild type strain,the fis deleted mutant had a decrease in persistence.7.Compared with the difference of persistence between wild type strain and fis deleted mutant in the LB medium,the difference in the special M9 medium was reduced.8.glt and fis double deleted mutant(ΔgltΔfis),glt complemented mutant(ΔgltΔfis+p BAD-glt)were successfully constructed.9.Compared with the fis deleted mutant,the rate of persisters in glt and fis double deleted mutant increased;The rate of persisters in glt complemented mutant decreased compared with the control strain.Conclusion:The persistence was detected in Salmonella enterica serovar Typhi and the formation of persisters was influenced by environmental factors.Fis played a positive role in the formation of persisters.This study showed that Fis mediated in the persisters formation by regulating the genes coding glutamate transporter,resulting in changes in the intracellular nutritional status of bacterial cells.This findings provide some information for exploring the specific mechanism of persisters formation in bacteria.