Influence of linear plasmid pBSSB1 gene LP002 on the pathogenicity of Salmonella enterica serovar Typhi
Objective To construct a mutant strain of linear plasmid pBSSB1 gene LP002(△LP002)of Salmonella enterica serovar Typhi(S.Typhi)and to investigate the role of LP002 in the pathogenicity of S.Typhi.Methods The homologous recombination mediated by suicide plasmid was used to construct △LP002.Two strains were cultured separately for 12 h.The values of optical density at 600 nm(OD600)were detected every hour,and the growth curves were plotted using spectrophotometer.The relative expression of LP002 mRNA was detected by real-time fluorescence quantitative PCR when the wild-type strain was cultured to OD600=0.2,0.4,0.6,0.8,1.2,and 1.8,respectively.The appropriate growth stages of △LP002 and wild-type strain were selected for phenotype experiments.Bacterial motility experiments were used to detect the diameters of the motility circles,and biofilm formation experiment was done to detect the amount of biofilm.After treatment with ampicillin and gentamicin,the number of viable bacterial colonies was detected,and the survival curves were plotted to observe the formation of persister.The persistent rates and the diameters of bacteriostatic circles were calculated after 5 h of antibiotic treatment.The invasion ability of HeLa cells and their viability in macrophages were detected to calculate the invasion index and proliferation index.Results After PCR verification,the amplification production of the wild strain genome was 1 450 bp,the length of amplification production of△LP002 genome was 888 bp,and the knockout length was 562 bp,which indicated that △LP002 was successfully constructed.At 1,2,3,4,5,6,7,8,9,10,11 and 12 h after culture,the OD600 values showed no significant differences between △LP002 and the wild strain(t=1.385,1.576,1.895,0.351,0.833,0.195,0.213,0.926,0.024,0.011,0.116,0.063;all P values＞0.05).There were no significant differences in the diameters of bacteriostatic circles and biofilm formation between △LP002[(35.67±1.15)mm,0.29±0.04]and the wild strain[(35.00±1.00)mm,0.36±0.04](t=0.756,P=0.492;t=2.196,P=0.093).The survival curves showed that persistent phenomenon existed in △LP002 and the wild strain,and the differences in persistent rates between △LP002[(0.300±0.200)％,(0.022±0.022)％]and the wild strain[(0.513±0.252)％,(0.063±0.044)％]were not statistically significant after 5-h treatment with ampicillin and gentamicin(t=1.143,P=0.317;t=1.464,P=0.217).The diameters of bacteriostatic circles showed no significant differences between △LP002 and the wild strain before and after treatment with ampicillin and gentamicin(P＞0.05),and showed no significant difference in △LP002 and the wild strain after treatment compared with those before treatment(P＞0.05).The invasion index and proliferation rate of △LP002 were lower[(28.65±4.73)％,1.10±0.17)than those of the wild strain[(41.35±3.59)％,2.63±0.18](t=5.230,P=0.001;t=15.550,P＜0.001).Conclusion The LP002 gene defect can inhibit the invasive ability of S.Typhi to HeLa cells and the survival ability in macrophages,but it does not affect the growth,motility,biofilm formation ability,persister formation ability,and sensitivities to ampicillin and gentamicin.
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