The Effect Of Salmonella Typhi Plasmid On The Formation Of Biofilm And Its Molecular Mechanisms

Objective:To study the effect of Salmonella enterica serovar Typhi (S. typhi) plasmid pRST98on the formation of biofilm (BF), and explore the molecular mechanisms how S. typhi plasmid pRST98interact with quorum-sensing (QS). Also it can provide the theoretical and experimental basis for controlling salmonella infection and discovering new drug target.Methods:1. The effect of Salmonella typhi plasmid pRST98on the formation of biofilm.(1) Determine the optimum temperature and time of biofilm formation.Wild-type S. Typhi strain carrying plasmid pRsT98ST8was cultured at30℃or37℃for3d to determine the optimum temperature of BF formation by using semi-quantitative method. Then the diluted bacteral was cultured at30℃for12h,1d,2d,3d,4d and5d to determine the BF mature time using semi-quantitative method.(2) Compare the formation of biofilm by the different strains of S. typhi.Wild-type S. typhi strain carrying plasmid pRST98ST8, pRST98-deletion S. typhi strain ST8-⊿pRST98and pRST98-complementsd S. typhi strain ST8-c-pRST98were used to build BF according to the conditions acquired by above experiments. Then the BF formation ability of these three strains was compared by using crystal violet staining method, semi-quantitative means, confocal laser scanning microscopy and scanning electron microscopy.(3) Comparison of the HeLa cell adhesion by various strains of S. typhi.These three strains (ST8, ST8-⊿pRST98and ST8-c-pRST98) were co-cultured with HeLa cell by100:1multiplicity of infection (MOI) for1h respectively. Then cell was collected and washed by PBS. Then destroy cell membrane and release bacterial. Plate culture count method was used to assess the adhesion ability. 2. Investigate the molecular mechanisms of S. typhi plasmid interacting with QS.The experimental strains were divided into two groups. The first group was added1μM/mL QS signal molecule C8-AHLs. The other group was added an equal amount of saline as control. Both groups were implemented following experiments.(1) The effect of AHLs to the adhesion ability of tested bacteriaThe Bacteria and HeLa cells were co-cultured by MOI=100:1for1hour; the HeLa cell was gathered and washed with PBS. After destroying the cell membrane, plate culture count method was employed to estimate the ability of adhesion of bacterial on HeLa cell.(2) The effect of AHLs to the anti-serum bactericidal ability of tested bacteriaAdjust the test bacterial concentration to104CFU/ml, bacterial co-culture together with rabbit serum and guinea pig serum respectively for2h in37℃. Then the bacterial survival rate was compared by plate culture count method.(3) The effect of AHLs to the level of mRNA transcription of tested bacteriaAfter adding Cg-AHLs, the two groups bacterial were cultured still for1h. Then total RNA were extracted from the two groups of bacteria, to conduct the reverse transcription-Polymerase Chain Reaction (RT-PCR) for the research of the AHLs’effect on the expression of mRNA of resistance to complement killing genes (rck).Results:1. Investigation of the effects of S. typhi plasmid pRST98on biofilm formation.(1) Determinate the suitable temperature and time for biofilm formation. The ability of ST8to form BF cultured at30℃for3d was higher compared with37℃. Therefore,30℃for3d was determined as the optimum condition to develop BF.(2) Compare the biofilm formated by different S.typhi strains. Both wild-type ST8and pRST98-complementsd S. typhi strain ST8-c-pRST98could develop the ability to form BF. However, pRST98-deletion S. typhi strain ST8-⊿pRST98failed to do it. Semi-quantitative method also showed that OD570value were significantly higher in detecting BF formation of Wild-type ST8and pRST98-complementsd S. typhi strain ST8-c-pRST98than pRST98-deletion S. typhi strain ST8-⊿pRST98(P<0.05); while there had no significantly difference between ST8and ST8-c-pRST98(P>0.05).(3) Compare the adhesion ability of S. typhi strains. It appeared that no difference was observed in the ability of adhesion of bacterial on HeLa cell between wild-type ST8, pRST98-deletion S. typhi strain ST8-⊿pRST98or pRST98-complementsd S. typhi strain ST8-c-pRST98(P>0.05).2. The investigation of interact molecular mechanisms between S. typhi plasmid pRST98and QS system.(1) The effect of AHLs to the adhesion ability of tested bacteria. Pretreated with AHLs enhanced the ability of adhesion of bacterial on HeLa cell of wild-type ST8and pRST98-complementsd S. typhi strain ST8-c-pRST98when compared with pRST98-deletion S. typhi strain ST8-⊿pRST98(P<0.05); While there was no significant difference between the three strains in the control group in the ability of adhesion of bacterial on HeLa cell (P>0.05). Between the two groups the ability of adhesion of bacterial on HeLa cell of ST8+AHLs and ST8-c-pRST98+AHLs were higher than ST8+NS and ST8-c-pRST98+NS (P<0.05), While there was no significant difference between ST8-⊿pRST98+AHLs and ST8-⊿pRST98+NS (P>0.05).(2) The effect of AHLs to the anti-serum bactericidal ability of tested bacteria. Pretreated with AHLs wild-type ST8and pRsT98-complementsd S. typhi strain ST8-c-pRST98were obviously resistent to rabbit and guinea pig serum than pRST98-deletion S. typhi strain ST8-⊿pRsT98(P<0.01). In the control group, the difference was not so significant (P<0.05). Between the two groups the anti-serum bactericidal ability of ST8+AHLs and ST8-c-pRST98+AHLs were higher than ST8+NS and ST8-c-pRST98+NS (P<0.05), While there was no significant difference between ST8-⊿pRST98+AHLs and ST8-⊿pRST98+NS (P>0.05).(3) The effect of AHLs to the level of mRNA transcription of tested bacteria. RT-PCR showed that both wild-type ST8and pRST98-complementsd S. Typhi strain ST8-c-pRST98showed positive when treated with AHLs. Target gene band of the three strains was negative without AHLs involved.Conclusions:1. The S. typhi plasmid pRsT98is closely related to BF formation and it can promote BF formation.2. The S. typhi plasmid pRST98promotes BF formation, a process in which AHLs-SdiA-rck act on QS system.