伤寒沙门菌质粒对细菌生物膜形成的影响及分子机制研究
发布时间：2018-08-04 13:54
【摘要】：目的： 研究伤寒沙门菌(Salmonella enterica serovar Typhi, S. typhi)质粒pRST98与细菌生物膜(biofilm,BF)形成的关系,探讨其与细菌密度感知系统(quorum-sensing,QS)相互作用的分子机制,为后续防治沙门菌感染及新药靶位的发现提供理论和实验依据。 方法： 1.伤寒沙门菌质粒对生物膜形成的影响 (1)生物膜形成适宜温度和时间的确定用携带pRST98的伤寒沙门菌野生株ST8于30℃和37℃培养3d,用半定量法确定BF形成的最适温度。将稀释的菌液于30℃分别培养12h、1d、2d、3d、4d和5d,用半定量法确定BF成熟的时间。 (2)伤寒沙门菌不同菌株生物膜形成的比较分别用携带pRST98的伤寒沙门菌野生株ST8、消除pRST98的突变株ST8-"縫RST98和将pRST98经接合转移重新导入ST8-"縫RST98所获得的回补株ST8-c-pRST98,按上述实验结果确定的条件建立BF模型。分别用结晶紫染色法、半定量法、扫描电镜(scanning electron microscopy, SEM)观察和共聚焦激光显微镜(confocal laser scanning microscopy, CLSM)断层扫描比较受试菌形成BF的差异。 (3)伤寒沙门菌不同菌株对HeLa细胞粘附的比较 将实验菌株ST8、ST8-"縫RST98和ST8-c-pRST98按感染复数(multiplicity of infection, MOI)100:1与HeLa细胞共培养1h后,收集细胞用PBS洗涤破膜后平板菌落计数法检测集落形成单位(colony-forming unit, CFU)比较受试菌对细胞的粘附力。 2.伤寒沙门菌质粒与细菌密度感知系统相互作用的分子机制 将实验菌株ST8、ST8-"縫RST98和ST8-c-pRST98分为两组：一组加入1μM/mL的QS系统信号分子酰基高丝氨酸内酯(N-acylhomoserine lactones, AHLs)商品化药物辛酰基高丝氨酸内酯(N-octanoyl-L-homoserine lactone, C8-AHLs);另一组加入等量的生理盐水(normal saline, NS)作为对照,分别进行下述实验。 (1)AHLs对受试菌粘附的影响 将受试菌按感染复数(multiplicity of infection, MOI)100:1与HeLa细胞共培养,1h后收集细胞用PBS洗涤破膜后用平板菌落计数法检测CFU比较受试菌对细胞的粘附力。 (2)AHLs对受试菌抗血清杀菌能力的影响 将受试菌液浓度调整至104CFU/ml,分别与兔和豚鼠血清37℃共作用2h后,用平板菌落计数法检测CFU比较受试菌的存活数。 (3)AHLs对受试菌rck基因转录水平的影响 将两组细菌37℃静置培养1h,药物和细菌充分作用后分别提取细菌的总RNA,进行逆转录聚合酶链反应(reverse transcription-Polymerase Chain Reaction, RT-PCR),检测AHLs对补体杀菌抗性基因(resistance to complement killing genes, rck) mRNA转录水平的影响。 结果： 1.沙门菌质粒对生物膜形成的影响 (1)生物膜形成适宜温度和时间的确定：菌株ST830℃培养比37℃形成BF能力强。30℃培养3d后BF生长趋于稳定,故选择30℃培养3d作为测定BF形成的条件。 (2)伤寒沙门菌不同菌株生物膜形成的比较：结晶紫染色、SEM及CLSM观察显示,伤寒沙门菌中野生株ST8和回补株ST8-c-pRST98均形成BF,而消除pRST98的伤寒沙门菌ST8-"縫RST98未见明显BF形成。半定量法测定显示,ST8和ST8-c-pRST98的0D570值均大于ST8-"縫RST98,差异有统计学意义(P0.05),ST8与ST8-c-pRST98的O0570值无统计学差异(P0.05)。 (3)伤寒沙门菌不同菌株对HeLa细胞的粘附比较：ST8、ST8-"縫RST98和ST8-C-pRsT98对HeLa细胞的粘附率未见明显差异(P0.05)。 2.伤寒沙门菌质粒与密度感知系统相互作用的分子机制 (1) AHLs对受试菌粘附的影响：AHLs干预组ST8及ST8-c-pRST98对HeLa细胞的粘附率均高于ST8-"縫RST98,差异有统计学意义(P0.05),ST8及ST8-c-pRST98对HeLa细胞的粘附率无统计学差异(P0.05);对照组ST8、ST8-"縫RST98和ST8-c-pRST98对HeLa细胞的粘附率无统计学差异；两组间ST8+AHLs与ST8-c-pRST98+AHLs对HeLa细胞的粘附率分别高于ST8+NS和ST8-c-pRST98+NS (P0.05),而ST8-"縫RST98+AHLs与ST8-"縫RST98+NS对HeLa细胞的粘附率无统计学差异(P0.05)。 (2) AHLs对受试菌抗血清杀菌能力的影响：AHLs干预组ST8和ST8-c-pRST98在兔和豚鼠血清中的存活率明显高于ST8-"縫RST98(P0.01);对照组ST8和STg-c-pRST98在兔和豚鼠血清中的存活率高于ST8-"縫RST98(P0.05);两组中ST8和ST8-c-pRST98在兔和豚鼠血清中的存活率均无统计学差异(P0.05)；两组间ST8+AHLs与ST8-c-pRST98+AHLs在兔和豚鼠血清中的存活率分别高于ST8+NS和ST8-c-pRST98+NS(P0.05),而ST8-"縫RST98+AHLs与ST8-"縫RST98+NS在兔和豚鼠血清中的存活率无统计学差异(P0.05)。 (3) AHLs对受试菌rck基因转录水平的影响：RT-PCR结果显示,AHLs干预组ST8和ST8-c-pRST98可见rck基因条带,ST8-"縫RST98未见该基因条带；对照组ST8、ST8-"縫RST98和ST8-c-pRST98均未见目的基因条带。 结论： 1.伤寒沙门菌质粒pRST98与该菌BF形成密切相关,能促进细菌BF的形成。 2.伤寒沙门菌质粒PRST98可通过AHLs-SdiA-rck信号通路作用于QS系统,促进细菌BF的形成。
[Abstract]:Objective:
The relationship between the plasmid pRST98 of Salmonella enterica serovar Typhi (S. typhi) and the formation of bacterial biofilm (biofilm, BF) was studied, and the molecular mechanism of its interaction with the bacterial density sensing system (quorum-sensing, QS) was explored to provide theoretical and experimental basis for the subsequent prevention and treatment of Salmonella infection and the discovery of new drug targets.
Method:
The effect of 1. Salmonella typhi plasmid on the formation of biofilm
(1) the optimum temperature and time of the biofilm were established to determine the optimum temperature of the ST8 at 30 and 37 C with the wild strain of Salmonella typhi carrying pRST98 at 30 and 37 C. The diluted bacterial liquid was cultured for 12h, 1D, 2D, 3D, 4D and 5D respectively at 30, and the period of BF maturation was determined by semi quantitative method.
(2) the comparison of the biofilm formation of different strains of Salmonella typhi was compared with the wild strain ST8 of Salmonella typhi carrying pRST98, respectively, to eliminate the ST8-c-pRST98 of the pRST98 mutant ST8- "sewn RST98 and pRST98 through the joint transfer to ST8-" RST98. The BF model was established according to the conditions determined above. The staining, semi quantitative, scanning electron microscopy (scanning electron microscopy, SEM) observation and confocal laser microscopy (confocal laser scanning microscopy, CLSM) scanning were used to compare the differences in the formation of BF of the tested bacteria.
(3) comparison of the adhesion of different strains of Salmonella typhi to HeLa cells
The experimental strain ST8, ST8- "sewn RST98 and ST8-c-pRST98" were co culture 1H on the multiplicity of infection, MOI) 100:1 and HeLa cells, and the cell colony formation unit was detected by the plate colony counting method after the scrubbing of the membrane.
2. molecular mechanism of interaction between Salmonella typhimurium plasmid and bacterial density sensing system
The experimental strain ST8, ST8- "sewn RST98 and ST8-c-pRST98" were divided into two groups: a group of QS system signaling molecules added to the QS system, the acyl high serine lactone (N-acylhomoserine lactones, AHLs), a commercialized drug of N-acylhomoserine lactones (N-octanoyl-L-homoserine lactone, C8-AHLs); the other group was added to the same amount of physiological saline. S) as a control, the following experiments were carried out respectively.
(1) the effect of AHLs on the adhesion of the tested bacteria
The tested bacteria were co cultured with the multiplicity of infection (MOI) 100:1 and HeLa cells. After 1h, the cells were washed and broken by PBS, and the adhesion of the tested bacteria to the cells was measured by the plate colony counting method.
(2) the effect of AHLs on the antisera bactericidal ability of the tested bacteria
After adjusting the concentration of the tested bacterial solution to 104 CFU/ml and co-acting with rabbit and guinea pig serum at 37 C for 2 hours, the survival number of the tested bacteria was compared with that of the CFU by plate colony counting method.
(3) the effect of AHLs on the transcriptional level of the RCK gene of the tested bacteria
Two groups of bacteria were incubated at 37 degrees centigrade for 1h, and the total RNA of bacteria was extracted after the full action of the drugs and bacteria, and the reverse transcriptase polymerase chain reaction (reverse transcription-Polymerase Chain Reaction, RT-PCR) was used to detect the effect of AHLs on the transcriptional level of the complement bactericidal resistance gene (resistance to complement killing).
Result:
The effect of 1. Salmonella plasmids on the formation of biofilm
(1) to determine the suitable temperature and time of biofilm, the growth of BF growth tends to be stable after the strain of ST830 C at 37 C and the ability to form BF at.30 C for 3D, so that 3D at 30 C is selected as the condition for determining BF formation.
(2) comparison of the biofilm formation of different strains of Salmonella typhi: crystal violet staining, SEM and CLSM observation showed that both ST8 and ST8-c-pRST98 of Salmonella typhi were BF, and ST8- "ST8-" of Salmonella typhi to eliminate pRST98 was not formed in BF. Semi quantitative determination showed that 0D570 values of ST8 and ST8-c-pRST98 were greater than those of pRST98. There was a significant difference in RST98 between the two joints (P0.05), and there was no significant difference in O0570 between ST8 and ST8-c-pRST98 (P0.05).
(3) Comparison of adhesion of different strains of Salmonella typhi to HeLa cells: There was no significant difference in adhesion rate of ST8, ST8-"suture RST98" and ST8-C-pRsT98 to HeLa cells (P 0.05).
2. molecular mechanism of interaction between plasmid and density sensing system of Salmonella typhi
(1) the effect of AHLs on the adhesion of the tested bacteria: the adhesion rate of ST8 and ST8-c-pRST98 to HeLa cells in the AHLs intervention group was higher than that of ST8- "RST98, the difference was statistically significant (P0.05), and there was no statistical difference between ST8 and ST8-c-pRST98 on HeLa cells (P0.05), but there was no statistical difference between the control group. The adhesion rates of ST8+AHLs and ST8-c-pRST98+AHLs between the two groups were higher than that of ST8+NS and ST8-c-pRST98+NS (P0.05), while ST8- "sewn RST98+AHLs and ST8-" slit RST98+NS had no significant difference between the adherence rate of HeLa cells (P0.05).
(2) the effect of AHLs on the antisera bactericidal ability of the tested bacteria: the survival rate of ST8 and ST8-c-pRST98 in the serum of rabbits and guinea pigs in the AHLs intervention group was significantly higher than that of ST8- "sewn RST98 (P0.01)", and the survival rate of ST8 and STg-c-pRST98 in the serum of rabbits and guinea pigs in the control group was higher than that of ST8- "ST8-" RST98 (P0.05), and the two groups were in the rabbit and guinea pig serum. There was no significant difference in survival rate (P0.05). The survival rate of ST8+AHLs and ST8-c-pRST98+AHLs in the serum of rabbits and guinea pigs between two groups was higher than that of ST8+NS and ST8-c-pRST98+NS (P0.05), while the survival rate of ST8- "sewn RST98+AHLs and ST8-" slit RST98+NS in rabbit and guinea pig serum had no statistical difference (P0.05).
(3) the effect of AHLs on the transcriptional level of the RCK gene of the tested bacteria: RT-PCR results showed that the RCK gene bands were found in ST8 and ST8-c-pRST98 in the AHLs intervention group, and ST8- "sewn RST98 was not the band of the gene, while the control group ST8, ST8-" suture RST98 and the target gene bands were not found.
Conclusion:
1. plasmid pRST98 of Salmonella typhi is closely related to the formation of BF, and can promote the formation of bacterial BF.
2. plasmid PRST98 of Salmonella typhi can promote the formation of bacterial BF through the AHLs-SdiA-rck signaling pathway in QS system.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.22
本文编号：2164092
[Abstract]:Objective:
The relationship between the plasmid pRST98 of Salmonella enterica serovar Typhi (S. typhi) and the formation of bacterial biofilm (biofilm, BF) was studied, and the molecular mechanism of its interaction with the bacterial density sensing system (quorum-sensing, QS) was explored to provide theoretical and experimental basis for the subsequent prevention and treatment of Salmonella infection and the discovery of new drug targets.
Method:
The effect of 1. Salmonella typhi plasmid on the formation of biofilm
(1) the optimum temperature and time of the biofilm were established to determine the optimum temperature of the ST8 at 30 and 37 C with the wild strain of Salmonella typhi carrying pRST98 at 30 and 37 C. The diluted bacterial liquid was cultured for 12h, 1D, 2D, 3D, 4D and 5D respectively at 30, and the period of BF maturation was determined by semi quantitative method.
(2) the comparison of the biofilm formation of different strains of Salmonella typhi was compared with the wild strain ST8 of Salmonella typhi carrying pRST98, respectively, to eliminate the ST8-c-pRST98 of the pRST98 mutant ST8- "sewn RST98 and pRST98 through the joint transfer to ST8-" RST98. The BF model was established according to the conditions determined above. The staining, semi quantitative, scanning electron microscopy (scanning electron microscopy, SEM) observation and confocal laser microscopy (confocal laser scanning microscopy, CLSM) scanning were used to compare the differences in the formation of BF of the tested bacteria.
(3) comparison of the adhesion of different strains of Salmonella typhi to HeLa cells
The experimental strain ST8, ST8- "sewn RST98 and ST8-c-pRST98" were co culture 1H on the multiplicity of infection, MOI) 100:1 and HeLa cells, and the cell colony formation unit was detected by the plate colony counting method after the scrubbing of the membrane.
2. molecular mechanism of interaction between Salmonella typhimurium plasmid and bacterial density sensing system
The experimental strain ST8, ST8- "sewn RST98 and ST8-c-pRST98" were divided into two groups: a group of QS system signaling molecules added to the QS system, the acyl high serine lactone (N-acylhomoserine lactones, AHLs), a commercialized drug of N-acylhomoserine lactones (N-octanoyl-L-homoserine lactone, C8-AHLs); the other group was added to the same amount of physiological saline. S) as a control, the following experiments were carried out respectively.
(1) the effect of AHLs on the adhesion of the tested bacteria
The tested bacteria were co cultured with the multiplicity of infection (MOI) 100:1 and HeLa cells. After 1h, the cells were washed and broken by PBS, and the adhesion of the tested bacteria to the cells was measured by the plate colony counting method.
(2) the effect of AHLs on the antisera bactericidal ability of the tested bacteria
After adjusting the concentration of the tested bacterial solution to 104 CFU/ml and co-acting with rabbit and guinea pig serum at 37 C for 2 hours, the survival number of the tested bacteria was compared with that of the CFU by plate colony counting method.
(3) the effect of AHLs on the transcriptional level of the RCK gene of the tested bacteria
Two groups of bacteria were incubated at 37 degrees centigrade for 1h, and the total RNA of bacteria was extracted after the full action of the drugs and bacteria, and the reverse transcriptase polymerase chain reaction (reverse transcription-Polymerase Chain Reaction, RT-PCR) was used to detect the effect of AHLs on the transcriptional level of the complement bactericidal resistance gene (resistance to complement killing).
Result:
The effect of 1. Salmonella plasmids on the formation of biofilm
(1) to determine the suitable temperature and time of biofilm, the growth of BF growth tends to be stable after the strain of ST830 C at 37 C and the ability to form BF at.30 C for 3D, so that 3D at 30 C is selected as the condition for determining BF formation.
(2) comparison of the biofilm formation of different strains of Salmonella typhi: crystal violet staining, SEM and CLSM observation showed that both ST8 and ST8-c-pRST98 of Salmonella typhi were BF, and ST8- "ST8-" of Salmonella typhi to eliminate pRST98 was not formed in BF. Semi quantitative determination showed that 0D570 values of ST8 and ST8-c-pRST98 were greater than those of pRST98. There was a significant difference in RST98 between the two joints (P0.05), and there was no significant difference in O0570 between ST8 and ST8-c-pRST98 (P0.05).
(3) Comparison of adhesion of different strains of Salmonella typhi to HeLa cells: There was no significant difference in adhesion rate of ST8, ST8-"suture RST98" and ST8-C-pRsT98 to HeLa cells (P 0.05).
2. molecular mechanism of interaction between plasmid and density sensing system of Salmonella typhi
(1) the effect of AHLs on the adhesion of the tested bacteria: the adhesion rate of ST8 and ST8-c-pRST98 to HeLa cells in the AHLs intervention group was higher than that of ST8- "RST98, the difference was statistically significant (P0.05), and there was no statistical difference between ST8 and ST8-c-pRST98 on HeLa cells (P0.05), but there was no statistical difference between the control group. The adhesion rates of ST8+AHLs and ST8-c-pRST98+AHLs between the two groups were higher than that of ST8+NS and ST8-c-pRST98+NS (P0.05), while ST8- "sewn RST98+AHLs and ST8-" slit RST98+NS had no significant difference between the adherence rate of HeLa cells (P0.05).
(2) the effect of AHLs on the antisera bactericidal ability of the tested bacteria: the survival rate of ST8 and ST8-c-pRST98 in the serum of rabbits and guinea pigs in the AHLs intervention group was significantly higher than that of ST8- "sewn RST98 (P0.01)", and the survival rate of ST8 and STg-c-pRST98 in the serum of rabbits and guinea pigs in the control group was higher than that of ST8- "ST8-" RST98 (P0.05), and the two groups were in the rabbit and guinea pig serum. There was no significant difference in survival rate (P0.05). The survival rate of ST8+AHLs and ST8-c-pRST98+AHLs in the serum of rabbits and guinea pigs between two groups was higher than that of ST8+NS and ST8-c-pRST98+NS (P0.05), while the survival rate of ST8- "sewn RST98+AHLs and ST8-" slit RST98+NS in rabbit and guinea pig serum had no statistical difference (P0.05).
(3) the effect of AHLs on the transcriptional level of the RCK gene of the tested bacteria: RT-PCR results showed that the RCK gene bands were found in ST8 and ST8-c-pRST98 in the AHLs intervention group, and ST8- "sewn RST98 was not the band of the gene, while the control group ST8, ST8-" suture RST98 and the target gene bands were not found.
Conclusion:
1. plasmid pRST98 of Salmonella typhi is closely related to the formation of BF, and can promote the formation of bacterial BF.
2. plasmid PRST98 of Salmonella typhi can promote the formation of bacterial BF through the AHLs-SdiA-rck signaling pathway in QS system.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.22
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