傷寒沙門菌質粒對細菌生物膜形成的影響及分子機制研究
發(fā)布時間：2018-08-04 13:54
【摘要】：目的： 研究傷寒沙門菌(Salmonella enterica serovar Typhi, S. typhi)質粒pRST98與細菌生物膜(biofilm,BF)形成的關系,探討其與細菌密度感知系統(tǒng)(quorum-sensing,QS)相互作用的分子機制,為后續(xù)防治沙門菌感染及新藥靶位的發(fā)現(xiàn)提供理論和實驗依據(jù)。 方法： 1.傷寒沙門菌質粒對生物膜形成的影響 (1)生物膜形成適宜溫度和時間的確定用攜帶pRST98的傷寒沙門菌野生株ST8于30℃和37℃培養(yǎng)3d,用半定量法確定BF形成的最適溫度。將稀釋的菌液于30℃分別培養(yǎng)12h、1d、2d、3d、4d和5d,用半定量法確定BF成熟的時間。 (2)傷寒沙門菌不同菌株生物膜形成的比較分別用攜帶pRST98的傷寒沙門菌野生株ST8、消除pRST98的突變株ST8-"縫RST98和將pRST98經接合轉移重新導入ST8-"縫RST98所獲得的回補株ST8-c-pRST98,按上述實驗結果確定的條件建立BF模型。分別用結晶紫染色法、半定量法、掃描電鏡(scanning electron microscopy, SEM)觀察和共聚焦激光顯微鏡(confocal laser scanning microscopy, CLSM)斷層掃描比較受試菌形成BF的差異。 (3)傷寒沙門菌不同菌株對HeLa細胞粘附的比較 將實驗菌株ST8、ST8-"縫RST98和ST8-c-pRST98按感染復數(shù)(multiplicity of infection, MOI)100:1與HeLa細胞共培養(yǎng)1h后,收集細胞用PBS洗滌破膜后平板菌落計數(shù)法檢測集落形成單位(colony-forming unit, CFU)比較受試菌對細胞的粘附力。 2.傷寒沙門菌質粒與細菌密度感知系統(tǒng)相互作用的分子機制 將實驗菌株ST8、ST8-"縫RST98和ST8-c-pRST98分為兩組：一組加入1μM/mL的QS系統(tǒng)信號分子�；呓z氨酸內酯(N-acylhomoserine lactones, AHLs)商品化藥物辛�；呓z氨酸內酯(N-octanoyl-L-homoserine lactone, C8-AHLs);另一組加入等量的生理鹽水(normal saline, NS)作為對照,分別進行下述實驗。 (1)AHLs對受試菌粘附的影響 將受試菌按感染復數(shù)(multiplicity of infection, MOI)100:1與HeLa細胞共培養(yǎng),1h后收集細胞用PBS洗滌破膜后用平板菌落計數(shù)法檢測CFU比較受試菌對細胞的粘附力。 (2)AHLs對受試菌抗血清殺菌能力的影響 將受試菌液濃度調整至104CFU/ml,分別與兔和豚鼠血清37℃共作用2h后,用平板菌落計數(shù)法檢測CFU比較受試菌的存活數(shù)。 (3)AHLs對受試菌rck基因轉錄水平的影響 將兩組細菌37℃靜置培養(yǎng)1h,藥物和細菌充分作用后分別提取細菌的總RNA,進行逆轉錄聚合酶鏈反應(reverse transcription-Polymerase Chain Reaction, RT-PCR),檢測AHLs對補體殺菌抗性基因(resistance to complement killing genes, rck) mRNA轉錄水平的影響。 結果： 1.沙門菌質粒對生物膜形成的影響 (1)生物膜形成適宜溫度和時間的確定：菌株ST830℃培養(yǎng)比37℃形成BF能力強。30℃培養(yǎng)3d后BF生長趨于穩(wěn)定,故選擇30℃培養(yǎng)3d作為測定BF形成的條件。 (2)傷寒沙門菌不同菌株生物膜形成的比較：結晶紫染色、SEM及CLSM觀察顯示,傷寒沙門菌中野生株ST8和回補株ST8-c-pRST98均形成BF,而消除pRST98的傷寒沙門菌ST8-"縫RST98未見明顯BF形成。半定量法測定顯示,ST8和ST8-c-pRST98的0D570值均大于ST8-"縫RST98,差異有統(tǒng)計學意義(P0.05),ST8與ST8-c-pRST98的O0570值無統(tǒng)計學差異(P0.05)。 (3)傷寒沙門菌不同菌株對HeLa細胞的粘附比較：ST8、ST8-"縫RST98和ST8-C-pRsT98對HeLa細胞的粘附率未見明顯差異(P0.05)。 2.傷寒沙門菌質粒與密度感知系統(tǒng)相互作用的分子機制 (1) AHLs對受試菌粘附的影響：AHLs干預組ST8及ST8-c-pRST98對HeLa細胞的粘附率均高于ST8-"縫RST98,差異有統(tǒng)計學意義(P0.05),ST8及ST8-c-pRST98對HeLa細胞的粘附率無統(tǒng)計學差異(P0.05);對照組ST8、ST8-"縫RST98和ST8-c-pRST98對HeLa細胞的粘附率無統(tǒng)計學差異；兩組間ST8+AHLs與ST8-c-pRST98+AHLs對HeLa細胞的粘附率分別高于ST8+NS和ST8-c-pRST98+NS (P0.05),而ST8-"縫RST98+AHLs與ST8-"縫RST98+NS對HeLa細胞的粘附率無統(tǒng)計學差異(P0.05)。 (2) AHLs對受試菌抗血清殺菌能力的影響：AHLs干預組ST8和ST8-c-pRST98在兔和豚鼠血清中的存活率明顯高于ST8-"縫RST98(P0.01);對照組ST8和STg-c-pRST98在兔和豚鼠血清中的存活率高于ST8-"縫RST98(P0.05);兩組中ST8和ST8-c-pRST98在兔和豚鼠血清中的存活率均無統(tǒng)計學差異(P0.05)；兩組間ST8+AHLs與ST8-c-pRST98+AHLs在兔和豚鼠血清中的存活率分別高于ST8+NS和ST8-c-pRST98+NS(P0.05),而ST8-"縫RST98+AHLs與ST8-"縫RST98+NS在兔和豚鼠血清中的存活率無統(tǒng)計學差異(P0.05)。 (3) AHLs對受試菌rck基因轉錄水平的影響：RT-PCR結果顯示,AHLs干預組ST8和ST8-c-pRST98可見rck基因條帶,ST8-"縫RST98未見該基因條帶；對照組ST8、ST8-"縫RST98和ST8-c-pRST98均未見目的基因條帶。 結論： 1.傷寒沙門菌質粒pRST98與該菌BF形成密切相關,能促進細菌BF的形成。 2.傷寒沙門菌質粒PRST98可通過AHLs-SdiA-rck信號通路作用于QS系統(tǒng),促進細菌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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378.22
本文編號：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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378.22
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