【作者】 李鹏成；

【导师】 杨倩；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2011， 博士

【摘要】 S-层蛋白(S-layer protein)是乳酸杆菌表面的一层蛋白分子,近年来发现该蛋白对肠道病原菌具有拮抗作用。肠道病原菌主要是通过粘附或入侵、诱导细胞骨架重排和信号转导通路、影响细胞凋亡达到入侵宿主上皮细胞的目的。拮抗和阻止病原菌的粘附及入侵作用是保护细胞免受感染的有效方法。本研究旨在探讨乳酸杆菌s-层蛋白拮抗肠道病原菌的作用机制。首先提取和纯化嗜酸乳酸杆菌的S-层蛋白,研究s-层蛋白对肠道病原菌的拮抗作用；然后应用人结肠腺癌细胞系(Caco-2细胞)作为体外模型,检测S-层蛋白拮抗鼠伤寒沙门氏菌粘附及入侵Caco-2细胞的影响,包括细菌的粘附入侵、细胞骨架的变化、信号转导通路和细胞凋亡中几种因子的变化等,以期揭示乳酸杆菌S-层蛋白拮抗肠道病原菌粘附或入侵宿主细胞机制。主要分为以下五个试验：试验一：嗜酸乳酸杆菌s-层蛋白的提取纯化本试验应用已知具有s-层蛋白的嗜酸乳酸杆菌ATCC4356作为对照,从蛋白和基因两个水平对36株猪十二肠分离得到的乳酸杆菌进行S-层蛋白菌株筛选,然后提取纯化S-层蛋白并制备抗血清。结果显示：36株乳酸杆菌均不存在S-层蛋白。本试验提取纯化得到嗜酸乳酸杆菌的S-层蛋白,应用该S-层蛋白接种新西兰兔,制备得到S-层蛋白抗血清,琼扩效价为1：16。本试验表明乳酸杆菌s-层蛋白只存在于某些菌株中。试验二：嗜酸乳酸杆菌S-层蛋白对肠道病原菌粘附或入侵的拮抗作用首先应用三种不同的粘附试验：竞争试验(competition assay)、排斥试验(exclusion assay)和置换试验(displacement assay),分别研究s-层蛋白对产肠毒素大肠杆菌K88和鼠伤寒沙门氏菌SL1344粘附或入侵Caco-2细胞的拮抗作用,然后应用Dot-blot方法检测S-层蛋白与两种细菌及Caco-2细胞的结合。结果显示：S-层蛋白能够显著协同产肠毒素大肠杆菌对Caco-2细胞的粘附。排斥试验和竞争试验的相对粘附力分别为452.30%±2.26%(P<0.01)和155.57%±5.81%(P<0.05)。S-层蛋白在排斥试验中,对产肠毒素大肠杆菌的协同粘附高于竞争试验。嗜酸乳酸杆菌也表现出明显的协同产肠毒素大肠杆菌粘附的效应。相反,S-层蛋白则能显著地拮抗鼠伤寒沙门氏菌的粘附及入侵。在竞争、排斥、置换三种粘附试验中,S-层蛋白可显著降低鼠伤寒沙门氏菌的粘附,其相对粘附力分别为1.17%±5.97%、8.71%±1.36%、10.56%±0.92%,差异极显著(P<0.01),其中竞争试验效果最好；并且S-层蛋白对鼠伤寒沙门氏菌粘附拮抗作用极显著高于嗜酸乳酸杆菌(P<0.01)；此外,S-层蛋白也能显著拮抗鼠伤寒沙门氏菌入侵。Dot-blot显示,S-层蛋白均可在两种细菌和Caco-2细胞表面自我组装。本试验表明,S-层蛋白协同增加产肠毒素性大肠杆菌对Caco-2细胞的粘附；却可显著拮抗鼠伤寒沙门氏菌对Caco-2细胞的粘附及入侵。S-层蛋白可能通过与鼠伤寒沙门氏菌竞争粘附受体,修饰细菌或Caco-2细胞表面相关结构介导拮抗作用。试验三：嗜酸乳酸杆菌S-层蛋白拮抗鼠伤寒沙门氏菌对细胞骨架的研究鼠伤寒沙门氏菌粘附或入侵宿主细胞过程中可显著引起细胞骨架变化。在试验二的基础上,本试验首先研究S-层蛋白对鼠伤寒沙门氏菌SL1344感染Caco-2细胞微绒毛和上皮完整性的变化；然后研究S-层蛋白抗感染过程中对Caco-2细胞微丝分布及其微丝相关调节蛋白Racl表达的影响。结果显示：鼠伤寒沙门氏菌感染Caco-2细胞后,可引起上皮细胞微绒毛变短,细胞微丝排列紊乱,细胞跨膜电阻下降。嗜酸乳酸杆菌S-层蛋白则能够拮抗鼠伤寒沙门氏菌对细胞微丝骨架的改变,并可减少鼠伤寒沙门氏菌引起Caco-2细胞跨膜电阻的破坏。鼠伤寒沙门氏菌可使Caco-2细胞Racl蛋白的表达量显著升高(0.39±0.02 VS 0.15±0.01；P<0.001),而S-层蛋白则可极显著上调感染鼠伤寒沙门氏菌后Caco-2细胞Rac1蛋白的表达(0.58±0.01 VS 0.39±0.02；P<0.01)。本试验表明嗜酸乳酸杆菌S-层蛋白可能通过拮抗鼠伤寒沙门氏菌对细胞微绒毛和微丝骨架的破坏；上调Rac1蛋白表达来拮抗鼠伤寒沙门氏菌的粘附及入侵。试验四：嗜酸乳酸杆菌S-层蛋白拮抗鼠伤寒沙门氏菌对细胞MAPK信号通路的研究丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)信号通路对病原菌的防御发挥重要作用。本试验研究了S-层蛋白拮抗鼠伤寒沙门氏菌SL1344过程中对Caco-2细胞MAPK信号通路三种重要亚族ERK、JNK和p38的影响。结果显示：鼠伤寒沙门氏菌感染Caco-2细胞后,ERK1/2 (0.34±0.02 VS 0.04±0.01; P<0.001)、JNK(1.77±0.19 VS 0.88±0.10；P<0.01)和p38(0.93±0.07 VS 0.72±0.02；P<0.05)的磷酸化水平均显著升高。S-层蛋白可明显增加Caco-2细胞ERK1/2的磷酸化(0.54±0.04；P<0.001),而不影响JNK与p38的活化。当S-层蛋白与鼠伤寒沙门氏菌共孵育后,细胞ERK1/2磷酸化水平(0.12±0.01)极显著低于分别单独作用细胞后的水平(P<0.001),并且S-层蛋白可以显著拮抗鼠伤寒沙门氏菌对Caco-2细胞JNK(0.64±0.05 VS1.77±0.19；P<0.01)和p38(0.61±0.04 VS 0.93±0.07；P<0.05)的磷酸化。本研究表明S-层蛋白可能阻断细菌对MAPK信号通路的活化发挥拮抗鼠伤寒沙门氏菌的作用。试验五：嗜酸乳酸杆菌S-层蛋白拮抗鼠伤寒沙门氏菌对细胞凋亡的研究病原菌可诱导上皮细胞凋亡,主要表现为推迟凋亡时间,引起凋亡损伤。本试验首先研究鼠伤寒沙门氏菌引起Caco-2细胞凋亡的途径；然后研究嗜酸乳酸杆菌S-层蛋白拮抗鼠伤寒沙门氏菌感染引起细胞凋亡的机理。结果显示：鼠伤寒沙门氏菌显著诱导Caco-2细胞的凋亡造成损伤。鼠伤寒沙门氏菌诱导Caco-2细胞凋亡并不激活Caspase-1,而是显著诱导Caspase-3的活化。S-层蛋白能够拮抗鼠伤寒沙门氏菌对Caco-2细胞凋亡引起的感染损伤。我们发现,S-层蛋白可以抑制Caco-2细胞Caspase-3的活化。本试验表明：鼠伤寒沙门氏菌是通过激活Caspase-3介导Caco-2细胞凋亡。S-层蛋白抑制Caspase-3的活化与试验四中S-层蛋白激活ERK1/2信号通路相符。嗜酸乳酸杆菌S-层蛋白可能通过抑制Caspase-3活性、激活ERK1/2信号通路,共同介导拮抗鼠伤寒沙门氏菌引起的细胞凋亡损伤。更多还原

【Abstract】 Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits present as the outermost component of the cell wall in several Lactobacillus species. In recent years, the S-layer proteins have showed the antagonistic activity on the enteropathogenic bacterias. Pathogens enter and invade host cell by adhesion, cell internalization, cytoskeleton disruption, interference of signalling pathways and cell apoptosis in their hosts. Inhibiting the bacterial pathogens adhesion is effective to protect host cell against infection. This study aims to investigate the possible antimicrobial mechanisms of S-layer proteins. Firstly Lactobacillus acidophilus (L. acidophilus) S-layer proteins were purified, antagonistic activity of L. acidophilus S-layer proteins on the adherence and invasion of enteropathogenic bacterias were detected. Thereafter, the influences of L. acidophilus S-layer proteins in Caco-2 cells (human colon cancer epithelial cells) are measured, including the pathogens adherence and invasion, cytoskeleton, the factors in signalling and apoptotic pathways. The results will reveal the mechanism of antagonistic activity of Lactobacillus S-layer proteins on pathogens. The details were divided into five experiments as follows:Experiment one:Purification of the S-layer protein from Lactobacillus acidophilusIn the present study,36 strains of Lactobacillus from pig duodenum were screened to extract S-layer proteins compared with L. acidophilus ATCC 4356 as associated the S-layer proteins. This screening work involved the two levels’examinations including protein and gene. The S-layer proteins were purified; then the rabbit-specific antiserum against the S-layer proteins was prepared. The results showed that 36 strains of Lactobacillus were not associated the S-layer proteins. To study the mechanism of Lactobacillus S-layer proteins in antimicrobial activity, S-layer proteins were purified from L. acidophilus ATCC 4356. Rabbit-anti-S-layer protein serum was obtained and its AGP (agar gel precipitation) titer was 1:16. The data supported that S-layer proteins only exist in certain lactobacilli.Experiment two:Antagonistic activity of S-layer proteins isolated from Lactobacillus acidophilus on the adherence and invasion of enteropathogenic bacteriasTo gain insight into the mechanism of Lactobacillus S-layer proteins in antimicrobial activity, we examined how L. acidophilus S-layer proteins inhibited the adherence and invasion of enterotoxigenic Escherichia coli K88 (ETEC K88) and Salmonella Typhimurium SL1344 (S. Typhimurium SL1344) in three different adhesive experiments (competition, exclusion, and displacement assays) in Caco-2 cells. Thereafter the binding between S-layer protein and the two pathogens or Caco-2 cells was detected by the Dot-blot. The results of adhesive experiments in Caco-2 cells demonstrated that compared with the control group, the S-layer proteins significantly increased the adherence of ETEC. The ability of adherence to Caco-2 cells was 452.30%±2.26% in the exclusion assay (P<0.01) and 155.57%±5.81% in the competition assay (P<0.05). Although this activity was demonstrated in the competition assay (coincubated with the S-layer proteins), a greater effect for S-layer proteins on ETEC was observed when ETEC was preincubated with the S-layer proteins. Furthermore, L. acidophilus also increased ETEC adherence in Caco-2 cells. In contrast, the S-layer proteins reduced S. Typhimurium association in Caco-2 cells. In the adhesive experiments (competitive, exclusive, displacement), the ability of adherence to Caco-2 cells were 1.17%±5.97%,8.71%±1.36% and 10.56%±0.92%, respectively (P< 0.01). The influence to inhibit the competitive adhesion of S. Typhimurium was optimal. Furthermore, the S-layer proteins showed a stronger effect than L. acidophilus to inhibit S. Typhimurium adhesion on Caco-2 monolayers (P<0.01). Moreover, Invasion of S. Typhimurium to Caco-2 monolayers was also inhibited by the S-layer proteins. Dot-blot showed that S-layer proteins could be bound directly to the Caco-2 cell line or be associated with ETEC and S. Typhimurium surface. The findings indicated that L. acidophilus S-layer proteins could increase the adherence of ETEC and inhibit the adherence and invasion of S. Typhimurium. The data support the antimicrobial mechanisms of L. acidophilus S-layer proteins, which are involved not only in a direct interaction between this protein and S. Typhimurium surface, but also in competition for binding sites on the surface of host epithelial cells. Experiment three:Study on Lactobacillus acidophilus S-layer protein-mediated inhibition of Salmonella Typhimurium-induced reorganization of cytoskeleton in vitroDuring S. Typhimurium adhere and invade to host-cell, reorganization of cytoskeleton is an important characteristic of infections. In the present study, effects of microvillous structure and epithelial cell integrity of S. Typhimurium SL1344-infected Caco-2 cells with S-layer proteins were detected. Thereafter, the influence of L. acidophilus S-layer proteins on Caco-2 cells was measured, including F-actin and the relative factor Racl. The results showed that shortened microvilli, F-actin rearrangement and transepithelial electrical resistance (TER) decrease were observed in Salmonella-infected Caco-2 cells. L. acidophilus S-layer proteins could inhibit these alternations in Caco-2 cells, which included that F-actin rearrangement was reduced and S. Typhimurium-induced microvilli damage was protected. In addition, S. Typhimurium-induced TER decrease was attenuated. The expression of Racl of S. Typhimurium-infected Caco-2 cells (0.39±0.02) was significantly increased compared with the uninfected cells (0.15±0.01; P<0.001). Interestingly, when Caco-2 infected with S. Typhimurium was coincubated with S-layer proteins, the expression of Racl (0.58±0.01) was also increased compared with the infected cells with S. Typhimurium alone (0.39±0.02; P<0.01). The study suggested that L. acidophilus S-layer proteins could be through blocking S. Typhimurium-induced microvilli damage and F-actin rearrangements, and up-regulating the expression of Racl to inhibit S. Typhimurium adherence and invasion in Caco-2 cells.Experiment Four:Study on Lactobacillus acidophilus S-layer protein-mediated inhibition of Salmonella Typhimurium-induced activation of MAPK signaling pathways in vitroMitogen-activated protein kinase (MAPK) signaling pathways are important in defending against pathogens. In this study, the extracellular signal-regulated kinases (ERKs) 1 and 2 (ERK1/2), c-Jun amino-terminal kinase (JNK) and p38 kinase MAPK activation were measured after S. Typhimurium SL1344 infection with L. acidophilus S-layer proteins in Caco-2 cells. The results showed that S. Typhimurium activated ERK1/2 (0.34±0.02 relative band density for infected cells versus 0.04±0.01 relative band density for control uninfected cells, P<0.001), JNK (1.77±0.19VS 0.88±0.10; P<0.01) and p38 (0.93±0.07 VS 0.72±0.02; P<0.05). We found that a significant increase in phospho-ERK1/2 activation after incubation with L. acidophilus S-layer proteins (0.54±0.04) compared with untreated Caco-2 cell monolayers (0.04±0.01; P< 0.001). However, S-layer proteins did not affect the JNK and p38 activation. Strikingly, when S-layer proteins and S. Typhimurium were coincubated with Caco-2 cells, the ERK1/2 phosphorylation (0.12±0.01) was markedly suppressed compared with each treatment alone (P<0.001). In addition, S-layer proteins could significantly suppress S. Typhimurium-induced JNK activation (0.64±0.05 for treated cells with S-layer proteins versus 1.77±0.19 for untreated cells, P<0.01) and p38 phosphorylation (0.61±0.04 for treated cells with S-layer proteins versus 0.93±0.07 for untreated cells, P<0.05). We demonstrated a novel role that L. acidophilus S-layer proteins were able to inhibit S. Typhimurium-induced ERK1/2, JNK and p38 activation.Experiment Five:Study on Lactobacillus acidophilus S-layer protein-mediated inhibition of Salmonella Typhimurium-induced apoptosis in vitroPathogens could induce epithelial cell apoptosis, which is linked to a delayed apoptotic response and cause cell damage. In the present study, firstly S. Typhimurium SL1344-induced apoptosis was detected in Caco-2 cells. Thereafter, the mechanism of L. acidophilus S-layer proteins inhibited against Salmonella-induced apoptosis was studied. The results showed that S. Typhimurium caused cell damage by the apoptotic pathway after infection in Caco-2 cells; and the apoptosis was mediated by activation of caspase-3, but not caspase-1. When S. Typhimurium and S-layer proteins were coincubated simultaneously, Caco-2 cell apoptosis was markedly decreased and the cell damage was modified. Detailed analyses showed that the S-layer proteins inhibited the caspase-3 activity. The results indicated that S. Typhimurium SL1344 induced Caco-2 cell apoptosis by the caspase-3 activation. Furthermore, S-layer protein-induced the inhibition of caspase-3 activation was consist with the results of S-layer protein-induced the ERK1/2 activation in experiment four. The findings support that S-layer proteins protected against Salmonella-induced apoptosis through reduced caspase-3 activation and increased ERK1/2 activation.更多还原