| 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. |
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