| Lactobacilli are physiological intestinal microbiota of human and animal. Due to their health-promoting functions in the host, lactobacilli have been widely used in food and pharmaceutical industries. Many studies have found that lactobacilli are wrapped by a kind of surface layer proteins(SLPs). They can be attached to archaea and bacteria cell walls by non-covalent interactions. In recent years, SLPs are found to have outstanding contributions to the prebiotic functions of lactobacilli. But not all lactobacilli have the surface protein structure. Moreover, the studies of SLPs are mainly concentrated in some strains. For the rich resources of lactobacilli, the data resources of SLPs available for research are very limited.In this paper, some lactobacilli carrying SLPs were screened from a lot of Lactobacillus strains. The SLPs were extracted and their physicochemical properties were determined. The influence of SLPs on the surface properties, gastrointestinal tolerance, adhesion properties and antibacterial activity of lactobacilli were investigated to confirm the biological contributions of SLPs to the Lactobacillus strains. Finally, in order to understand the biological functions of Lactobacillus SLPs, the inhibition mechanism of different SLPs were further studied. All the findings would provide a theoretical basis for the applications of lactobacilli and their SLPs in the food and pharmaceutical industries.L. acidophilus NCFM was set as a reference strain, as it had been confirmed to possess SLPs. Thirty Lactobacillus strains to be screened were treated by Li Cl and the Li Cl extracts were analyzed by SDS-PAGE. The results showed that only six of the lactobacilli extracts had SLP bands. They were L. bulgaricus fb04, L. casei fb05, L. rhamnosus fb06, L. gasseri fb07, L. gasseri fb08 and L. acidophilus fb11. The relative molecular weights of the six SLPs were in the range of 45,000-47,000. Using transmission electron microscopy to observe the changes of the surface structures of Lactobacillus before and after Li Cl treatment, the surface structures of the above Lactobacillus were further confirmed.Ion exchange chromatography was used to purify the seven SLPs to obtain high purity SLPs for physiochemical properties analysis. Amino acid composition analysis showed that the amino acid compositions of the seven SLPs were similar. The seven SLPs all contained a large number of hydrophobic amino acids(31%-49%) and had more basic amino acids than acidic amino acids. All the results agreed with the general characteristics of SLPs reported in previous literatures. Circular dichroism was used to determine the secondary structure of the seven SLPs, wherein the secondary structures of the two L. gasseri and two L. acidophilus SLPs were similar, respectively. The secondary structures of other Lactobacillus SLPs were quite different. It was speculated that the secondary structures of L. gasseri and L. acidophilus SLPs were related with the strain affinity.Differential scanning calorimetry was used to measure the thermal denaturation temperature of the SLPs. The results showed that all SLPs had only one thermal denaturation temperature. L. bulgaricus fb04 SLP showed the lowest thermal denaturation temperature, 43.14 ± 0.82°C, while L. rhamnosus fb06 SLP showed the highest thermal denaturation temperature, 72.83 ± 0.60°C. The thermal denaturation temperatures of the other five SLPs were 59-71°C. From the comprehensive results of the secondary structure and thermal denaturation temperature, it could be found that the SLPs with high content of β-sheet would have a high thermal denaturation temperature.The biological contributions of SLPs to Lactobacillus strains were studied in many perspectives, such as surface hydrophobicity, surface charge, aggregation ability and gastrointestinal tolerance. After the SLPs were stripped, the surface hydrophobicity, surface charge, aggregation ability and gastrointestinal tolerance of the seven lactobacilli were decreased significantly(P <0.05). Under bile salt stress conditions, except L. bulgaricus fb04, other six Lactobacillus strains expressed 1.5-2.8 times SLPs than the control group. These results indicated that SLPs had positive contributions to the surface properties of these strains and could help lactobacilli resist adverse environment.Type I, type V collagens and HT-29 cells were set as intestinal tissue models to study the effects of SLPs on the adhesion property and antibacterial activity of Lactobacillus. After losing the SLPs, the adhesion ability of the seven lactobacilli to collagens and HT-29 cells was decreased by 37% to 95% and 63% to 94%, and their ability to inhibit Escherichia coli and Salmonella typhimurium invading HT-29 cells was also significantly weakened. In addition, the seven Lactobacillus SLPs could reduce the adhesion of E. coli and S. typhimurium to type I and type V collagen. The seven SLPs could also decline the adhesion of E. coli and S. typhimurium to HT-29 cells through exclusion, competition and displacement ways, and the exclusion way had the strongest effects. All the results suggested that the SLPs not only helped lactobacilli adhere to intestine and suppress intestinal bacterial infection, but also could be used as anti-adhesion agents to reduce the adhesion of E. coli and S. typhimurium to intestinal tissue.In order to reveal the inhibition mechanism of SLPs to pathogens infection, the hydrolase activities of the seven SLPs on E. coli and S. typhimurium cell wall peptidoglycan were studied. By measuring the growth curve and counting colony of pathogens, we found that only the SLPs of L. acidophilus NCFM and L. acidophilus fb11 could directly inhibit the growth of E. coli and S. typhimurium. Zymography results showed that the two L. acidophilus SLPs could hydrolyze the cell wall peptidoglycan of E. coli and S. typhimurium, suggesting that L. acidophilus SLPs may be a kind of peptidoglycan hydrolase. Transmission electron microscopy and flow cytometry analysis were used to detect the impact of the hydrolase activities on the morphological and physiological states of pathogens. The results showed that the murein hydrolase activity of SLPs damaged the cell wall of E. coli and S. typhimurium but the intracellular enzyme activity in the two pathogens still could be detected, which indicated a sublethal state. In the seven SLPs, this was a unique mechanism for L. acidophilus SLPs to inhibit pathogens.The inhibition mechanism of Lactobacillus SLPs to bacterial infection was investigated from a cell apoptosis perspective. First, by Hoechst 33342/PI double staining, L. casei fb05, L. rhamnosus fb06, L. acidophilus fb11 and L. acidophilus NCFM SLPs were observed to have the ability to reduce E. coli and S. typhimurium-caused chromatin condensation in HT-29 cells under fluorescence microscopy. Flow cytometry was used to analyze Annexin V-FITC/PI double staining cells, and the results showed that the four SLPs could reduce the early apoptosis in HT-29 cells caused by E. coli and S. typhimurium. The key factors in apoptosis pathway were measured. The results showed that the four SLPs could inhibit the E. coli and S. typhimurium-induced apoptosis in HT-29 cells by stabilizing mitochondrial membrane potential and intracellular Ca2+ levels, suppressing the activation of Caspase-1, Caspase-9 and Caspase-3. These results indicated that the four Lactobacillus SLPs could reduce the intestinal bacteria-induced apoptosis and damage in epithelial cells by regulating the mitochondria-mediated apoptotic pathway.In summary, seven lactobacilli carrying SLPs were selected and the physiochemical properties of the SLPs were analyzed. The positive contributions of SLPs to the surface properties, gastrointestinal tolerance, adhesion properties and antibacterial activity of lactobacilli were confirmed. The inhibition mechanism of different SLPs were explored on the cellular and molecular level. All the researches could provide a theoretical support for the practical application of lactobacilli and their SLPs in the field of health interventions. |
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