Fig 1.
The hydrolysis of TJs in Caco-2 cells by rTsASP2 enhanced paracellular permeability.
(A) The purified rTsASP2 was analysed by SDS-PAGE. M: protein molecular weight marker; 1: rTsASP2 purified by Ni-NTA Spin Kit. (B) The level of Occludin and Claudin-1 in rTsASP2-treated Caco-2 cell lysates was analysed by Western blot and quantified by ImageJ (n = 3). (C) The level of Occludin and Claudin-1 in rTsASP2-treated Caco-2 monolayer was analysed by Western blot and quantified by ImageJ. GAPDH served as the loading control (n = 3). (D) TEER of cell monolayers pre- and post-rTsASP2 treatment (n = 7–9). (E) The permeability of 4 kDa-FITC dextran was increased after Caco-2 monolayer treated with rTsASP2, representative results from one out of three independent experiments with n = 3. The data shown are means ± SD. *P < 0.05, **P < 0.01 versus the PBS group.
Fig 2.
Construction and identification of the recombinant L. lactis- pNZ8149/TsASP2/NZ3900.
(A) Schematic of the pNZ8149/TsASP2 plasmid. (B) Positive clones selected as yellow colonies on Elliker selective agar. (C) PCR verification of the plasmid pNZ8149/TsASP2. M: DNA marker; 1–3: PCR product of TsASP2. (D) Restriction enzyme digestion verification of the plasmid pNZ8149/TsASP2. M: DNA marker; 1–3: digested products of pNZ8149/TsASP2 with NcoI and XbaI; the arrow indicated TsASP2. (E) SDS-PAGE and (F) Western blot analysis of rTsASP2 expression in recombinant L. lactis. SDS-PAGE revealed a protein of approximately 43.4 kDa specifically in recombinant L. lactis (E, 3), identified as rTsASP2 by Western blot (F, 3). This band was absent in L. lactis with pNZ8149 vector (1) and the non-induced recombinant L. lactis (2), confirming successful induction and specific immunodetection. the arrow indicated the rTsASP2.
Fig 3.
Biological characteristics of recombinant L. lactis.
(A) The growth curves of NZ3900, pNZ8149/NZ3900, and pNZ8149/TsASP2/NZ3900. Data are shown as mean ± SD (n = 3). (B) PCR products of 5, 10, 15, 20, 25, 30 generations of recombinant L. lactis. M: DNA marker. (C) PCR products from 20 individual colonies of the 30th generation. M: DNA marker. (D) Tolerance of recombinant L. lactis in acid environment. Data are shown as mean ± SD (n = 3). Results were from one of two experiments performed showing similar results.
Fig 4.
Colonization characteristics of recombinant L. lactis.
(A) Schematic of recombinant L. lactis- treated and sample- collected process. (B) PCR products amplified from fecal DNA of normal mice. (C) PCR products amplified from ileal (1), cecal (2), and colonic (3) contents DNA of normal mice. (D) PCR products amplified from fecal DNA of mice with flora clearance. (E) PCR products amplified from ileal (1), cecal (2), and colonic (3) contents DNA of mice with flora clearance. (F) Positive green fluorescence staining was clearly observed in intestinal epithelium of mice immunized with recombinant L. lactis. There was no positive fluorescence staining in two control groups. The nuclei of intestinal cells were stained blue by DAPI. Scale bar = 100 μm. Results were from one of three experiments performed showing similar results.
Fig 5.
Recombinant L. lactis destroy the intestinal barrier.
(A) Schematic of recombinant L. lactis- treated process. (B) The histopathological changes in the duodenum were examined by H&E staining. Scale bars = 200 μm. (C) Width of gut villus in each high-power field was determined using the ImageJ software for the duodenum samples. Data shown are means ± SD (n = 4) from one of two experiments performed showing similar results. (D) Transmission electron microscopy was used to observe the junction complex between intestinal epithelial cells. T: tight junction, ZA: intermediate junction, Des: desmosome, M: microvilli, Mito: mitochondria. Damaged T and ZA were indicated by green dashed circles. Scale bars = 500 nm. (E) Mice were gavaged with 50 mg/mL FD4, and its level in the serum was assessed 4 hours later. Data shown are means ± SD (n = 3–4) from one of two experiments performed showing similar results. (F) Invaded larvae in the monolayer. Arrows indicated the larval migratory traces. (G) Non-invaded larvae. Scale bars = 100 μm. (H) Facilitation of rTsASP2 on larval invasion. Data shown are means ± SD (n = 3) from one of two experiments performed showing similar results. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus the PBS group. A-E: PBS indicates PBS treated; pNZ indicates pNZ8149/NZ3900 treated; TsASP2 indicates pNZ8149/TsASP2/NZ3900 treated.
Fig 6.
Recombinant L. lactis exacerbated DSS-induced colitis.
(A) Schedule of recombinant L. lactis- treated and DSS-induced colitis process. (B) Weight change in percent. (C) The changes in disease activity index (DAI), which included diarrhea, bleeding and body weight loss. (D) Average daily water intake per group of mice during DSS treatment. (E) Macroscopic appearance of the colon. (F) The colon length in each group. (G) The histopathological changes in the colon tissues were examined by H&E staining. Scale bars = 200 μm. (H) Histopathological scores were determined for the colon tissue samples. (I) Gating strategy of peripheral blood (CD45+Ly6G+CD11b+) in flow cytometry analysis (FACS). (J) The percentage of neutrophils in peripheral blood. (K) Gating strategy of spleen in FACS. (L)The percentage of neutrophils in spleen. The data shown are means ± SD. Representative results from one out of two independent experiments with n = 5–6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the pNZ-DSS group; #P < 0.05, ##P < 0.01, ####P < 0.0001 versus the respective control group; ns: no significant difference. pNZ indicates pNZ8149/NZ3900 treated; TsASP2 indicates pNZ8149/TsASP2/NZ3900 treated; pNZ-DSS indicates pNZ8149/NZ3900 treated and DSS induced colitis; TsASP2-DSS indicates pNZ8149/TsASP2/NZ3900 treated and DSS induced colitis.
Table 1.
Sequences of peptides of rhOccludin generated by hydrolysis using rTsASP2.
Fig 7.
Proteolytic activity of rTsASP2 on rhOccludin.
(A) Molecular docking model of TsASP2-Occludin interaction. TsASP2 was shown in cyan and Occludin in green; key interfacial residues were represented as sticks within the dashed box; hydrogen bonds were indicated by yellow dashed lines. (B) SDS-PAGE analysis of the co-incubation protein products of rTsASP and rhOccludin. M: protein molecular weight marker. Arrows indicated the two major protein bands specific to the hydrolysis products (absent in both the rhOccludin and rTsASP2 controls): the blue arrow indicated Band 1, and the orange arrow indicated Band 2. (C) Mapping rTsASP2 cleavage sites on rhOccludin. Black text denoted rhOccludin amino acid sequence; the blue arrow indicated the hydrolysis site identified by integrating sequencing data from Band 1; the orange arrow indicated the hydrolysis site identified by integrating sequencing data from Band 2.