Bioinformatic analyses on CshA

The whole genome of Lp. plantarum SK156 (Genbank Accession No. CP059473) was analyzed by Hwang et al. [38]. Identification of classical anchor domains was performed using the Pfam [39] and InterPro [40] databases. Subcellular localization and the presence of signal peptides (SP) were predicted using SignalP v 6.0 [41]. Protein structural and functional predictions were performed using the I-TASSER online server [42,43,44]. BLASTp was used to compare the amino acid sequences against those of other bacteria (https://blast.ncbi.nlm.nih.gov/BLAST).

Bacterial strains and culture conditions

The bacterial strains used in this study are listed in Table 1. E. coli DH5α and E. coli BL21 (DE3) were used as cloning and expression hosts, respectively. E. coli strains were grown in Luria–Bertani (LB) broth (BD Difco, USA) supplemented with ampicillin (100 µg/mL) at 37 °C with aeration. LAB strains were cultured in Man Rogosa Sharpe (MRS) broth (BD Difco, USA) at 37 °C without aeration.

Table 1 Bacterial strains, plasmids, and primers used in this studyMolecular cloning

The plasmids and PCR primers used in this study are listed in Table 1. All PCRs were performed using Taq polymerase (TaKaRa, Tokyo, Japan). The cshA and sfGFP genes were amplified from the chromosomal DNA of Lp. plantarum SK156 and pCB4270B-sfGFP plasmids [45], respectively, using primers C1 and C2 (cshA), CS1 and CS2 (sfGFP with overlap), and S1 and S2 (sfGFP only). Amplicons were excised and cleaned from the agar gel using a NucleoSpin® Gel and PCR Clean-up Kit (Machery-Nagel, Düren, Germany). To generate cshA-sfGFP, purified cshA and sfGFP amplicons were used as templates for overlap PCR using the primers C1 and CS2. The enzyme restriction and ligation (T4 ligase) reactions were performed according to the manufacturer’s instructions (TaKaRa, Tokyo, Japan). The PCR products, sfGFP and cshA-sfGFP, were digested with NheI and XhoI, and then ligated into the NheI/XhoI sites of pET21b ( +) to construct pSfGFP and pCSHA-sfGFP, respectively. To check for sequence correctness, E. coli DH5α was transformed with either pSfGFP or pCSHA-sfGFP, according to the manufacturer’s protocol (Biofact, Daejeon, Republic of Korea). For protein overexpression, E. coli BL21 (DE3) was transformed with either pSfGFP or pCSHA-sfGFP according to the manufacturer’s protocol (Real BioTech, Taipei, Taiwan).

Protein overexpression and purification

E. coli BL21 (DE3) cells harboring either pSfGFP or pCSHA-sfGFP were grown overnight in LB broth supplemented with ampicillin (100 µg/mL) at 37 °C with aeration. Overnight cultures were then diluted 1:100 in LB broth with ampicillin and allowed to grow to an OD600 of 0.6. Protein overexpression was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture. After incubation at 25 °C for 6 h, cells were harvested by centrifugation at 10,000 × g for 10 min and then washed twice with phosphate buffer saline (PBS; pH 7). Cell pellets were resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 8) and disrupted using a sonicator for 6–7 cycles (10 s sonication, 15 s pause) on ice. After sonication, the clear lysate (for sfGFP protein) or pellet (for CshA-sfGFP protein) was collected by centrifugation at 13,000 × g for 20 min. The clear lysate was filtered using a 0.22-µm filter to remove cell debris, and the cell pellet was first solubilized with 8 M urea and then passed through a 0.22-µm filter. His-tag protein purification was performed as described by Spriestersbach et al. [46] under native conditions for the sfGFP protein or denaturing conditions for the CshA-sfGFP protein. Purified proteins were dialyzed in a protein storage buffer (50 mM Tris, 150 mM NaCl, 1 mM dithiothreitol, 30% glycerol, pH 8). The purified proteins were stored at − 20 °C until further use.

SDS-PAGE and western blotting

Bradford protein assay was performed to determine protein concentration (Bio-Rad, Germany). Protein expression was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomassie blue or transferred onto a 0.45-µm nitrocellulose membrane (Bio-Rad, Germany) at 400 mA for 90 min for western blot analysis. After transfer, the membrane was washed thrice with TBST (1 × Tris-buffered saline 0.1% Tween 20) and blocked with 5% bovine serum albumin (BSA) in TBST for 1 h at room temperature. Anti-His antibody (1:10,000 dilution in TBST with 2% BSA) was added as the primary antibody and incubated overnight at 4 °C with slight agitation. After exposure to the primary antibody, the membrane was washed thrice before incubation with HRP-conjugated anti-His antibody (Thermo Scientific, USA) for 1 h at room temperature. Detection was carried out using the SuperSignal® West Pico Chemiluminescent Substrate kit (Thermo Scientific, USA), following the manufacturer’s instructions, and then visualized with ChemiDoc™ XRS + and Image Lab™ software (Bio-Rad, Germany).

Surface display of CshA-sfGFP on LAB

Overnight cultures of LAB species were prepared for the binding experiments. One milliliter of each LAB culture was collected, centrifuged at 8000 × g for 10 min, and washed twice with PBS (pH 7). Harvested cells were incubated with either purified CshA-sfGFP or sfGFP proteins in binding buffer (1 × PBS, pH 7) at 37 °C for 2 h. Next, cells were collected by centrifugation at 10,000 × g for 5 min and washed twice with the binding buffer. The fluorescence intensity was determined using a spectrophotometer (SpectraMax, Molecular Diagnostics, USA) with excitation at 485 nm and emission at 511 nm. Cell background fluorescence was determined as relative fluorescence units (RFU). The fluorescence intensity was normalized by dividing the RFU values by OD600. The cell-surface display was visualized using a Nikon Eclipse 80i with a GFP filter (Nikon, New York, USA).

Surface display of CshA-sfGFP on pretreated Lm. fermentum cells

Chemical pretreatment of the cell surface of L. fermentum was performed according to previously described methods [22, 23, 47]. Briefly, 1 mL of overnight Lm. fermentum cultures were harvested by centrifugation at 8000 × g for 10 min and washed twice with PBS (pH 7). Harvested cells were treated with the following chemicals and conditions: 5 M LiCl and 10% TCA at 37 °C for 1 h; 10% TCA, 5% TCA, 0.01 M HCl, 5.6 M acetic acid, 0.72 M lactic acid and 10% SDS at 100 °C for 10 min; and 90% acetone at room temperature for 10 min. Cells were collected and washed twice with PBS to remove residual chemicals prior to binding experiments.

Factors affecting the display of CshA-sfGFP on Lm. fermentum

Lm. fermentum was grown overnight in MRS broth until it reached an OD600 of ~ 1.8. Cell cultures were prepared and pretreated with 5% TCA as described above. To investigate the effect of NaCl concentration and pH on the display of CshA-sfGFP, a binding experiment was performed using binding buffer with either varying concentrations of NaCl (0, 100, 200, 300, 400, and 500 mM) or varying pH levels (4.5–11). To determine the optimal binding temperature and time, binding experiments were performed at different temperatures (25, 30, and 37 °C) at different time points (0.5, 1, 1.5, 2, and 3 h).

Binding capacity of CshA on Lm. fermentum

To determine the binding capacity of CshA to Lm. fermentum, the methods from Tay et al. [22] were adapted for this study. Briefly, the binding experiment was performed with different concentrations of CshA-sfGFP protein (0, 0.5, 1, 2, 3, 4, and 5 µM). The relative fluorescence values for each point were determined and fitted to a nonlinear curve, and the Bmax and R2 values were calculated. A standard curve using the free CshA-sfGFP protein was created to determine the protein concentration at a specific Bmax value. Uniformity of the distribution of bound proteins in the cells was assumed.

Surface display retention of CshA-sfGFP on Lm. fermentum under various conditions

To test the display retention of CshA-sfGFP on Lm. fermentum, the method described by Gordillo et al. [37] was performed with modifications. The binding experiments were performed as described above. Pretreated Lm. fermentum cells displaying CshA-sfGFP were collected and subsequently incubated in PBS at varying pH levels (3–5) or bile salt concentrations (0.25, 0.50, and 1%) at 37 °C for 2 h to simulate the conditions of the GIT. As a control, the CshA-decorated Lm. fermentum were incubated in PBS at pH 7 without bile salts. After incubation, the cells were washed twice and collected to determine the fluorescence intensity.

Statistical analyses

All statistical analyses in this study were performed using GraphPad Prism version 8.4.2 for Windows (GraphPad Software, San Diego, California, USA). One-way ANOVA with Tukey’s test was performed to determine significant differences in the binding studies. Differences were considered statistically significant at P < 0.05. Nonlinear regression was performed to calculate Bmax using the one-site binding model in GraphPad Prism. All experimental assays were performed in triplicate. All values are reported as mean ± standard deviation (SD).