The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158–172aa and UreB349–363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.


Introduction
Bacterial cell surface display allows for the production of target biomolecules, such as peptides or proteins on the bacterial surface.This technology has been investigated and developed for several applications, including peptide library screening and whole-cell biocatalysts, especially for vaccine delivery with improvements in immune effectiveness.Surface display vaccines can increase the immunogenicity of foreign antigens by facilitating their recognition by the immune system.For example, the surface display of antigens derived from Mycobacterium tuberculosis on Salmonella typhimurium [1] or Lactiplantibacillus plantarum [2] evokes a stronger immune response compared to that with cytoplasmic delivery.Surface display vaccines also have advantages such as their ease of use and low manufacturing costs.
Trimeric autotransporter adhesins (TAAs) are type V secretion system subtype C (T5cSS) proteins, which are involved in several aspects of the infection process in gramnegative bacteria and developed as candidates for recombinant subunit vaccines [11].TAAs are composed of three identical polypeptide chains and share a common N-terminus-headneck-stalk-membrane anchor-C-terminus architecture, varying greatly in length, from 23 nm [12] to 240 nm [13].Nakatani reported the trimeric autotransporter adhesin (AtaA) from Acinetobacter sp.Tol 5 could be used to display a His-tag in Escherichia coli cells.This onfiber display system could change the distance between the cell surface and the displayed biomolecule [14].Phan et al. used the truncated mutant of trimeric autotransporter UpaG in uropathogenic E. coli to display calmodulin and a nanobody binding to a green fluorescent protein on E. coli cells with the overexpression of the BAM (β-barrel-assembly machinery) complex [15].The length, trimeric form, and autodisplay characteristics indicate that TAAs could serve as display platforms for efficient vaccine development.
Attenuated Salmonella spp.have been developed to deliver heterologous antigens from viruses, bacteria, protozoans, and fungi to induce immune responses against pathogenic infections [3,7,16,17].SadA, the TAA that has been characterized in S. typhimurium, consists of polypeptides comprising 1461 amino acids and forms a 108 nm long model [18,19], whereas SadB, a small inner membrane lipoprotein in Salmonella spp., is important for SadA translocation.Grin et al. reported a significantly higher immunofluorescence signal level in a P BAD ::sadBA Salmonella enterica strain than in a P BAD ::sadA S. enterica strain upon L-arabinose addition, suggesting the direct involvement of SadB in the biogenesis of SadA [20].In this study, we first utilized full-length and truncated SadAs to display epitope peptides on the surface of S. typhimurium with the assistance of SadB.Furthermore, truncated SadAs were employed to display heterologous proteins of different sizes to test the passenger protein capacity.We evaluated the immunogenicity of the heterologous antigen surface displayed on S. typhimurium using SadA as an anchoring motif in a mouse model.Our work indicates that SadA from S. typhimurium could be developed into a novel antigen surface expression system for studying attenuated live vector vaccines.

Chromosomal Deletion of sadA, ygeA, and murI Genes from S. typhimurium
The S. typhimurium 1.1174 parental strain was used for the construction of the Stm∆sadA strain.All genetic manipulations were performed using the CRISPR-Cas9 system [21].Briefly, at an optical density (OD) 600 = 0.6~0.7,bacterial cultures were placed on ice for 30 min and centrifugated at 3500× g for 8 min.After washing with ice-cold ddH 2 O, S. typhimurium cells were washed three times with 10% glycerol to make electrocompetent cells.Then, pCas was transformed into S. typhimurium cells.The N 20 sequence was designed using the online design tool (https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM, accessed on 9 March 2022), and inserted into pTargetF to obtain recombinant plasmid named pTargetF-sadA.Next, 517 bp upstream and 493 bp downstream sequences of the targeted region were cloned to obtain donor DNA using the primers shown in Table S2.The pCas/S.typhimurium was made into electrocompetent cells by adding arabinose at a final concentration of 10 mM for λ-Red induction.Then, 100 ng of pTargetF-sadA and 1 µg of donor DNA were co-transformed into fleshly grown electrocompetent pCas/S.typhimurium cells.The sadA-deleted clones were verified using PCR and DNA sequencing after overnight culture on Luria-Bertani (LB) plates containing kanamycin and spectinomycin (Table S2).The correct edited clone was cultured in LB with kanamycin and isopropyl-β-D-thiogalactopyranosid (IPTG) overnight at 30 • C to remove pTargetF.Subsequently, pCas was removed by culturing the strains overnight at 42 • C in LB medium without any antibiotics.
ygeA and murI genes were deleted from the chromosomal of the S. typhimurium 1.1174 parental strain according to the method mentioned above.

Strains and Growth Conditions
Escherichia coli strain DH5α used for cloning, and Stm∆sadA used for expression, were routinely cultured in LB broth with shaking or on agar supplemented with 100 µg/mL ampicillin and/or IPTG, where appropriate, at 37 • C. S. typhimurium, Stm∆sadA, and B. anthracis A16R were grown in LB broth at 37 • C for DNA isolation.The ∆ygeA and ∆murI double-deleted strain (Stm∆ygeA∆murI) was cultured in LB broth supplemented with 100 µg/mL ampicillin and 5 mM D-glutamic acid (D-Glu) at 37 • C. H. pylori SS1 was cultured on Campylobacter Ager Base plates (CDRC, Shanghai, China) containing 7% fetal bovine serum in a microaerophilic atmosphere (80% N 2 , 5% O 2 , 15% CO 2 ) for 3 days at 37 • C to extract genomic DNA.

Protein Expression and Analysis
Stm∆sadA and Stm∆ygeA∆murI strains with the recombinant plasmid were grown aerobically in 5 mL of LB liquid medium (200 µg/mL ampicillin) and LB liquid medium (200 µg/mL ampicillin and 5 mM D-Glu) overnight at 37 • C, respectively.The next day, the cells were inoculated and induced with the addition of 1 mM IPTG when the OD 600 value reached about 0.6, and then incubated for 15 h at 16 • C. 1 OD 600 bacterial cells were harvested, washed twice with PBS, and resuspended in 200 µL of SDS sample buffer.A 30 µL sample was subjected to 4~12% SDS-PAGE (GenScript, Nanjing, China).The transferred PVDF membrane was blocked in PBST containing 5% skim milk at 4 • C overnight.Subsequently, the membrane was incubated with anti-Flag tag rabbit polyclonal antibody (diluted to 1:3000 with 5% skim milk in PBST; Easybio, Beijing, China), or A1H10 or A3C10 (1:2000 dilution) for 1 h at 37 • C.After three washes with PBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution; Easybio, Beijing, China) or HRP-conjugated goat anti-mouse IgG (1:5000 dilution; Abcam, Cambridge, UK) for 1 h at 37 • C.An ECL-enhanced Western Blot Analysis Kit (Easybio, Beijing, China) was used to detect the binding reactions.

Proteinase K Treatment of Bacterial Cells
Proteinase K digestion of the surface-exposed domains was performed as described by Xu et al. [6].Briefly, the 1 OD 600 -induced bacterial cells were incubated with proteinase K at the final concentration of 400 µg/mL at 37 • C, and then we added 2 mM PMSF to stop the reaction.Aliquots were washed three times with PBS before protein electrophoresis, and immunofluorescence analyses were performed.

Immunofluorescence and Flow Cytometric Analyses
Immunofluorescence analysis was performed as described previously.Briefly, 1 OD 600 cell was immobilized with 4% paraformaldehyde and blocked in PBS containing 1% bovine serum albumin (BSA) for 30 min.A rabbit anti-Flag antibody (diluted to 1:50 with 1% BSA in PBS) and an Alexa Fluor 488 goat anti-rabbit IgG antibody (1:50 dilution; Easybio, Beijing, China) were used for immunostaining.After washing with PBS, the cells were resuspended in 1 mL of PBS and transferred to a microplate (Nunc MicroWell 96, Thermo Fisher Scientific, New York, NY, USA).A SpectraMax i3× microplate reader was used to measure the fluorescence intensity using wavelengths of E x : 490 nm and E m : 535 nm.The amount of recombinant proteins displayed on the cell surface was quantified by dividing the fluorescence intensity of each sample by its OD 600 value.Finally, the cells were mounted on poly-L-lysine-coated coverslips and dyed with DAPI, and immunofluorescence images were captured with a Nikon Ti2 inverted fluorescence microscope.
Flow cytometric analysis of the samples was performed.Each sample was labeled as described previously herein, using a rabbit anti-Flag antibody as the primary antibody (1:50 dilution) and Alexa Fluor 488 goat anti-rabbit IgG antibody (1:50 dilution) as the secondary antibody.Surface expression levels were evaluated by measuring fluorescence using a Northern Lights-CLC flow cytometer (CYTEK, Shanghai, China).

Whole-Cell Dot Blot
Recombinant S. typhimurium was immobilized in 4% paraformaldehyde and 0.04 OD 600 cells were dropped onto the nitrocellulose membrane (NC).After drying for 30 min at 37 • C, the dot blot protocol proceeded as described previously herein using the rabbit anti-Flag-tag antibody and A3C10 as the primary antibody.As the whole-cell dot blot did not destroy the cell membrane and the cell membrane remained intact, the results showed whether the recombinant protein was displayed on the surface of the bacteria.

Immunization, Sample Collection, and Specific Antibody Detection by ELISA
For cultivation of pSadBA 1292 -FUM/Stm∆ygeA∆murI and pSadBA 877 -FUM/Stm∆ygeA ∆murI strains, LB liquid medium was supplemented with 200 µg/mL ampicillin and 5 mM D-Glu.After inducing by 1 mM IPTG overnight at 16 • C, the cells were obtained by centrifugation and washed with PBS twice.
Three groups of 6-to-8-week-old BALB/c female mice (N = 10/group) were purchased from the Vital River Laboratory (Beijing, China) and immunized three times on days 0, 10, and 25.Group A and group B received oral pSadBA 1292 -FUM/Stm∆ygeA∆murI and pSadBA 877 -FUM/Stm∆ygeA∆murI with 10 9 CFU for all three immunizations, respectively.Group C received oral PBS for all three immunizations as the control group.On day 31, sera and fecal samples from all mice were collected for the detection of antigen-specific IgG and secretory IgA (sIgA) levels.The fecal samples were treated according to the method reported by Zhang et al. [22].
Antigen-specific IgG levels in the serum and sIgA levels in the fecal samples were determined by ELISA according to the protocol published previously [22].

Statistical Analysis
All data were analyzed using GraphPad Prism 8.0.2 and are presented as the mean ± standard deviation.One-way ANOVA was employed to determine significant differences, and a p-value < 0.05 was considered statistically significant.

Full-Length SadA Can Be Displayed on the Surface of S. typhimurium
To evaluate the suitability of SadA protein for surface display, a sadA-deleted mutant strain of S. typhimurium was constructed using the CRISPR-Cas9 genome editing system and selected as the expression host bacterium.The sadA-deleted mutant strain was confirmed by PCR and sequencing (Figure S1A).
Then, we constructed a Flag×3-tagged full-length SadA to confirm its surface display on Stm∆sadA cells.The recombinant protein was expressed in Stm∆sadA and the Flag-tag display on the surface was tested.We confirmed the production of the Flag-tagged SadA in Stm∆sadA through immunoblotting using the anti-Flag-tag antibody.The expressed protein was detected as a band of approximately 185 kDa on a PVDF membrane (Figure 1A).
ampicillin and 5 mM D-Glu.After inducing by 1mM IPTG overnight at 16 °C, the cells were obtained by centrifugation and washed with PBS twice.
Three groups of 6-to-8-week-old BALB/c female mice (N = 10/group) were purchased from the Vital River Laboratory (Beijing, China) and immunized three times on days 0, 10, and 25.Group A and group B received oral pSadBA 1292 -FUM/StmΔygeAΔmurI and pSadBA 877 -FUM/StmΔygeAΔmurI with 10 9 CFU for all three immunizations, respectively.Group C received oral PBS for all three immunizations as the control group.On day 31, sera and fecal samples from all mice were collected for the detection of antigen-specific IgG and secretory IgA (sIgA) levels.The fecal samples were treated according to the method reported by Zhang et al. [22].
Antigen-specific IgG levels in the serum and sIgA levels in the fecal samples were determined by ELISA according to the protocol published previously [22].

Statistical Analysis
All data were analyzed using GraphPad Prism 8.0.2 and are presented as the mean ± standard deviation.One-way ANOVA was employed to determine significant differences, and a p-value < 0.05 was considered statistically significant.

Full-Length SadA Can Be Displayed on the Surface of S. typhimurium
To evaluate the suitability of SadA protein for surface display, a sadA-deleted mutant strain of S. typhimurium was constructed using the CRISPR-Cas9 genome editing system and selected as the expression host bacterium.The sadA-deleted mutant strain was confirmed by PCR and sequencing (Figure S1A).
Then, we constructed a Flag×3-tagged full-length SadA to confirm its surface display on StmΔsadA cells.The recombinant protein was expressed in StmΔsadA and the Flag-tag display on the surface was tested.We confirmed the production of the Flag-tagged SadA in StmΔsadA through immunoblotting using the anti-Flag-tag antibody.The expressed protein was detected as a band of approximately 185 kDa on a PVDF membrane (Figure 1A).To confirm that the SadA-Flag×3 protein was exposed on the surface of intact bacterial cells, a whole-cell dot blot assay was performed by incubating bacterial suspensions spotted on NCs with an anti-Flag-tag antibody.The anti-Flag-tag antibody reacted strongly with pSadA-Flag×3/Stm∆sadA, but not with the control group cells (Figure 1B).Since the dot blot assay did not destroy the cell membrane and the cell membrane remained intact, the result showed that the recombinant protein was successfully displayed on the surface of Stm∆sadA cells.At the same time, the immunofluorescence staining also confirmed that the SadA-Flag×3 protein was located on the cell surface (Figure 1C).In contrast, no fluorescence of the Flag tag was detected for pTrc99A/Stm∆sadA.

SadB Enhances the Surface Display of SadA in S. typhimurium
The sadB gene is located upstream of sadA and in an operon containing sadA.We created inducible overexpression constructs for the entire sadBA operon to improve the SadA surface display.Western blot analysis showed that the recombinant protein was successfully expressed in the presence of SadB, and the protein expression level of SadBA was significantly higher than that of SadA alone (Figure 2A).Whole-cell dot blotting and immunofluorescence analyses showed that the recombinant proteins were located on bacterial surfaces (Figure 2B,C).Only proteins displayed on the bacterial surface can be digested by proteinase K, which cannot enter the cells.A significant decrease in the fluorescence intensity of pSadA-Flag×3/Stm∆sadA or pSadBA-Flag×3/Stm∆sadA was observed after treatment with proteinase K compared to that without treatment.Although SadA could be used for surface display, this occurred at a low level according to the fluorescence intensity (Figure 2D).The fluorescence intensity of recombinant cells in the presence of SadB was approximately three-fold higher than that in the absence of SadB (Figure 2D).These results indicated that the Flag tag was exported to the cell surface, confirming the feasibility of SadBA as a surface display vector.

Epitopes Can Be Displayed on the Cell Surface Using SadAs with Different Sizes as Anchoring Motifs
UreB is the subunit responsible for the enzymatic activity of urease, and is considered to be an excellent candidate antigen for the development of vaccines against H. pylori infection.UreB158-172aa and UreB349-363aa have been identified as B-cell epitopes of UreB [23].We fused Flag tags and the two previously mentioned epitopes of UreB with six SadA derivatives, as shown in Figure 3.These constructs were expressed in Stm∆sadA, and the epitopes displayed on these derivatives were examined.The expression of different SadA derivatives was detected via Western blotting using an antibody against the Flagtag and monoclonal antibodies for UreB158-172aa (A1H10) and UreB349-363aa (A3C10), resulting in the display of monomer bands at ~40, ~50, ~80, ~115, ~160, and ~185 kDa (Figure 4A and Figure S2A,B).The bands at higher positions on the PVDF membrane could represent multimeric forms of the constructs, with the stronger reacting bands likely representing multiple conformations of dimeric and trimeric forms, which have been observed for the trimeric autotransporter YadA (Yersinia sp.) [24] and truncated UpaG [25].Trimeric autotransporters have been reported to be resistant to denaturation during SDS-PAGE [26].Expression of the trimeric form was significantly greater in the pSadBA 877 -FU2/Stm∆sadA strain than in the other recombinant cells (Figure 4A).The disappearance of bands corresponding to the trimeric form in pSadBA 1292 -FU2/Stm∆sadA, pSadBA 1171 -FU2/Stm∆sadA, pSadBA 877 -FU2/Stm∆sadA, and pSadBA 644 -FU2/Stm∆sadA after proteinase K treatment indicated that the recombinant proteins were displayed on the bacterial surface using SadA derivatives as the membrane anchor (Figure 4A).However, no trimer protein bands from pSadBA 269 -FU2/Stm∆sadA and pSadBA-FU2/Stm∆sadA were observed on PVDF membranes.No dot was detected in the pTrc99A/Stm∆sadA control group, but the specific response signal was observed with the six SadA derivatives when using the anti-Flag-tag antibody, A1H10, or A3C10, which confirmed that epitopes were successfully displayed on the surface of bacteria using the six SadA derivatives as anchoring motifs (Figure S2C).
pSadBA 877 -FU2/StmΔsadA, pSadBA 1171 -FU2/StmΔsadA, and pSadBA 1292 -FU2/StmΔsadA cells (Figure 4B).The display capacity was further compared by quantifying Alexa Fluor 488 fluorescence using a microplate reader.Figure 4C shows that the fluorescence intensity of pSadBA 877 -FU2/StmΔsadA, pSadBA 269 -FU2/StmΔsadA, and pSadBA-FU2/StmΔsadA was significantly higher than that of pSadBA 1292 -FU2/StmΔsadA, pSadBA 1171 -FU2/StmΔsadA, and pSadBA 644 -FU2/StmΔsadA, which was in line with the high rate of positivity observed in these three cell lines, according to the flow cytometric analysis (Table 1).Further, the induced recombinant S. typhimurium treated with proteinase K showed a significant decrease in fluorescence intensity compared to that in the absence of proteinase K (Figure 4C).These data demonstrated that epitopes were displayed on the surface of the transformed cells using truncated and full-length SadAs as the anchoring motif.The data were presented as mean ± SD, and differences between groups were tested using one-way

Truncated SadAs Can Mediate the Surface Display of Heterologous Proteins on the S. typhimurium Mutant
The decoration of bacteria with multiple fused antigens can be used as a tool to increase the efficacy of antigen delivery.Because SadA is composed of 1462 amino acids, its full-length expression with a heterologous protein imposes a heavy burden on the cell, resulting in low expression and suboptimal surface display.Therefore, we used the truncated SadAs (SadA 1292 and SadA 877 ) to export the heterologous protein on the surface of Stm∆sadA cells.We chose the fluorescent protein mScarlet, the UreB fragment (UreB111-377aa, containing UreB158-172aa and UreB349-363aa), and the PAD4 fragment (139 aa), which could be used as the vaccine candidate against B. anthracis [27], to analyze the ability of this system to express foreign proteins.For this, we designed two truncated SadAs fused with an N-terminally Flag×3-mScarlet (~30 kDa, SadBA 877 -FM, and SadBA 1292 -FM) and Flag×3-UreBm-mScarlet (~58 kDa, SadBA 877 -FUM, and SadBA 1292 -FUM) or Flag×3-UreBm-PAD4-mScarlet (~75 kDa, SadBA 877 -FUPM, and SadBA 1292 -FUPM) (Figure 5).

Truncated SadAs Can Mediate the Surface Display of Heterologous Proteins on the S. typhimurium Mutant
The decoration of bacteria with multiple fused antigens can be used as a tool to increase the efficacy of antigen delivery.Because SadA is composed of 1462 amino acids, its full-length expression with a heterologous protein imposes a heavy burden on the cell, resulting in low expression and suboptimal surface display.Therefore, we used the truncated SadAs (SadA 1292 and SadA 877 ) to export the heterologous protein on the surface of StmΔsadA cells.We chose the fluorescent protein mScarlet, the UreB fragment (UreB111-377aa, containing UreB158-172aa and UreB349-363aa), and the PAD4 fragment (139 aa), which could be used as the vaccine candidate against B. anthracis [27], to analyze the ability of this system to express foreign proteins.For this, we designed two truncated SadAs fused with an N-terminally Flag×3-mScarlet (~30 kDa, SadBA 877 -FM, and SadBA 1292 -FM) and Flag×3-UreBm-mScarlet (~58 kDa, SadBA 877 -FUM, and SadBA 1292 -FUM) or Flag×3-UreBm-PAD4-mScarlet (~75 kDa, SadBA 877 -FUPM, and SadBA 1292 -FUPM) (Figure 5).As expected, the expression of recombinant proteins resulted in a pink color when StmΔsadA was induced (Figure S3A).The expression of the fusion proteins was analyzed via Western blotting using a polyclonal antibody against the Flag tag and the A1H10 monoclonal antibody (Figures 6A and S3B).The monomeric bands formed by pSadBA 1292 -Figure 5. Schematic representation of genetic constructs for heterologous proteins display using truncated SadAs as an anchoring motif.SP, signal peptide of SadA; 43 bp gene fragment, the gene fragment between sadB and sadA in the chromosome of S. typhimurium; gray, the GGGGS linker.
Whole-cell dot blot and immunofluorescence analysis indicated that the recombinant proteins were located on the surface of pSadBA 1292 -FM/Stm∆sadA, pSadBA 1292 -FUM/Stm∆sadA, pSadBA 1292 -FUPM/Stm∆sadA, pSadBA 877 -FM/Stm∆sadA, and pSadBA 877 -FUM/Stm∆sadA (Figures S3C and 6B).However, a small amount of green fluorescence was observed on pSadBA 877 -FUPM/Stm∆sadA, indicating that FUPM display on the cell surface was marginal when using SadBA 877 as the anchoring motif.No fluorescence was detected for pFM/Stm∆sadA, which is a control strain, expressing FM with no membrane anchor.
The fluorescent intensity and rates of positivity of SadBA 877 -FUM/Stm∆sadA were significantly lower than those of pSadBA 877 -FM/Stm∆sadA (Figure 6C, Table 1).Meanwhile, the fluorescence intensity of pSadBA 1292 -FM/Stm∆sadA was higher than that of pSadBA 1292 -FUM/Stm∆sadA and pSadBA 1292 -FUPM/Stm∆sadA.These results indicated that the SadA display was decreased as the molecular weight of the foreign protein was increased.
The fluorescence intensity and rate of positivity of pSadA 877 -FM/Stm∆sadA were higher than those of pSadBA 1292 -FM/Stm∆sadA (Figure 6C, Table 1), which was consistent with the improved ability of SadBA 877 to facilitate epitope surface display compared to that of SadBA 1292 .
When the recombinant cells were treated with proteinase K, the fluorescence of pSadBA 1292 -FM/Stm∆sadA, pSadBA 1292 -FUM/Stm∆sadA, pSadBA 1292 -FUPM/Stm∆sadA, pSadBA 877 -FM/Stm∆sadA, and pSadBA 877 -FUM/Stm∆sadA was decreased significantly (Figure 6C).These data indicated that the heterologous proteins FM, FUM, and FUPM could be displayed on the surfaces of transformant cells using SadBA 1292 as an anchoring motif and that SadA 877 could help to expose FM and FUM on the surfaces of cells.We transferred pSadBA 1292 -FUM and pSadBA 877 -FUM into competence cells of Stm∆yg eA∆murI, which had been confirmed by PCR and sequencing (Figure S1B).The heterologous proteins were expressed and the surface displayed successfully in Stm∆ygeA∆murI (Figures S4 and S5).In order to determine whether the recombinant protein was able to elicit antibodies, BALB/c mice were immunized orally with pSadBA 1292 -FUM/Stm∆ygeA∆murI and pSadBA 877 -FUM/Stm∆ygeA∆murI (Figure 7A).Antigen-specific IgGs of groups A and B were significantly higher than those of group C in the same dilution.Meanwhile, differences in serum IgG levels for groups A and B were not significant in the same dilution (Figure 7B).Compared with the control group C, groups A and B both produced the antigen-specific mucosal sIgA, but there was no difference between groups A and B (Figure 7C).-FUM/Stm∆ygeA∆murI on days 0, 10, 25, respectively; Group C mice received oral administration of PBS.On the sixth day after the final immunization, serum and feces of the mice were collected and the UreB-specific IgG (B) and sIgA (C) levels were measured by ELISA.Differences between groups were tested using one-way ANOVA.**** p < 0.0001, ns p > 0.05.

Discussion
SadA was the first reported TAA exposed on the surface of Salmonella spp.cells and it is highly conserved among S. enterica strains [18].SadA is a positional ortholog of UpaG and EhaG in E. coli, but with different functions.Specifically, it promotes biofilm formation and host cell adherence but does not bind extracellular matrix molecules or mediate serum resistance.Although the exact mechanism of TAA secretion remains unclear, many important proteins involved in TAA biogenesis have been found, such as BAM [28] and chaperones [29].During the biogenesis of SadA, a trimeric protein, SadB, for which the encoding gene is located upstream of sadA, facilitates the export of SadA to the cell surface.pSadBA-Flag×3/Stm∆sadA had an approximately three-fold higher mean fluorescence than pSadA-Flag×3/Stm∆sadA in this study (Figure 2D), which is in accord with a previous report [15].Western blot results showed that SadA monomer expression in pSadBA-Flag×3/Stm∆sadA was significantly higher than that in pSadA-Flag×3/Stm∆sadA in this study, suggesting that SadB might enhance the surface display of SadA by increasing its expression or preventing its degradation when it is not yet at the cell surface, in the periplasm.During TAA biogenesis, the N-terminal signal sequence mediates translocation of the subunit into the periplasm in a Sec-dependent manner.The Sec system also transports several proteins, such as lipoproteins, periplasmic proteins, and outer membrane proteins (OMPs), into the periplasm [30].When heterologous OMPs are over-expressed, the Sec machinery is prone to saturation.To solve this problem, the most abundant OMPs can be knocked out to alleviate the burden on the Sec system and provide a larger membrane area for foreign proteins.Meuskens found that the expression of the membrane anchor domain of the trimeric autotransporter YadA (YadAM) is higher in ompA-ompC-ompF-lamB quadruple mutant BL21 Gold (DE3) cells than in BL21 (DE3) cells.This provides ideas for further improving the SadA display [31].
Nakatani et al. found that in bacteria, a His-tag displayed by full-length AtaA could bind to Ni-Sepharose beads more rapidly than other truncated AtaAs, confirming that a certain distance from the bacterial surface could help to overcome steric hindrance and maintain the function of foreign proteins [14].Possibly for the same reason, pSadBA 1292 -FU2/Stm∆sadA, pSadBA 1171 -FU2/Stm∆sadA, and pSadBA 644 -FU2/Stm∆sadA exhibited a significantly lower fluorescence intensity than pSadBA-FU2/Stm∆sadA and pSadBA 269 -FU2/Stm∆sadA in this study (Figure 4C).This indicates that the exogenous peptides displayed by full-length SadA are more easily recognized by antibodies.However, although SadA 877 is shorter, the expression of its trimer was higher (Figure 4A).Accordingly, the fluorescence intensity of pSadBA 877 -FU2/Stm∆sadA was significantly enhanced, which was similar to that with pSadBA-FU2/Stm∆sadA and pSadBA 269 -FU2/Stm∆sadA (Figure 4C).
An autotransporter-based display system can display a protein of interest (POI) on the surface of gram-negative bacteria through the exchange of all or part of the native passenger domain with the POI.However, one limiting factor for the successful translocation of autotransporters is the final dimensions of the fused protein [32].For example, the maximum size limit of heterologous proteins using AIDA-I of E. coli, MisL of S. typhimurium, IcsA of Shigella spp., and Hbp of E. coli is 130 kDa [33], 58 kDa [34], 57 kDa [35], and 50 kDa [36], respectively.In this study, SadBA 1292 could be used to display FUPM (~75 kDa) on the surface of S. typhimurium.However, with SadBA 877 , the display of FUPM (~75 kDa), which is larger than FUM (~58 kDa), on the bacterial surface, failed (Figure 6B).Moreover, the fluorescence intensity of pSadBA 877 -FUM/Stm∆sadA and pSadBA 1292 -FUM/Stm∆sadA was significantly lower than that of pSadBA 877 -FM/Stm∆sadA and pSadBA 1292 -FM/Stm∆sadA, respectively.As the molecular weight of the foreign protein increases, it becomes more difficult to display the recombinant protein on the cell surface.The complex structure of for-eign proteins is another limiting factor for the successful exposure of autotransporters [37].When autotransporter Hbp was used to display calmodulin or a nanobody, secretion was hindered because the recombinant protein could not pass through the β-barrel due to the form of a stable fold in the presence of calcium ions or the form of two disulfide bonds [38].BamA and BamD are required for the secretion and function of SadA [39].In contrast, SadA can be displayed on the surfaces of mutant cells in which bamB, bamC, or bamE are deleted, suggesting that these three lipoproteins are not essential for SadA secretion.Trang et al. found that overproduction of the BAM complex can increase the surface display of Hbp fusions and the trimeric autotransporter UpaG [15].This provides a solution for the secretion of heterologous proteins with complex structures using SadA as a surface display tool.
Autotransporters (type Va secretion systems) have been widely used to display foreign proteins on cell surfaces, especially for vaccine development.AIDA-I, MisL, ShdA, and Hbp have been successfully used to display different antigens on the surfaces of Salmonella spp. as vaccine candidates.Moreover, auto-displayed vaccines can effectively increase the immunogenicity of antigens [40].TAA belongs to the T5cSS family and exhibits autodisplay characteristics similar to those of autotransporters.In contrast to autotransporters, TAAs can display stable trimeric polymers on gram-negative bacterial surfaces owing to the structure of T5cSS.Trimeric proteins exhibited a stronger immunogenic response than their monomer forms [41].
Genetically attenuated Salmonella spp.can be engineered by deleting important virulence genes and the key enzyme genes of metabolic pathways to deliver recombinant heterologous antigens to elicit the host immune system [42].D-Glu, the major component of peptidoglycan, is synthesized by MurI and YgeA in Salmonella spp.A murI-deleted S. typhimurium displayed a lower level of virulence than the wild-type strain and preserved pathogen-associated molecular patterns [43].Cabral et al. found that the D-Glu auxotrophic Pseudomonas aeruginosa could evoke an immune response with intranasal administration and protect the mice infected with two cytotoxic P. aeruginosa strains [44].In this work, a mutant strain Stm∆ygeA∆murI was constructed and employed to confirm the immunogenicity of heterologous antigen displayed by SadA.Orally administrated pSadBA 1292 -FUM/Stm∆ygeA∆murI or pSadBA 877 -FUM/Stm∆ygeA∆murI can elicit a mucosal and humoral immune response.Therefore, the surface-displayed antigen using truncated SadA as an anchoring motif kept its antigenicity and evoked host immunity reactivity.SadA could be exploited to construct a novel antigen surface expression system for attenuated bacterial vaccine development.

Conclusions
In summary, the SadA display was tested in terms of its length and the heterologous protein load.Both full-length and truncated SadAs could display exogenous proteins.In vivo test, the heterologous antigen surface displayed on Stm∆ygeA∆murI using truncated SadA as an anchoring motif could elicit a significant mucosal and humoral immune response.These findings suggest that SadA anchoring proteins are suitable for developing an attenuated bacterial vaccine.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines12040399/s1,Table S1: bacterial strains and plasmids; Table S2: oligonucleotides used in this study; Table S3: the gene sequences of commercial synthesis and amino acid sequences of functional proteins; Figure S1: the sadA-deleted S. typhimurium and ygeA-murI-double-deleted S. typhimurium were confirmed by PCR; Figure S2: Western blot and dot blot of the displaying epitopes on the cell surface using full-length and truncated SadAs; Figure S3: confirmation of recombinant proteins displaying on the surface of cells using truncated SadAs as an anchoring motif; Figure S4: Western blot of heterologous proteins expressed in Stm∆ygeA∆murI using the antibodies against Flag tag (A) and UreB158-172aa (B); Figure S5: cell surface display of Flagtagged SadA derivatives on Stm∆ygeA∆murI by immunofluorescence staining using anti-Flag-tag primary antibody and Alexa Fluor 488 conjugated secondary antibody.

Figure 1 .Figure 1 .
Figure 1.Displaying of the full-length SadA on the surface of S. typhimurium mutant cells.(A) Western blot of whole cell lysates to analyze expression of SadA in StmΔsadA.The Flag tag was inserted at the N-terminus of SadA passenger.The recombinant protein bands were detected by incubating the PVDF using anti-Flag-tag antibody.The putative position of monomeric form (*) was indicated on the right side of the panel.Lane1, pTrc99A/StmΔsadA; lane 2, pSadA-Flag×3/StmΔsadA.Dot blot of whole cells showed the surface display of Flag-tagged SadA on StmΔsadA (B).Lane1, Figure 1.Displaying of the full-length SadA on the surface of S. typhimurium mutant cells.(A) Western blot of whole cell lysates to analyze expression of SadA in Stm∆sadA.The Flag tag was inserted at the N-terminus of SadA passenger.The recombinant protein bands were detected by incubating the PVDF using anti-Flag-tag antibody.The putative position of monomeric form (*) was indicated on the right side of the panel.Lane1, pTrc99A/Stm∆sadA; lane 2, pSadA-Flag×3/Stm∆sadA.Dot blot of whole cells showed the surface display of Flag-tagged SadA on Stm∆sadA (B).Lane1, pTrc99A/Stm∆sadA; lane2, pSadA-Flag×3/Stm∆sadA.(C) Cell surface display of Flag-tagged SadA by immunofluorescence staining using anti-Flag-tag primary antibody and AlexaFluor 488 conjugated secondary antibody (Objective, 100×; Magnification, 1000×).

Figure 3 .
Figure 3. Schematic representation of genetic constructs for epitopes display using truncated and full-length SadA derivatives as an anchoring motif.SP, signal peptide of SadA; 43bp gene fragment, the gene fragment between sadB and sadA in the chromosome of S. typhimurium; gray, the GGGGS linker.

Figure 3 .
Figure 3. Schematic representation of genetic constructs for epitopes display using truncated and fulllength SadA derivatives as an anchoring motif.SP, signal peptide of SadA; 43bp gene fragment, the gene fragment between sadB and sadA in the chromosome of S. typhimurium; gray, the GGGGS linker.Vaccines 2024, 12, x FOR PEER REVIEW 9 of 17

Figure 5 .
Figure 5. Schematic representation of genetic constructs for heterologous proteins display using truncated SadAs as an anchoring motif.SP, signal peptide of SadA; 43 bp gene fragment, the gene fragment between sadB and sadA in the chromosome of S. typhimurium; gray, the GGGGS linker.

Figure 6 .Figure 6 .
Figure 6.Recombinant proteins displaying on the surface of cells using truncated SadA as an anchoring motif.Western blot showed that fused proteins could be expressed in the StmΔsadA using the antibodies against Flag tag (A).The putative positions of monomeric (*), dimeric (**), and trimeric (***) complexes were indicated under the bands.(1) pSadBA 1292 -FM/StmΔsadA; (2) pSadBA 1292 -Figure 6.Recombinant proteins displaying on the surface of cells using truncated SadA as an anchoring motif.Western blot showed that fused proteins could be expressed in the Stm∆sadA using

Vaccines 2024 , 17 Figure 7 .
Figure 7. Immunization schedule and immune responses induced by immunization.(A) Group A and B mice received oral administration of 10 9 CFU pSadBA 1292 -FUM/StmΔygeAΔmurI and pSadBA 877 -FUM/StmΔygeAΔmurI on days 0, 10, 25, respectively; Group C mice received oral administration of PBS.On the sixth day after the final immunization, serum and feces of the mice were collected and the UreB-specific IgG (B) and sIgA (C) levels were measured by ELISA.Differences between groups were tested using one-way ANOVA.**** p < 0.0001, ns p > 0.05.

Table 1 .
Flow cytometric analysis of different SadA derivatives.