An Engineered Outer Membrane-Defective Escherichia coli Secreting Protective Antigens against Streptococcus suis via the Twin-Arginine Translocation Pathway as a Vaccine

Abstract

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.

Introduction

Non-pathogenic or virulence-attenuated bacteria, such as Escherichia coli, Lactobacillus, Bacillus, Salmonella, and Listeria, when genetically engineered to secrete heterologous proteins, can be exploited as tools for antigen delivery [1]. Among the prominent advantages of live bacterial vaccines are low cost, flexible vaccination routes, and enhanced induction of immune response [1-3]. E. coli is the most widely used “workhorse” due to its high simplicity in genetic manipulations and high efficiency [4-6]. However, as a gram-negative bacterium, the secretion of heterologous proteins from the cytoplasm to the extracellular environment is challenging due to the presence of an outer membrane. One of the strategies that have been applied for heterologous protein secretion by E. coli is the utilization of bacterial protein secretion systems [7, 8]. So far, several bacterial protein secretion systems have been devised to facilitate heterologous proteins to be anchored to the cytoplasmic membrane [9], translocated to the periplasm [10, 11], displayed on the bacterial surface [12], secreted to the extracellular environment [13], or even injected into the eukaryotic cells [14]. By employing these secretion systems, several live bacterial vector vaccines have been developed. By using an attenuated enteropathogenic E. coli (EPEC) as a live vaccine vector, secretion of Shiga toxin B subunit was achieved via an E. coli hemolysin secretion (type I secretion) apparatus, and the rabbits inoculated with the recombinant live vaccine showed partial protection from infection of a virulent EPEC strain [15]. Staphylococcus aureus antigens SaEsxA and SaEsxB were successfully delivered into the cytosol of host cells when incorporated into the Salmonella type III secretion system, which elicited both humoral and cellular immune response, providing protection from S. aureus infection [16].

The twin-arginine translocation (Tat) system is a protein export system that recognizes its cargo proteins through the Tat signal peptide featured with an S/T-R-R-x-F-L-K motif, and exports them to the periplasm, or inserts them into the membrane [11, 17]. A distinct feature of the Tat system is that it allows the folded protein to be exported. The Tat system has been shown to have the potential for exporting therapeutic and industrial proteins, including growth hormones, interferon, single-chain fragment variable (scFV) [18, 19]. Recently, Albiniak et al. reported a high-level secretion of GFP using an E. coli strain in which its native Tat system was replaced with a Bacillus Tat system [20]. Moreover, the Tat system has been used to construct a multivalent vaccine [21].

Streptococcus suis is a zoonotic bacterial pathogen that causes huge economic loss while also posing a threat to human health [22, 23]. As carriage of S. suis is prevalent in pig herds and antimicrobial resistance is severe, controlling S. suis infection will still hinge on the use of vaccines. Both bacterins and subunit vaccines have been developed [24]. However, due to the complexity of S. suis epidemiology in swine, there is still a lack of universal vaccines providing cross-protection and effective control of S. suis infection. Therefore, developing novel vaccines is critical.

In this study, we show that protective antigen proteins of S. suis, including SLY, Enolase, and Sbp, when fused with a Tat signal peptide, can be secreted to the extracellular environment by using E. coli MC4100 ΔamiAΔamiC, a commensal E. coli strain with compromised outer membrane integrity. Using the antigen-secreting E. coli strains to orally inoculate mice, we show that the vaccinated mice displayed milder clinical symptoms in the lung, brain, and spleen, lower bacteria load, and a higher survival rate after S. suis challenge.

Materials and Methods

Bacterial Strains and Plasmids

Bacterial strains and plasmids used in this study are listed in Table 1. The DNA sequence of primers was listed in Table S1. E. coli was normally grown in lysogeny broth (LB) or on LB agar at 37°C. When necessary, chloramphenicol, ampicillin, and spectinomycin were used at a final concentration of 25 μg/ml, 100 μg/ml, and 100 μg/ml, respectively. E. coli MC4100 ΔtatABC and E. coli ΔamiAΔamiC strains were constructed using CRISPR/Cas9 technology as previously described with some modifications [25]. Briefly, the pREDCas9 plasmid was used to transform E. coli MC4100 competent cells. The pREDCas9-containing E. coli strain was cultured until the absorbance at OD₆₀₀ reached 0.3 in LB containing 0.1 mM IPTG to induce the expression of the λ RED recombination system, and then competent cells were prepared. A mixture containing the pgRNA-bacteria plasmid expressing sgRNA targeting the gene of interest and a DNA fragment containing the 500 bp upstream and the 500 bp downstream regions of the target gene was used for transformation. The colonies were selected and PCR was performed to test the presence of the target gene. The pgRNA was then eliminated by culturing the cells in the presence of 0.2% (w/v) arabinose at 30°C, and the pREDCas9 plasmid was cured by growing the cells at 42°C.

Table 1.

Bacterial strains and plasmids used in this study.

Strain/plasmid	Description	Source
Strains
E. coli MC4100	A commensal E. coli K-12 strain, wild type	[26]
E. coli MC4100 ΔamiAΔamiC	As E. coli MC4100, amiA and amiC deleted	This study
E. coli MC4100 ΔtatABC	As E. coli MC4100, tatABC deleted	This study
Plasmids
pgRNA-bacteria	Plasmid for sgRNA expression, Ampr	[27]
pREDCas9	Plasmid for constitutive expression of Cas9 and inducible expression of the λ Red recombineering system, Spcr	[28]
pgRNA-tatABC	As pgRNA-bacteria, expressing the sgRNA targeting the tatABC operon. Used in deleting tatABC.	This study
pgRNA-amiA	As pgRNA-bacteria, expressing the sgRNA targeting amiA. Used in deleting amiA.	This study
pgRNA-amiC	As pgRNA-bacteria, expressing the sgRNA targeting amiC. Used in deleting amiC.	This study
pQE80-SufIss-GFPhis	As pQE80, a DNA fragment encoding GFP with a SufI signal peptide fused to its N-terminus and a C-terminal hexahistidine tag inserted in the MCS, Ampr	This study
pQE-SufIss-SLYhis	As pQE80, a DNA fragment encoding S. suis SLY with a SufI signal peptide fused to its N-terminus and a C-terminal hexahistidine tag inserted in the MCS, Ampr	This study
pQE-SufIss-Enohis	As pQE80, a DNA fragment encoding S. suis enolase with a SufI signal peptide fused to its N-terminus and a C-terminal hexahistidine tag inserted in the MCS, Ampr	This study
pQE-SufIss-Sbphis	As pQE80, a DNA fragment encoding S. suis Sbp with a SufI signal peptide fused to its N-terminus and a C-terminal hexahistidine tag inserted in the MCS, Ampr	This study
pJ23	As pQE80, the promoter was replaced with a constitutive J23119 promoter	This study
pJ23-SufIss-SLYhis	As pQE-SufIss-SLYhis, the promoter was replaced with a constitutive J23119 promoter	This study
pJ23-SufIss-Enohis	As pQE-SufIss-Enohis, the promoter was replaced with a constitutive J23119 promoter	This study
pJ23-SufIss-Sbphis	As pQE-SufIss-Sbphis, the promoter was replaced with a constitutive J23119 promoter	This study

Plasmids were constructed by seamless cloning using the ClonExpress MultiS One Step Cloning Kit (Cat. No. C113, Vazyme Biotech Co., Ltd., China). pQE80-SufIss-GFPhis was constructed by ligating the EcoRI- and HindIII-digested pQE80, the SufI signal peptide-encoding DNA, and a DNA fragment containing the GFP-encoding sequence by seamless cloning. pJ23 was constructed by replacing the T5 promoter and lac operator with the constitutive promoter J23119 (Part: BBa_J23119). pJ23-SufIss-SLYhis plasmid was constructed by ligating the EcoRI and HindIII-digested pJ23, the SufI signal peptide-encoding DNA, and a DNA fragment containing the signal peptide-less SLY-encoding sequence with His tag by seamless cloning. pJ23-SufIss-Enohis and pJ23-SufIss-Sbphis were constructed in the same way. pgRNA-tatABC was constructed by replacing the fragment between AatII and SalI sites of pgRNA-bacteria with the fragment expressing the crRNA targeting tatABC. pgRNA-amiA and pgRNA-amiC were constructed in the same way.

Protein Methods

Subcellular fractionation was carried out as described previously [29]. Briefly, overnight cultures of bacteria were subcultured 1:100 to 100 ml LB and incubated at 37°C with shaking until the absorbance at OD₆₀₀ reached 1. To prepare whole-cell samples, 5 ml of culture was pelleted and the cells were resuspended with 250 μl of resuspension buffer (50 mM Tris-HCl, pH 7.6, 2 mM EDTA) followed by ultrasonic fragmentation for 10 min. To prepare periplasmic samples, cells were pelleted from 25 ml of culture and resuspended with 500 μl of fractionation buffer (20 mM Tris-HCl, pH 7.6, 2 mM EDTA, 20% sucrose). Then, lysozyme (final concentration 0.6 mg/ml) was added, and the supernatant was collected after 20 min incubation at room temperature, which was the periplasmic sample. The prepared whole-cell samples and the periplasmic fraction samples were subjected to Western blotting. To test the secretion of antigen proteins to the culture medium, cells of E. coli MC4100 strain or ΔamiAΔamiC strain expressing the indicated SufI signal peptide-fused antigen proteins were grown to the mid-log phase. Then, the same amount of culture supernatant was taken and concentrated by ultrafiltration and the same amount of sample was analyzed by Western blotting. RNA polymerase was used as the control.

Immunization and Challenge

All animal experiments were approved by the Laboratory Animal Monitoring Committee of Huazhong Agricultural University and performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of Hubei Province, China (Approval No. HZAUMO-2020-0070). Female Kunming mice were purchased from the Experimental Animal Center, Huazhong Agricultural University, Wuhan, China. Three strains containing plasmid pJ23-SufIss-SLYhis, pJ23-SufIss-Enohis, and pJ23-SufIss-Sbphis, respectively, were cultured to the mid-log phase at 37°C with shaking. The cells of each strain were mixed at a 1:1:1 ratio, pelleted, and washed three times with sterile saline. Then, 4- to 5-week-old Kunming mice were used for vaccination in which 100 μl of the bacteria mixture containing 10¹⁰ CFU cells was administered by oral gavage. A vector group was set in parallel which was administered with100 μl of the bacterial mixture containing 10¹⁰ CFU of E. coli ΔamiAΔamiC cells. The mice were vaccinated again after two weeks. Seven days after secondary immunization, both groups of mice were injected intraperitoneally with 1.5 × 10⁹ S. suis SC19 strain. Clinical symptoms were recorded. At 24 h post-infection, 4 mice were euthanized, and the brain, spleen, and lung were collected, homogenized, diluted, and plated on 5% bovine fetal serum-containing TSA plates for bacteria enumeration.

Histopathological Examination

The brain, spleen, and lung tissues of the mice in each group were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. The samples were cut into thin sections, stained with haematoxylin/eosin (H&E), and examined under a light microscope.

Results

Secretion of Tat Signal Peptide-Fused GFP to the Extracellular Milieus Is Enhanced in the Outer Membrane-Defective E. coli Strain

The Tat system can transport correctly folded proteins into the periplasm by recognizing the amino-terminal signal peptide of the substrate proteins [11]. Due to the presence of the outer membrane, it is normally difficult for the periplasmic proteins to be further secreted to the extracellular environment. However, it has been shown that the deletion of amiA and amiC, which encode two Tat-dependent amidases, compromises the integrity of the outer membrane [30]. This enlightened us to test whether proteins exported to the periplasm via the Tat system could be further secreted to the extracellular environment by such an outer membrane-defective strain. To test this, SufIss-GFP, a fusion containing SufI signal peptide and green fluorescent protein, which has been shown as able to be exported by the Tat system, was expressed from pQE80-SufIssGFPhis plasmid in E. coli MC4100 (WT) and E. coli MC4100 ΔamiAΔamiC strains, respectively. By collecting the culture supernatant and measuring green fluorescence intensity, it was shown in Fig. 1A that the green fluorescence intensity in the supernatant of the ΔamiAΔamiC strain was significantly higher than that of the WT strain. Western blot results also showed that much more GFP was present in the culture supernatant of the ΔamiAΔamiC strain than the WT strain (Fig. 1B), while a comparable amount of RNA polymerase was detected in the culture supernatant for the two strains (Fig. 1C). These results suggest that the Tat signal peptide-fused protein could be secreted to the extracellular environment by the outer membrane-defective E. coli strain.

Fig. 1. Secretion of SufI signal peptide-fused GFP.

Cells of E. coli MC4100 (WT) or E. coli MC4100 ΔamiAΔamiC strain containing pQE80-SufIss-GFPhis were grown overnight at 37°C in the presence of 1 mM IPTG. The culture was centrifuged and the supernatant was harvested. The green fluorescence intensity was measured by using a plate reader (A). The supernatant was concentrated by ultrafiltration and the sample was analyzed by Western blotting using anti-GFP antibody and anti-RNA polymerase (RNAP) antibody, respectively (B).

The SufI Signal Peptide-Fused S. suis Antigen Proteins Can Be Transported via the Tat Pathway

We next investigated whether S. suis antigen proteins could be transported to the periplasm via the Tat system when fused with a SufI signal peptide. We first showed that SufI signal peptide-fused S. suis immunoprotective proteins SLY, Sbp, and Enolase could be successfully expressed in the soluble fraction of E. coli MC4100 (Figs. 2A and 2B). To check whether the signal peptide-containing antigen proteins could be exported by the Tat pathway, the proteins were expressed in the WT strain and the Tat system-deficient strain (E. coli MC4100 ΔtatABC), respectively. Whole-cell samples and periplasmic fractions were prepared and analyzed by Western blot. It was shown in Fig. 2C that a smaller band corresponding to the mature form of SLY could be seen in the WT strain, but not in the ΔtatABC strain, indicating that SLY could be exported to the periplasm through the Tat system. These results suggest that S. suis antigen proteins, at least SLY, could be exported in a Tat-dependent manner.

Fig. 2. Expression and secretion of S. suis antigen proteins.

(A-B) E. coli BL21(DE3) containing pQE80-SufIss- SLYhis, pQE80-SufIss-Enohis, and pQE80-SufIss-Sbphis, respectively, was subcultured overnight in LB. IPTG was added with a final concentration of 1 mM when the absorbance at OD₆₀₀ reached 0.3, and the cells were cultured for another 3 h for protein expression induction. The cells were pelleted and the protein expression was analyzed by SDS-PAGE (A) and Western blot using anti-His antibody (B). (C) Cells of E. coli MC4100 (WT) and E. coli MC4100 Δtat strain (Δtat) containing pJ23-SufIss- SLYhis plasmid were subcultured 1:100 in LB overnight and incubated at 37°C until the absorbance at OD₆₀₀ reached 1. 5 ml and each culture was pelleted by centrifugation. The cell pellet was resuspended with 250 μl of resuspension buffer (50 mM Tris- HCl, pH 7.6, 2 mM EDTA), which was the whole-cell sample (W). Cells were pelleted from 25 ml of each culture and resuspended with 500 μl of fractionation buffer (20 mM Tris-HCl, pH 7.6, 2 mM EDTA, 20% sucrose). Lysozyme (final concentration 0.6 mg/ml) was added, and the supernatant was collected after 20 min incubation at room temperature, which was the periplasmic fraction (P). The whole-cell samples and the periplasmic fraction samples were analyzed by SDS-PAGE followed by Western blotting using anti-His antibody.

Secretion of the S. suis Antigen Proteins to the Extracellular Environment

Given that foreign proteins containing a SufI signal peptide can be transported to the periplasm via the Tat system, we next tested whether the S. suis antigen proteins that were exported to the periplasm could be further released to the extracellular environment by using an outer membrane-defective strain. N-terminally SufI signal peptide-fused SLY, Sbp, and Enolase of S. suis were individually expressed from pJ23 plasmid in the WT and the ΔamiAΔamiC strain, respectively. As shown in Fig. 3A, a significantly higher amount of the proteins could be detected in the culture supernatant of the ΔamiAΔamiC strain than that of the WT strain, while a comparable amount of RNA polymerase was detected in the culture supernatant of both strains. These results suggest that by exploiting the Tat pathway, the S. suis antigen proteins can be successfully secreted to the extracellular environment by using the outer membrane-defective E. coli.

Fig. 3. Secretion of the S. suis antigens by the outer membrane-defective E. coli.

E. coli ΔamiAΔamiC strain containing pJ23-SufIss-SLYhis, pJ23-SufIss-Enohis, and pJ23-SufIss-Sbphis, respectively, was grown overnight in LB with shaking. The same amount of the culture supernatant was collected and concentrated by ultracentrifugation. The samples were then analyzed by SDS-PAGE followed by Western blotting using anti-His antibody (A) and anti-RNA polymerase (RNAP) antibody (B).

Mice Vaccinated with the E. coli Secreting the Antigen Proteins Showed Higher Survival and Milder Clinical Symptoms after the S. suis Challenge

Based on the above results, we constructed three ΔamiAΔamiC strains which constitutively expressed SufI signal peptide-fused antigen proteins SLY, Enolase, and Sbp, respectively, from a plasmid pJ23 containing the constitutive promoter J23119, which were used to inoculate mice. The mice of the vaccinated and vector groups were challenged one week after the second vaccination with S. suis SC19, which is a virulent clinical isolate [31]. As shown in Fig. 4A, the vector group mice showed obvious clinical signs, including clustering, depression, shortness of breath, ragged dorsal hair, and copious eyelid discharge after challenge, while the mice in the immunized group were in good mental condition and only showed slightly ragged dorsal hair. The survival rate at 24 h after the challenge was only 62.5% (5/8) for the vector group, while no mice died in the immunized group (Fig. 4B). These results showed that vaccination with the antigen-secreting E. coli can provide immune protection against S. suis challenge.

Fig. 4. Clinical symptoms and survival.

Four-to-five-week-old Kunming mice were randomly divided into 3 groups with 8 mice in each group. The mice of the vaccinated group were inoculated twice with the vaccine by oral gavage. The vector group was administered with the same amount of bacterial cells of the vector stain. The blank group was neither inoculated nor challenged. Seven days after the second vaccination, the mice in the vaccinated group and the vector group were challenged by intraperitoneal injection with 1.5 × 10⁹ CFU of S. suis SC19. The clinical symptoms (A) and survival (B) were recorded.

Lower Bacteria Load and Milder Pathological Changes Were Observed in the Organs of Vaccinated Mice

To further assess the immune protection efficacy, we analyzed the bacteria loads as well as pathological injury in mice organs including the brain, spleen, and lung after the S. suis challenge. As shown in Fig. 5A, the bacteria loads were significantly lower in the organs of the vaccinated group than those of the vector group. In addition, three mice in each group were dissected 24 h after the challenge and the organs were subjected to histopathological examination. As shown in Fig. 5B, the mice in the vector group showed much more severe pathological changes compared to the immunized mice, including large areas of edema and hemorrhage in brain tissues, collapsed alveoli, and thickened alveolar walls, with lymphocyte necrosis, neutrophilia, and hemorrhage in the spleen.

Fig. 5. Analysis of bacteria burdens and pathological changes.

(A) At 24 h after the challenge, 4 mice in each group were euthanized and the organs were collected which were homogenized, diluted with PBS, and plated on TSA plate containing 5% fetal bovine serum for bacteria enumeration. (B) 3 mice in each group were dissected 24 h after the challenge, and the brain, spleen, and lung tissues of the mice in each group were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. The samples were cut into thin sections, stained with haematoxylin/eosin (H & E), and examined under a microscope.

Discussion

Live vector vaccines have the advantages of low cost, flexibility, and enhanced induction of immune response [1-3]. The most commonly used bacterial vectors to deliver antigens include E. coli, Salmonella, and lactic acid bacteria as well as virulence-attenuated pathogen bacteria [2, 32-34]. Salmonella can elicit an efficient host immune response as it can invade the M cells of the intestine and present antigens effectively [35, 36]. However, in food-producing animals including pigs and poultry, where control and prevention of Salmonella is of urgency, using live Salmonella as vaccine vectors comes with difficulties in differentiating Salmonella infection and Salmonella vaccine. E. coli, which is frequently used for heterologous protein expression, has also been exploited as a vector for antigen protein delivery [33, 37]. Due to the presence of the cell wall, including the inner membrane and the outer membrane in gram-negative bacteria, the cytoplasmically expressed proteins are difficult to be secreted or exposed at the surface of the bacteria. Therefore, protein secretion systems have been exploited to present antigens on the bacterial surface or for antigen secretion. Byrd et al. constructed an attenuated E. coli strain secreting Shiga toxin B subunit via the type V secretion system and showed that this strain provided robust immune protection against challenge with enterohemorrhagic E. coli (EHEC) [38]. The type III secretion system has also been used to deliver antigens that can be directly injected into the host cells. Salmonella delivering PcrV protein through the type III secretion system was used as a vaccine that was shown to be able to provide protection against Pseudomonas aeruginosa [39].

In our study, we constructed a live vector vaccine using an engineered commensal E. coli strain. To achieve protein export from the cytoplasm, the Tat pathway was exploited. The Tat pathway is unique in that it can transport fully folded proteins, therefore enabling the export of proteins with correct conformation [40, 41]. Targeting heterologous proteins to the Tat pathway can be easily accomplished by fusing a Tat signal peptide to the N-terminus of the protein of interest. We used a SufI signal peptide in which SufI is a well-studied Tat substrate without the need for chaperone proteins for its export [42, 43]. Our results showed that SufI signal peptide can effectively guide the S. suis antigen protein to be exported by the Tat pathway (Figs. 1 and 2). Effective targeting of foreign antigen proteins to the Tat pathway by SufI signal peptide has also been reported previously [21].

Another barrier for foreign proteins to be secreted to the extracellular environment is the outer membrane of E. coli. It has been reported that antigen proteins present in the periplasm can not elicit robust immune responses [38]. Thus, we constructed an E. coli ΔamiAΔamiC strain which has been previously shown to be defective in the outer membrane [30]. Our results showed that secretion of the SufI signal peptide-fused S. suis antigen proteins was significantly enhanced by such an E. coli strain (Fig. 3). In a previous study, it was shown that high-level secretion of a Tat signal peptide-fused foreign protein to the culture medium was feasible by using a Bacillus subtilis Tat system in E. coli [20]. In the study, the secretion of foreign protein was achieved in an E. coli strain in which the native Tat system was absent. The possible mechanism could be that the outer membrane was leaky in the absence of the E. coli Tat system. In our study, the native E. coli Tat system is still present and able to export its substrate proteins, and at the same time, deletion of the Tat-dependent amidases leads to a leaky outer membrane, which enhances the release of periplasmic proteins to the extracellular environment. Therefore, our study provides a simple method for protein secretion in E. coli.

S. suis, which has worldwide distribution, is an important bacterial pathogen threatening the pig industry as well as public health [22, 23]. The most commonly used vaccine in controlling S. suis infection in pigs consists of bacterins. However, due to the high diversity in serotypes and sequence types, it is difficult to use bacterins to provide cross-protection [22]. To overcome this problem, subunit vaccines have been studied [44, 45]. Antigen proteins, including SLY [46], Enolase [47], and Sbp [48], have been revealed to provide immune protection against S. suis infection. However, so far, no live vector vaccine against S. suis has been reported. In this study, we reported that by using the outer membrane-defective E. coli, the S. suis antigen proteins SLY, Enolase, and Sbp can be effectively secreted via the Tat pathway. This live vector vaccine showed immune protection against S. suis infection in a mice model. It should be pointed out that although the vaccine showed protection against S. suis infection, the protective efficacy is not very high as the vaccinated group still showed mild clinical signs and bacterial colonization, indicating that further optimizations are needed. Also, by using live engineered bacteria as vaccine, risks including lateral transfer of genes and environmental contamination should be considered. In addition, we use a plasmid to constitutively express the antigen proteins in which plasmid stability and antibiotics resistance concerns need to be considered. Integrating the antigen expression cassette into the chromosome may be applied to solve this problem.