Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

doi:10.21203/rs.2.13690/v1

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Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

https://doi.org/10.21203/rs.2.13690/v1

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published 07 May, 2020

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Background: Recombinant Salmonella enterica serotype Choleraesuis (S. Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S. Choleraesuis vaccine candidate strain rSC0011 (ΔPcrp527::TT araC PBAD crp Δpmi-2426 ΔrelA199::araC PBAD lacI TT ΔasdA33, Δ,deletion, TT, terminator) delivering SaoA, a conserved surface protein that is present in many S. suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein, we described another recombinant attenuated S. Choleraesuis vector, rSC0012 (ΔPfur88:: TT araC PBAD fur Δpmi-2426 ΔrelA199:: araC PBAD lacI TT ΔasdA33) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results:The strain rSC0012 strain with the ΔPfur88::TT araC PBAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔPcrp527::TT araC PBAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S. Choleraesuis challenge in BALB/C mice. Conclusions: The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S. Choleraesuis and S. suis. Keywords: Salmonella Choleraesuis, virulence, immunogenicity, Fur, inflammatory

Animal Science

General Microbiology

Salmonella Choleraesuis

virulence

immunogenicity

Fur

inflammatory

Streptococcus suis is a pandemic pathogen responsible for a wide range of invasive diseases such as pneumonia, meningitis and bacteraemia in both humans and pigs[1,2]. S. suis type 2 (SS2) is the most frequently and virulent isolated from both humans and pigs among all serotypes reported to date [1,3]. The surface-anchored protein (Sao) is a highly conserved membrane-anchored protein and proved to be a immunogenic vaccine candidate[4].However,Sao formulated with Emulsigen-Plus® provides only partial protection to mice against the SS2 infection [3].In our previous study,a recombinant attenuated Salmonella enterica serotype Choleraesuis vaccine strain rSC0016 carrying saoA gene, provided full protection to mice against the SS2 challenge[5]. From the above,an effective delivery system such as live Salmonella enterica serotype Choleraesuis play a crucial role to the effectiveness of Sao.

The use of intracellular Salmonella enterica as a vehicle to deliver heterologous protective antigens against pathogens is an attractive strategy. Curtiss et al. developed the RDAS (Regulated Delayed Attenuated Strategies), which enable live salmonella vaccine effectively colonize lymphoid tissues during the invasion stage because of its wild-type aggressiveness and then be full attenuated by silencing the virulence factor, while stimulate both strong cellular and humoral immunity in the immunized mice[6]. Several ways were used to implement this strategy (RDAS). One way is the reverse synthesis of lipopolysaccharide O-antigen by pmi mutation [7]. Another way is to replace the upstream regulatory and promoter sequences of virulence genes with a tightly regulated araC P_BAD activator–promoter [8]. This strategy has been successfully used for S. Typhimurium and S. Typhi[6,7,8]. With this strategy, we construct a regulated delayed S. Choleraesuis vaccine strain rSC0011 with regulated crp and pmi mutations[9]. rSC0011 delivering S. suis antigens were effective to induce protective immunity against SS2 in mice, but it occasionally caused enteritidis.

We sought to improve our S. Choleraesuis candidate vector vaccine by using alternative mutation or introducing new mutation to decreasing its potential to induce enteritidis and enhance immunogenicity. Fur is an important regulatory protein in Salmonella. In the presence of iron, Fur acts as a repressor of iron-controlled genes and mounts an adaptive acid tolerance response [8]. Synthesis of Fur in a vaccine strain during growth confers acid tolerance and maintains iron homeostasis. A decrease of Fur synthesis in Salmonella leads to acid sensitivity and iron acquisition [9]. Curtiss et al. reported that a S. Typhimuriumstrain with an arabinose regulated fur mutation is highly immunogenic [10]. In these consideration, an arabinose regulated fur mutation (ΔP_fur::TT araC P_BAD fur) was introduced into a S. Choleraesuis vaccine strain with multiple preexist mutations (Δpmi–2426 ΔrelA199::araC P_BAD lacI TT ΔasdA33) to generate strain rSC0012. A plasmid pS-SaoA[5], encoding saoA from SS2, was transformed into this strain. We evaluated the virulence, immunogenicity and protection against challenge with virulent SS2 or S. Choleraesuis C78–3.

Construction and characterization of the S. Choleraesuisvaccine strain rSC0012

Fur is a ferric uptake regulator that is involved not only in iron metabolism, uptake, and transport, but also invasion and survivalof S. Typhimurium in the hosts [11–13]. The absence of Fur attenuates S. Typhimurium6,14]. To improve the safety and increase the immunogenicity of S. Choleraesuis vector, a new strain, rSC0012, was generated with an arabinose regulated fur, ΔP_fur::TT araC P_BAD fur (Figure 1A).

The phenotypes of the mutations ΔP_fur::TT araC P_BAD fur and ΔrelA:::araC P^BADlacI TT were confirmed by western blot analysis (Figure 1B). The level of Fur synthesis decreased with arabinose dilution (Figure 1B). The presence of mutation ΔrelA:::araC P^BADlacI TT in rSC0012(pS-SaoA) were confirmed by the increased synthesis of SaoA (Figure 1B) due to the derepression of P_trc promoter on plasmid in rSC0012, which resulted from reduced LacI production whose production was controlled by arabinose. The pmi gene encodes 6-phosphomannose isomerase that interconverts fructose–6-phosphate and mannose–6-phosphatein in Salmonella [7].Because mannose is required for O-antigen synthesis, the Δpmi mutation enables the strain rSC0012 to display a smooth LPS pattern in nutrient broth in the presence of mannose and a rough pattern in the absence of mannose (Figure 1C). The ΔasdA mutation enables the strain rSC0012 to have an obligate requirement for DAP [18], which can be complemented with a vector harboring the asd gene then eliminates the need for antibiotic resistance genes for plasmid maintenance.[19]. The growth rates of rSC0012 with DAP, rSC0012(pS-SaoA), and rSC0012(pYA3493) were similar (Figure 1D). rSC0012 could grow only with DAP (Figure 1D).

Antigen synthesis and plasmid stability in S. Choleraesuis rSC0012

Stable maintenance of plasmids and the production of heterologous antigens are critical to ensure efficacy of recombinant live vaccines. The SaoA protein is a highly conserved surface protective antigen among S. suis serotypes [2, 5]. Using live attenuated S. Choleraesuis vector delivering SaoA antigen from S. suis will allow to develop a bivalent vaccine against both S. Choleraesuis and S. suis. The stabilities of pS-SaoA and pYA3493 in rSC0012 were evaluated by continuous culturing for 50 generations. The stabilities of both Asd⁺ plasmids, pS-SaoA and pYA3493, were 100% in rSC0012 (data not shown). All rSC0012 colonies examined (100 clones/generation) by endonuclease digestion possessed the Asd+ plasmid pS-SaoA or pYA3493. The 34-kDa SaoA protein was detected in cells obtained from both the first and 50th generations of rSC0012(pS-SaoA) (data not shown), indicating the stability of plasmid and stable synthesis of SaoA.

Distribution of secreted SaoA in S. Choleraesuis rSC0012

The production levels of SaoA in various subcellular fractions from pS-SaoA-carrying strains, rSC0011, rSC0012, and rSC0018, were determined (Figure 2A-B). rSC0018 is a derivative of C500 [5,20], a licensed live S. Choleraesuis vaccine attenuated by chemical methods in China, with an asdA mutation. The results showed that the level of the SaoA protein produced in the cytoplasm by strain rSC0012(pS-SaoA) was significantly lower than strain rSC0011(pS-SaoA) (Figure 2B; #, P<0.05), but significantly higher in supernatant when compared with rSC0011(pS-SaoA) and rSC0018(pS-SaoA) (Figure 2B; **, P<0.01). The SaoA protein produced in the periplasm fraction of rSC0012(pS-SaoA) was significantly higher than strain rSC0018(pS-SaoA)(Figure 2A-B).

Lower virulence of S. CholeraesuisrSC0012 in vivo

To evaluate the virulence of strains with mutations ΔP_fur::TT araC P_BAD fur or ΔP_crp::TT araC P_BAD crp, the LD₅₀ values of rSC0012 and rSC0011 were tested in 3- -week-old BALB/c mice, which represents young mice. rSC0018 was used as an attenuation control. All strains carried the expression plasmid pS-SaoA. The results revealed that the LD₅₀s of rSC0011(pS-SaoA), rSC0012(pS-SaoA), and rSC0018(pS-SaoA) were at least 10⁹ CFU by oral inoculation; whereas, the LD₅₀of wild-type C78–3 was 9.5×10² CFU (Table 2). Following intraperitoneal infection, the LD₅₀ of rSC0012(pS-SaoA) was 38.89-fold higher than that of rSC0011(pS-SaoA) and 3.2-fold higher than that of rSC0018(pS-SaoA) in 3-week-old mice. The LD₅₀ of rSC0012(pS-SaoA) was significantly higher than that of C78–3 and rSC0011(pS-SaoA), but similar to rSC0018(pS-SaoA) in 3-week-old mice (Table 2). These results indicated that the virulence of rSC0012(pS-SaoA) harboring ΔP_fur::TT araC P_BAD fur was significantly lower than that of rSC0011(pS-SaoA) harboring ΔP_crp::TT araC P_BAD crp by intraperitoneal route in young mice.

Tissue distribution of S. Choleraesuisstrains in BALB/c mice

Fur and Crp are important regulatory proteins in Salmonella. Inactivation of the fur and crp genes, attenuates the organism [6,21]. To quantitatively the colonization of the S. Choleraesuisstrains containing regulated fur or crp mutations, 3-week-old BALB/c mice were orally inoculated with rSC0011(pYA3493), rSC0012(pYA3493), rSC0018(pYA3493), rSC0011(pS-SaoA), rSC0012(pS-SaoA), rSC0018(pS-SaoA), or wild-type C78–3 (Figure 3A–F). The mice inoculated with wild-type S. Choleraesuis C78–3 died 3–5 days after inoculation, whereas the mice infected orally with vaccine strains rSC0011, rSC0012, rSC0018 containing either plasmid pS-SaoA or pYA3493 survived. The bacteria titers of wild-type strain C78–3 in Peyer’s patches, spleen, and liver were significantly higher than those of vaccine strains rSC0011, rSC0012, rSC0018 containing either plasmid pS-SaoA or pYA3493 at 3 days after inoculation. (Figure 3A–C). The titers of bacteria in Peyer’s patches were similar for strains rSC0012(pYA3493), rSC0012(pS-SaoA), rSC0011(pYA3493), and rSC0011(pS-SaoA) at 3–21 days post-inoculation(Figure 3A). At 3d, 7 d, 14 d, 21 d and 28 d, the numbers of rSC0012(pS-SaoA) were signiﬁcantly higher than those of attenuated vaccine strains rSC0018(pS-SaoA), same for two control vector, the numbers of rSC0012(pYA3493) were signiﬁcantly higher than those of attenuated vaccine strains rSC0018(p YA3493) and there was no significant difference with same strain with expression or control vector.respectively (P < 0.01; Fig. 3A), indicating that the colonization abilities of strains rSC0012(pS-SaoA) and rSC0012(pYA3493) in Peyer’s patches were higher than that of rSC0018 with either pYA3493 or pS-SaoA.

In spleen, the titers of rSC0012(pYA3493) and rSC0012(pS-SaoA) were similar to those of vaccine strains rSC0018(pYA3493) and rSC0018(pS-SaoA) at 7, 14, 21, 28 days after inoculation (Figure 3B). The titers of strains rSC0011(pYA3493) and rSC0011(pS-SaoA) were significantly higher than those of rSC0012(pYA3493), rSC0012(pS-SaoA), rSC0018(pYA3493), and rSC0018(pS-SaoA), at 3, 7, 14, and 21 days, respectively (Figure 3B).

In liver, the titers of rSC0012(pYA3493) and rSC0012(pS-SaoA) were similar to those of vaccine strains rSC0018(pYA3493) and rSC0018(pS-SaoA) at 3, 14, 21, 28 days after inoculation (Figure 3C). At 3d, 7 d, 14 d, 21 d and 28 d post-inoculation, the titers of rSC0012(pS-SaoA) were significantly lower than those of rSC0011(pS-SaoA), same for two control vector, the numbers of rSC0012(pYA3493) were signiﬁcantly lower than those of strains rSC0011(pYA3493).respectively(P < 0.01; Fig. 3C), whereas the strain rSC0012(pYA3493) was the fastest to be cleared in liver (Figure 3C). These results indicated that the ΔP_fur::TT araC P_BAD fur mutation impaired the colonization in the liver of S. Choleraesuis vaccine strains. In a summary, the ΔP_fur::TT araC P_BAD fur mutation reduced the colonization ability of S. Choleraesuis vaccine strains in mice spleen and liver but not in the Peyer’s patches.

Antibody responses in mice immunized with S. Choleraesuisstrains

All of the mice immunized with strains containing pS-SaoA developed anti-SaoA antibodies (Figure 4A). rSC0012(pS-SaoA) induced significantly higher anti-SaoA IgG titer than did rSC0011(pS-SaoA) 3 weeks after the immunization in 3-week-old mice (Figure 4A; *, P<0.05). Although the anti-SaoA IgG titer of rSC0012(pS-SaoA) were slightly higher than that of rSC0011(pS-SaoA) 5 weeks after immunization, they were not significantly different (Figures 4A). Compared to mice immunized with rSC0018(pS-SaoA), higher serum IgG titers against SaoA were detected in mice immunized with both rSC0011(pS-SaoA) and rSC0012(pS-SaoA) at 3 weeks and 5 weeks postimmunization (Figure 4A, *, P<0.05, **, P<0.01). After boosting, higher titers of anti-SaoA IgG were observed in mice immunized with all three strains containing pS-SaoA (Figure 4A, #, P<0.05). All three attenuated recombinant Salmonella strains induced significant OMP titers after the first immunization in mice (Figure 4B). A significant boosting of serum antibody responses to OMPs was observed after the second immunization, (Figure 4B; #, P<0.05).

Mucosal IgA anti-SaoA responses were detected at week 3 in mice immunized with all three attenuated strains containing pS-SaoA. rSC0018(pS-SaoA) induced lower titers of anti-SaoA IgA than rSC0012(pS-SaoA) or rSC0011(pS-SaoA) did in mice (Figure 4C, *, P<0.05, **, P<0.01). Anti-SaoA IgA levels detected in the rSC0012(pS-SaoA) immunized group were significantly higher than those induced in the rSC0011(pS-SaoA) immunized group at 3 and 5 weeks after the immunization (Figure 4C, *, P<0.05, **, P<0.01). These results indicated that strain rSC0012(pS-SaoA) harboring ΔP_fur::TT araC P_BAD fur elicited a stronger immune response than strain rSC0011(pS-SaoA) harboring ΔP_crp::TT araC P_BAD crp did, especially elicit a stronger mucosal immune response.

IFN-γ or IL–4 production induced by S. Choleraesuisstrains

To further evaluate the effect of the ΔP_fur::TT araC P_BAD fur and ΔP_crp::TT araC P_BAD crp mutations in a strain with multiple preexist mutations on Th1/Th2 immune responses, the levels of IFN-γ and IL–4 in the spleen tissues of mice 7 days and 14 days after booster immunization were measured. The results showed that the titers of IFN-γ and IL–4 induced in mice immunized with rSC0012, and rSC0011 with either pYA3493 or pS-SaoA were significantly higher than those induced in mice inoculated by strain rSC0018 with either pYA3493 or pS-SaoA at both 7 and 14 days after booster (Figure 5A, B; *, P<0.01,**, P<0.05). Although rSC0011(pS-SaoA) induced a slightly higher level of IFN-γ in mice than rSC0012(pS-SaoA) did, no significant difference was observed in 3-week-old mice at both 7 and 14 days after booster (Figure 5A-C). However, rSC0011(pYA3493) elicited a higher level of IFN-γ than rSC0012(pYA3493) did at 7 days after booster. The titers of IL–4 induced by rSC0012 with either pS-SaoA or pYA3493 in spleen tissues were significantly higher than those induced by rSC0011 with either pS-SaoA or pYA3493 in 3-week-old mice (Figure 5B; *, P<0.05, **, P<0.01) at 7 and 14 days after booster. Of note, rSC0012(pS-SaoA) induced a dominant Th2 immune response (IFN-γ < IL–4, IFN/IL–4<1) in 3-week-old mice (Figure 5C); whereas, rSC0011(pS-SaoA) elicited a moderately dominant Th1 immune response (IFN-γ > IL–4, IFN/IL–4>1) in 3-week-old mice (Figure 5C, *, P<0.05, **, P<0.01). These results indicated the isogenic strain with two different mutations, ΔP_fur::TT araC P_BAD fur or ΔP_crp::TT araC P_BAD crp, affected different branches of immune responses. In additional, the titers of IFN-γ and IL–4 induced by the three attenuated strains harboring pS-SaoA in immunized mice were higher than those induced by the same strains harboring the emptor vector pYA3493 (Figure 5A–C; #, P<0.05, ##, P<0.01), suggesting that the protein, SaoA, might augment the immune responses in mice.

Induction of inflammation in mice

The inflammatory properties of intestinal tissue were investigated in mice after immuned vaccine strains and wild type strain,C78–3.The expression of cytokine genes, TNFα, IL–1β, IL–6 and IL–8, were assessed by quantitative real -time PCR in gut tissue samples at 6 hours and 12 hours postinfection. All strains with mutation ΔP_fur::TT araC P_BAD fur showed significantly lower transcription levels of cytokine genes IL–1β, IL–6, IL–8 and TNFα than the wild -type strain, C78–3 at both 6 h and 12 h postinfection (Fig 6A-D,*, P<0.05, **, P<0.01). The C78–3 with mutation ΔP_fur::TT araC P_BAD furinduced significantly lower transcription levels of cytokine genes IL–1β, IL–6,IL–8 and TNFα than the same strain with ΔP_crpTT araC P_BADcrpmutation at both 6 hours and 12 hours postinfection (Fig 6 C and D, &, P<0.05, &&, P<0.01). At 6 hours postinfection, strain rSC0012( pS-SaoA) showed significantly lower transcription levels of cytokine genes IL–1β, IL–6, IL–8 and TNFα than strain rSC0011( pS-SaoA) (Fig 6A), a similar trend was seen with IL–1β, IL–6, IL–8 and TNFα at 12 h, although the differences were not significant for IL–6 and TNFα (Fig 6B,#, P<0.05, ##, P<0.01). These results suggest that the addition of the ΔP_fur::TT araC P_BAD fur mutations could decreased the inflammatory potential of strains rSC0012( pS-SaoA).

Comparison of the protective immunity induced by S. Choleraesuisstrains

To evaluate the protective immunity conferred by rSC0012(pS-SaoA), the mice in each immunized group were challenged orally with 50×LD₅₀ of the virulent S. Choleraesuis C78–3 strain or 20×LD₅₀ of the virulent SS2 strain at 14 days post-boost immunization. After challenge with C78–3, the results revealed 100% protection in mice immunized with either strain rSC0011(pS-SaoA) or strain rSC0012(pS-SaoA), suggesting full protection. Mice immunized with the rSC0018(pS-SaoA) strains resulted in 20% survival. In contrast, all the mice in the PBS group succumbed to the challenge after 4 days. There were no significant difference between the groups immunized with rSC0012(pS-SaoA) and rSC0011(pS-SaoA), though both displayed significantly higher levels of protection than the group immunized with rSC0018(pS-SaoA) (Figure 7A). After the SS2 challenge, immunization with the rSC0011(pS-SaoA), rSC0012(pS-SaoA) and rSC0018(pS-SaoA) strains resulted in 80% survival, 95% survival and 16.7% survival with the lethal SS2 challenge, respectively. In contrast, all the mice in the PBS group succumbed to the challenge after 2 days.

The ultimate goal of an engineered live vaccine strain relys on achieving the proper balance between immunogenicity and attenuation [22,23]. Achieving that goal will restrict unacceptable reactogenicity to avoid over-excitation of inflammatory responses, but sufficient metabolic activity should be maintained to enble the live vaccine to reach deep lymphatic tissues and induce protective immunity[24].Modern industry vaccine development relies on the genetic engineering of defined mutations in bacterial strains[23,25,26].In order to enhancing the immunogenicity of vaccine strains or to disarm them, multiple independent defined mutations were introduced into Salmonella to generate a new recombinant attenuated vaccine strains[6,25,23,27]. By incorporating proper mutations,vaccine strains can be rationally designed to to avoid unacceptable reactogenicity and enhanced immunogenicity [21]. Our previous S. Choleraesuis live attenuated vaccine vector rSC0011 occasionally caused enteritidis in mice[9]. One of the ways to address this problem is by incorporating a sopB mutation [5,25]. In this paper, another way to improve the S. Choleraesuis vector was reported.

Fur is an important regulatory proteins of Salmonella, which has been implicated in the acid tolerance response since fur mutants are acid sensitive and cause altered expression of several acid shock proteins [28,29]. A S. Typhimuriumstrain with an arabinose regulated fur mutation is adequately attenuated and highly immunogenic [6,30]. However, S. Typhimurium belong to group B, while S. Choleraesuis group C. Whether the ΔP_fur::TT araC P_BAD fur mutation that results in the proper balance between attenuation and immunogenicity of S. Typhimurium is also appropriate for attenuated S. Choleraesuis vaccine has not been reported previously.

We corroborated the S. Choleraesuis strain rSC0012 (ΔP_fur::TT araC P_BAD fur Δpmi ΔrelA:::araC P_BAD lacI TT ΔasdA)displayed a regulated decrease of Fur production in the absence of arabinose. In a previous publication, S. Typhimurium strain with a single ΔP_fur::TT araC P_BAD fur mutation has shown higher virulent than S. Typhimurium with a single ΔP_crp527::TT araC P_BAD crp mutation by an oral immunization. Unlike report from Curtiss et al in S. Typhimurium [6], our studies did not showed that the ΔP_fur::TT araC P_BAD fur mutation has higher virulent than ΔP_crp527::TT araC P_BAD crp mutation in S. Choleraesuis.In fact,the LD₅₀of rSC0012(pS-SaoA) was 38-fold higher than rSC0011(pS-SaoA) for 3-weeks-old mice with intraperitoneal injection. This result suggest that the fur mutation does different attenuation with S. Choleraesuis or S. Typhimurium,which may due to its complex role as a transcriptional activator of virulence in different strains [31].

The ΔP_fur::TT araC P_BAD fur mutation can modify the iron regulated outer membrane protein,then strain with ΔP_fur::TT araC P_BAD fur induced less inflammatory cytokine production in mice than the isogenic strain with ΔP_crp527::TT araC P_BAD crp mutation or wild type strain. Theses results suggest that the S. Choleraesuis withΔP_fur88::TT araC P_BAD fur mutation exhibits a lower propensity to elicit over-inflammatory responses while attenuated sufficiently.

Through the oral vaccination route, a vaccine strain will endure the challenges of acid, bile, and antimicrobial peptides existing in the gastrointestinal tract. Once inside Peyer’s patch, it will face macrophagocytes and T cells during the process transferring to deep lymphatic tissues [33]. Fe²⁺is required for cellular function within nearly all cells, limiting the availability of Fe²⁺ starves pathogens for Fe²⁺and weakens the pathogens’ ability to combat antimicrobial responses by the host[31,33]. Fur is a transcription factor which utilizes Fe²⁺as a corepressor and represses siderophore synthesis in pathogens[34].Thus, regulated delayed fur mutation may affect the growth of S. Choleraesuis in the deep tissue of the mice. rSC0012, harboring mutation ΔP_fur::TT araC P_BAD fur, was cleared more rapidly than rSC0011, harboring mutation ΔP_crp::TT araC P_BAD crp, in the deeper lymphatic tissues, spleen, and liver of the immunized mice though both rSC0011 and rSC0012 had similar levels of Peyer’s patch colonization by oral immunization. The clearance of rSC0012 in liver was similar to the attenuation control rSC0018. This result indicates that strain rSC0012 was more attenuated than rSC0011 in deep lymphoid tissue, which maybe due to preventive rSC0012 from getting iron in deep lymphoid tissue with regulated decreasing levels of Fur protein.

Both rSC0012 and rSC0011 induced higher levels of IgA, IgG, IFN-γ, and IL4 responses with great colonization compared to strain rSC0018 with less colonization in mice.In general,the live Salmonella vaccines with RDAS display superior colonization level in lymphoid tissues during the invasion stage,leading to enhanced protection by exposed to more Salmonella mycoprotein and heterologous antigens[10,35].However,there are do exist high levels of colonization but low immunogenicity.A strain with the regulated delayed rfc mutation exhibits superior colonization and yet does not stimulate higher heterologous protection than a Δrfc strain without RDAS[36].Thus, in addition to colonization level, other determining factors may exist to induce the enhanced protection achieved by regulated delayed attenuation. From the above, Selecting the proper mutation is critical for vaccine development with RDAS.This study conﬁrmed above statement.Although the colonization of strain rSC0012(pS-SaoA) in mice was less than that of strain rSC0011(pS-SaoA), strain rSC0012(pS-SaoA) stimulated stronger serum IgG and mucosal IgA responses than the strain rSC0011(pS-SaoA). This phenomenon suggests that strain with regulated delayed fur mutation may stimulate stronger antibody response with fewer bacteria than strain with regulated delayed crp mutation.

Both rSC0012 and rSC0011 aroused a Th1 cell-mediated response, as ostensived by the significant up-regulation of imprint Th1 cytokines, IFN-γ, the stimulator of Th1-type T cell immune response.It could partially be that the intracellular characteristics of Salmonella enterica cause them to be detected on the surface of APC through MHCⅠ.This phenomenon is consistent with previous studies with S. Typhi [6].Strain rSC0012 induced higher Th2 cell-mediated response than strain rSC0011, as aroused by the significant up-regulation of imprint Th2 cytokines, IL–4 and greater humoral responses than rSC0011 in mice. The presence of S. suis-specific IgA serves to promptly deliver the antigen to Peyer’s patch dendritic cells or phagocytes and also promptly excite the adaptive immunity during secondary exposure [37]. Quaintly, the more attenuated rSC0012 strain induced significantly higher IgA and IgG antibody responses to SaoA than the rSC0011 at 3 weeks after the initial immunization in juvenile mice,while in general live vaccine strains that are more attenuated are less immunogenic[21,27].This may be due to a balance between attenuation and the ability to participation immune components and activate a controlled over-inflammatory response by the finely crafted strain[23].

The results presented herein highlight that strain rSC0012(pS-SaoA) with regulated delayed fur mutation has retained vaccine efficacy and adequate immunogenicity whilst being safer than previous strain rSC0011(pS-SaoA).Furthermore, the inclusion of the ΔP_fur88::TTaraCP_BADfur mutation may be able to decrease inflammation caused by live-attenuated Salmonella enterica serotype Choleraesuis vaccine, which has been an imperfection for other live-attenuated Salmonella enterica serotype Choleraesuis vaccine which transformed from wild type virulent strain. Both S. Choleraesuis and S. suis are major swine pathogens. Currently, there exist license live vaccines against S. Choleraesuis for swine, including Entersol® Salmonella T/C and SC–54 manufactured by Boehringer Ingelheim and Arugs SC/ST, respectively [38]. However, there is no licensed vaccine against S. suis. Preventing the diseases caused by SS2 in swine with a combined vaccine is a long-sought goal.

Our results have shown the strains rSC0012(pS-SaoA) with regulated delayed fur mutation could confer higher protection against challenges with lethal doses of SS2 or S. Choleraesuis C78–3. Thus,the use of attenuated S. Choleraesuis to develop a vaccine against S. suis will have the great potential to ease the burden of both pathogens. The regulated delayed fur mutation in the novel vaccine rSC0012 resulted in a well-safety, highly immunogenic, and effective vaccine in mice, this study has paved the way for testing in piglets. Our findings will aid the optimal of a S. Choleraesuis vaccine vector capable of eliciting a suitable immune response against other pathogens.

Animals

All animal experiments were authorized by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU–2007–0005) and accorded to the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals.All surgery was performed under anesthesia intraperitoneally injected with sodium pentobarbital,675μg per mouse.All the animals were humanely euthanized after the study by inhalation of CO₂,while injection with sodium pentobarbital,675μg per mouse, and all efforts were made to minimize suffering.

Strains, plasmids, and culture conditions

The strains, plasmids, used in this study are described in Table 1. C500, an approved live S. Choleraesuis vaccine strain attenuated by thallium compound in China, was used as an attenuation control [39]. The genetic characterization of this strain has been reported [40]. S. suis serotype 2 (SS2, CVCC3928) and S. Choleraesuis C78–3 (CVCC79103) were purchased from China Institute of Veterinary Drug Control.Plasmid pYA3493 is an Asd⁺ vector with a P_trc promoter. The asd gene from Salmonella was used as a unique plasmid marker to be used in asd mutants to constitute a balanced-lethal system [18]. LB medium[5],Nutrient broth (NB) and MacConkey agar (Difco) were used for phenotype characterization.When required, media were supplemented with 2,6-diaminopimelic acid (DAP;50 µg/mL),chloramphenicol (Cm; 25µg/mL),L-arabinose (0.2% wt/vol), D-mannose (0.2% wt/vol)or sucrose (5% wt/vol).Plasmid pS-SaoA were described on previously study[5]. The saoA gene is under the control of the P_trc promoter (Table 1). S. Choleraesuis vaccine vector strain rSC0012 harboring plasmid pS-SaoA (expression vector) or pYA3493 (control vector) were grown in LB broth with both 0.2% arabinose and 0.2% mannose. Selenite broth was used for enrichment of S. Choleraesuis from mice tissues. Strains were prepared as previously described [5,9,10,30]. Bacterial growth was monitored with a spectrophotometer at OD₆₀₀ and by direct plating for colony counts.

Construction of S. Choleraesuis mutant strains.

Four mutations ΔP_fur::TT araC P_BAD fur, Δpmi, ΔrelA:::araC P_BAD lacI TT, and ΔasdA were introduced into S. Choleraesuis C78–3 by conjugation with E. coli χ7213 harboring aforementioned suicide vectors as previously described [41]. The suicide vectors used are listed in Table 1 and Fig. 1A. To construct mutation ΔP_fur::TT araC P_BAD fur, a 1,335-bp TT araC P_BAD cassette were used to replace the 239-bp promoter sequence of the fur gene to achieve arabinose-regulated Fur synthesis (Figure 1A). The araC P_BADcassette contains a transcription terminator (TT) sequence to prevent araC transcription reading through adjoining genes. Plasmid for ΔP_fur::TT araC P_BAD fur were confirmed by DNA sequencing. All the primers used have been reported [26].

Characterization of S. Choleraesuismutations in vitro

All mutations were confirmed by colony PCR using homologous primers [30]. The ΔasdA mutation was verified by growth with or without DAP in LB broth [18,19].Lipopolysaccharide (LPS) profiles were examined by silver staining in 12% sodium dodecyl sulfate-polyacrylamide (SDS) gel for the Δpmi mutation [41,42]. The ΔP_fur::TT araC P_BAD fur deletion-insertion mutation was verified by reduced production of Fur protein as arabinose concentrations decreased with the increased bacterial growth by western blot using anti-Fur antiserum[30]. The production of SaoA was verified by western blot using anti-SaoA antiserum, respectively [9,18,19,20,].

Salmonella subcellular fractionation

To evaluate the subcellular localization of synthesized SaoA in the live attenuated S. Choleraesuis vaccine, cultures were grown in NB to an OD₆₀₀ of 0.8 and centrifuged at 13,200 × g for 5 min to collect supernatant and pellet. The culture supernatant was saved for later analysis of the bacterial secreted proteins. Periplasmic fractions were prepared using the lysozyme-osmotic shock method as previously described [43,44]. Equal volumes of supernatant,periplasmic, cytoplasmic samples were separated by SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride membrane for western blot analysis using anti-SaoA antiserum [5]. The gel band were analyzed with ImageJ software (NIH) [45].

Preparation of SaoA and Salmonella outer membrane proteins (SOMPs)

His-tagged SaoA fusion protein and Salmonella outer membrane proteins (SOMPs) were prepared as previous described [5].

Determination of virulence in mice.

Three-week-old female BALB/c mice were obtained from Animal Center of Yangzhou University. Mice were fasting for 6 h before inoculation.Recombination S. Choleraesuis vector strains were cultured in LB broth with D–0.2% mannose and 0.2% L-arabinose for 12 hours at 37°C as standing cultures. The cultures were diluted 1:50 in the same media and cultured at 37°C to an OD₆₀₀ of 0.9. Bacteria were collected by centrifugation at 13,200 × g for 5 min at room temperature and resuspended in PBS to densities suitable for the inoculation.Serial dilutions of the S. Choleraesuis strains were plated onto LB agar supplemented with 0.2% D-mannose and 0.2% L-arabinose to measure the actual densities. Groups of five mice were inoculated with different doses in 20 μl (oral immunization) or 100 μl (intraperitoneal immunization). The mice were observed for 4 weeks for death. The LD₅₀s were calculated according the method of Reed and Muench [46].

Distribution of Salmonella in BALB/c mice

A colonization assay for recombination S. Choleraesuis vector strains was performed as described previously [9, 11]. Three-week-old female BALB/c mice were divided into 7 groups with 25 mice in each group.Each mouse was orally inoculated with 1±0.2×10⁹ CFU of S. Choleraesuis strains.Peyer’s patches, spleen, and liver of the mice were collected on days 3, 7, 14, 21 and 28 post-inoculated. The densities of bacteria in the tissues were determined using the method reported in previous studies[5,9,11,47]. The assay was performed twice, and the data were similar and pooled for analysis.

Immunization of mice

Bacteria were grown and collected as above.Serial dilutions of the S. Choleraesuis vaccine strains were plated onto LB agar supplemented with 0.2% D-mannose and 0.2% L-arabinose to determine the actual dose.Three-week-old female BALB/c mice were orally inoculated with 1±0.2×10⁹ CFU of S. Choleraesuis vaccine strains containing either pS-SaoA or pYA3493.Mice were boosted inoculated with the same dose of the same strain after three weeks. About 50 μL of whole blood was collected by tail vein three weeks after primary inoculation and two weeks after boosting. Serum was separated from the whole-blood samples and stored at −70°C. Vaginal-wash samples in mice were collected at the indicated time and stored at −70 °C[9,35,48,49].This experiment was performed in triplicate with each group receiving a similar dose of the vaccine strains.

Tissue collection after Salmonella infection of mice

Three-week-old female BALB/c mice were divided into 5 groups with 10 mice in each group. Groups of mice were orally inoculated with 1±0.3×10⁹ CFU of Salmonella strains. Spleen and intestinum tenue of the mice were collected at 6 h and 12 h postinfection.The tissues were frozen with liquid nitrogen and then transferred to –70°C.

Enzyme-linked immunosorbent assay (ELISA)

Serum IgG antibody production against S. Choleraesuisouter membrane proteins (OMPs) and SS2 SaoA,and vaginal-wash IgA antibody production against SaoA in mice were evaluated by Enzyme-linked immunosorbent assay (ELISA) [5].Cytokines in tissues were analyzed by sandwich ELISA using commercial kits (BD Biosciences) according to manufacturer’s instructions. The results from the two experiments were pooled for statistical analysis.

Quantitative real -time PCR (qPCR) for cytokines

For RNA isolation, gut tissues were homogenized and suspended in TRIzol® (Thermo Fisher Scientific,USA). Tubes were vortexed for 3 min to disrupt the tissues. Chloroform was added to TRIzol® -treated samples and the samples centrifuged at 13200×g for 10 min. The aqueous phase was separated out, and the RNA precipitated using precooled isopropanol. For quantitative real-time PCR, 1 µg of RNA was then reverse transcribed to cDNA. The primers were designed using Primer Blast (NCBI net) and synthesized by TSINGKE Biological Technology Co., Ltd. The sequences of the primers are listed in Table 3.Each sample were amplified using 7500 Fast Real -Time PCR Instrument (ABI,US) using Fast SYBR Green Master Mix (Thermo -Fisher Scientific). The results were using internal reference GAPDH as control for normalization, and the 2 ^^-ΔΔCt method was used to estimate the relative expression level of the mRNAs of target genes.

Challenge with S. suis serotype 2 (SS2)and S. Choleraesuis (C78–3) in mice

Twenty mice in each group were challenged with SS2 by intraperitoneal (i.p.) injection with 2.4 × 10⁸CFU of SS2 in 100 μl PBS at five weeks after primary immunization . The 50% lethal dose (LD₅₀) of SS2 in BALB/c mice was 1.2 × 10⁷CFU. Another twenty mice in each group were challenged orally with 4.8 × 10⁴CFU of C78–3 in 20 μl PBS. The LD₅₀ of C78–3 in 3-week-old BALB/c mice was 9.5 × 10²CFU. Challenged mice were monitored for death daily for 15 days [9,25,26,35].

Statistical analysis

Statistical analyses on ELISA were presented as the geometric means and standard deviations for all assays. A Mann-Whitney U Test ( GraphPad Software, Inc.) was applied to contrasting the distribution of the S. Choleraesuis in tissues of mice. The Kaplan-Meier method (SPSS Software) was used for obtain the survival fractions following i.p. challenge of immunized mice. A P value of 0.05 was considered statistically significant.

Salmonella enterica serotype Choleraesuis:S.Choleraesuis;Streptococcus suis: S. suis;Sao:Surface-anchored protein;SS2:S.suis serotype 2;RDAS:Regulated delayed attenuated strategies;LB: Luria broth;LD50: 50% lethal dose. Antigen-Presenting Cells: APC; Major Histocompatibility ComplexⅠ: MHCⅠ; T helper type 1: Th1; Phosphate buffer saline:PBS; Lipopolysaccharide:LPS;Sodium dodecyl sulfate-polyacrylamide: SDS.

Ethics approval and consent to participate

Procedures involving the care and use of animals were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU–2007–0005) and complied with the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analysed during this study will be available from the corresponding author on reasonable request

Competing interests

None of the authors of this manuscript have any competing or financial interests.

Funding

This work was supported by the National Natural Science Foundation of China (31172300, 31672516), supported by the Grant No. BE2016343 from Jiangsu province and the Priority Academic Program Development of Jiangsu Higher Education Institutions, supported by State Key Laboratory of Genetically Engineered Veterinary Vaccines (No.AGVSKL-ZD/ZY–201807).The funding bodies have not been involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Authors’ contributions

Design of the study: YA-L, HY-S and SF-W. Performed experiments: YA-L,YY-C, YZ-D, WW-G,J-F. Wrote the manuscript: YA-L, MB, SF-W. All authors have read and approved the manuscript.

Acknowledgements

The authors thank to Dr. Curtiss, III for kindly providing χ7213, pRE112, pYA3493, and the antibody of Fur.

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Table 1. Strains and plasmids

Strain or plasmid	Relevant characteristics or genotype	Source or reference
E. coli strains
BL21	F- pT hsdSB(rB- mB-al dcm (DE3)	Invitrogen
χ7213	thi-1 thr-1 leuB6 fhuA21 lacY1 glnV44 asdA4 recA1 RP4 2-Tc::Mu pir; Km^r	Gift from Dr. Curtiss, III
Salmonella Choleraesuis
C78-3	Wild type, virulent, CVCC79103	Lab stock
C500	S. Choleraesuis vaccine strain attenuated by chemical mutation , CVCC79500	[40]
rSC0005	Δpmi-2426, C78-3	[9]
rSC0008	Δpmi-2426 ΔrelA199::araC P_BAD lacI TT	This study
rSC0009	Δpmi-2426 ΔrelA199::araC P_BAD lacI TT ΔP_fur88::TT araC P_BAD fur	This study
rSC0011	ΔP_crp527::TT araC P_BAD crp Δpmi-2426 ΔrelA199::araC P_BAD lacI TT ΔasdA33	[9]
rSC0012	Δpmi-2426 ΔrelA199::araC P_BAD lacI TT ΔP_fur88::TT araC P_BAD fur ΔasdA33	This study
rSC0018	ΔasdA33	C500
	C78-3 ΔP_fur88::TT araC P_BAD fur	C78-3
	C78-3 ΔP_crp527::TT araC P_BAD crp	C78-3
Streptococcus suis serotype 2	Wild type, virulent, CVCC3928	Lab stock
Plasmids
pYA3493	Asd⁺; pBR ori , P_trc promoter, β-lactamase signal sequence-based periplasmic secretion plasmid	[19]
pS-SaoA	pYA3493 with SaoA, P_trc promoter	[5]
pET28a	expression vector, T7 promoter; Km^r	Novagen
Suicide vector
pRE112	sacB mobRP4 R6K oriV oriT Cm^r	[50]
pDMS197	tet^r sacB mobRP4 R6K oriV oriT	[50]
pYA3832	ΔP_crp527::TT araC P_BAD crp, pRE112	[46]
pYA3546	Δpmi-2426, pDMS197	[46]
pS003	ΔrelA199::araC P_BAD lacI TT, pRE112	[9]
pS005	ΔP_fur88::TT araC P_BAD fur	This study
pYA3736	ΔasdA33, pRE112	[9]

Table 2. Virulence of rSC0012(pS-SaoA) in 3-week-old BALB/c mice

Strain	Description	LD₅₀ (CFU)
		Oral	i.p
C78-3	Wild type	9.5×10²	<10
rSC0018(pS-SaoA)	ΔasdA33 in a live attenuated S. Choleraesuis vaccine strain C500	>2.8×10⁹	6.6×10⁶ **, ##
rSC0012(pS-SaoA)	ΔP_fur88::TT araC P_BAD fur Δpmi-2426 ΔrelA199::araC P_BAD lacI TT ΔasdA33 in C78-3	>5.8×10^9**	2.1×10⁷ **, ##
rSC0011(pS-SaoA)	ΔP_crp527::TT araC P_BAD crp Δpmi-2426 ΔrelA199::araC P_BAD lacI TT ΔasdA33 in C78-3	>1.0×10^9**	5.4×10⁵ **

**, P<0.01, compared with C78-3; ##, P<0.01, compared with rSC0011(pS-SaoA)

Table 3 Primer sequences

Gene		Sequence （5’-3’）
GAPDH	Forward	CTT AGC ACC CCT GGC CAA G
	Reverse	GAT GTT CTG GAG AGC CCC G
IL-1β	Forward	GTG TCT TTC CCG TGG ACC TT
	Reverse	AAT GGG AAC GTC ACA CAC CA
IL-6	Forward	GGC GGA TCG GAT GTT GTG AT
	Reverse	GGA CCC CAG ACA ATC GGT TG
TNF-α	Forward	ATG AGC ACA GAA AGC ATG A
	Reverse	AAG AGG CTG AGA CAT AGG C
IL-8	Forward	CTG CAA GAG ACT TCC ATC CAG
	Reverse	AGT GGT ATA GAC AGG TCT GTT GG

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Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

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