Multiple immunodominant O-epitopes co-expression in live attenuated Salmonella serovars induce cross-protective immune responses against S. Paratyphi A, S. Typhimurium and S. Enteritidis

Pei Li; Ke Zhang; Ting Lei; Zuoyong Zhou; Hongyan Luo

doi:10.1371/journal.pntd.0010866

Abstract

Salmonella enterica subsp. enterica (S. enterica) is a significant public health concern and is estimated to cause more than 300,000 deaths annually. Nowadays, the vaccines available for human Salmonellosis prevention are all targeting just one serovar, i.e., S. Typhi, leaving a huge potential risk of Salmonella disease epidemiology change. In this study, we explored the strategy of multiple immunodominant O-epitopes co-expression in S. enterica serovars and evaluated their immunogenicity to induce cross-immune responses and cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis. We found that nucleotide sugar precursors CDP-Abe and CDP-Par (or CDP-Tyv) could be utilized by S. enterica serovars simultaneously, exhibiting O2&O4 (or O4&O9) double immunodominant O-serotypes without obvious growth defects. More importantly, a triple immunodominant O2&O4&O9 O-serotypes could be achieved in S. Typhimurium by improving the substrate pool of CDP-Par, glycosyltransferase WbaV and flippase Wzx via a dual-plasmid overexpressing system. Through immunization in a murine model, we found that double or triple O-serotypes live attenuated vaccine candidates could induce significantly higher heterologous serovar-specific antibodies than their wild-type parent strain. Meanwhile, the bacterial agglutination, serum bactericidal assays and protection efficacy experiments had all shown that these elicited serum antibodies are cross-reactive and cross-protective. Our work highlights the potential of developing a new type of live attenuated Salmonella vaccines against S. Paratyphi A, S. Typhimurium and S. Enteritidis simultaneously.

Author summary

Currently, vaccines available for human Salmonellosis prevention are mostly against one serovar, i.e., S. Typhi, leaving a significant risk of Salmonella disease epidemiology change. Consequently, more efforts are needed to develop vaccine candidates against other medically important but somehow less concerned S. enterica serovars, such as S. Paratyphi A, S. Typhimurium and S. Enteritidis. Evidence reveals that S. Paratyphi A is taking the place of S. Typhi as the leading cause of “enteric fever” in South Asia, and S. Typhimurium and S. Enteritidis are the leading causing of “Invasive Non-typhoidal Salmonella disease” in sub-Saharan Africa. One of the solutions is to increase the cross-immunogenicity and cross-protections of potential S. enterica vaccine candidates. Salmonella outer membrane O-antigen polysaccharide is a well-known protective antigen, and its diverse structure is serotype specific. More precisely, S. Paratyphi A, S. Typhimurium and S. Enteritidis are characterized by O2, O4 and O9 O-serotypes. In this study, we aimed to combine the immunodominant O2, O4 and O9 O-epitopes into one S. enterica serovar and evaluated its cross-immune responses in a murine model. Our results indicated that the rational design of O-antigen structure in live attenuated Salmonella vaccines is a promising strategy to induce effective cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis.

Citation: Li P, Zhang K, Lei T, Zhou Z, Luo H (2022) Multiple immunodominant O-epitopes co-expression in live attenuated Salmonella serovars induce cross-protective immune responses against S. Paratyphi A, S. Typhimurium and S. Enteritidis. PLoS Negl Trop Dis 16(10): e0010866. https://doi.org/10.1371/journal.pntd.0010866

Editor: Sharon M. Tennant, University of Maryland School of Medicine, UNITED STATES

Received: May 15, 2022; Accepted: October 4, 2022; Published: October 13, 2022

Copyright: © 2022 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 31902340 to HL) and the Natural Science Foundation of Chongqing, China (Grant No. cstc2019jcyjmsxmX0532 to PL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Salmonella belongs to the family Enterobacteriaceae and is a medically important pathogen for both humans and animals. Based on its surface antigenic composition, Salmonella is currently divided into more than 2600 serotypes [1]. However, 99% of human and animal infections are caused solely by one subspecies, Salmonella enterica subsp. enterica (S. enterica). S. enterica is estimated to cause more than 300,000 deaths annually [2,3], mostly in developing countries. According to their clinical manifestations and presentations, S. enterica has traditionally been divided into ‘typhoidal serovars’ and ‘non-typhoidal serovars’ [4,5]. For example, human host-restricted S. enterica serovar Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the leading causes of systemic infections known as typhoid and paratyphoid fever [2], respectively. In contrast, broad host-ranged S. enterica serovar Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) generally induce self-limiting gastroenteritis in healthy individuals [6]. However, non-typhoidal serovars may become invasive when the host are infants, young children or immunocompromised adults, causing a life-threatening infection involving the bloodstream, meninges, and other normally sterile sites [7]. Invasive Non-typhoidal Salmonella (iNTS) disease is a severe illness with a case fatality ratio of approximately 15%. In sub-Saharan Africa, where the iNTS is observed to be a particular threat [8–10], S. Typhimurium and S. Enteritidis were the most frequently isolated iNTS pathogens [8], accounting for more than 80%. Unfortunately, widespread antimicrobial resistance among iNTS isolates is threatening the effectiveness of amenable antibiotic treatments [11]. To date, vaccines are regarded as one of the most economical and effective ways to prevent salmonellosis.

Immunity to Salmonella, induced by natural infection or vaccination, is serotype-specific [12]. In S. enterica serovar, this serotype specificity is largely determined by the O-antigen polysaccharide [13] or Vi capsule [14]. The Vi capsule is produced by S. Typhi, while the O-antigen is widely found in other S. enterica serovars. The O-antigen polysaccharide is the outermost part of the lipopolysaccharides (LPS), which is a structurally diverse polymer and repeats in a diverse range of numbers. The LPS is found exclusively in the outer leaflet of the Salmonella membrane [15]. Approximately 2×10⁶ LPS molecules cover ∼75% of the cell surface, thus resulting in a formidable barrier limiting the antibodies from accessing the bacterial surface [16]. Nowadays, the only licensed single antigen vaccine against Salmonella infections is rationally based on their surface polysaccharides, i.e., the Vi capsule [17] or Vi capsule glycoconjugate vaccines [18]. However, multi-valent strategies are being explored in clinical development. For example, a bivalent outer membrane vesicle approach, also referred to as Generalized Modules for Membrane Antigens (GMMA) [19], targets S. Enteritidis and S. Typhimurium, and a trivalent glycoconjugate approach targets S. Enteritidis, S. Typhimurium, and S. Typhi [20,21]. Both of them involve in O-antigens formulation. Consequently, an OAg-based vaccine covering the other frequently isolated strains (i.e., S. Paratyphi A, S. Typhimurium and S. Enteritidis) is predictably desirable.

Thanks to the research work of monoclonal antibodies against S. enterica serogroups A to E, the immunogenic properties of Salmonella O-antigen are now clear [22]. The non-specific O-epitopes 1 and 12 are mainly attributed to the O-antigen common trisaccharide backbone 2)-α-Man(1→4)-α-Rha-(1→3)-α-Gal-(1→, which is shared by S. Paratyphi A, S. Typhimurium and S. Enteritidis [23]. However, the immunodominant serovar-specific O-epitopes are largely confined to the paratose (Par), abequose (Abe) and tyvelose (Tyv) side-branch sugars (Fig 1A), namely, O2 (S. Paratyphi A, serogroup A1, α-Par(1→3)Man), O4 (S. Typhimurium, serogroup B1, α-Abe(1→3)Man) and O9 (S. Enteritidis or S. Typhi, serogroup D1, α-Tyv(1→3)Man). Passive protection studies demonstrated that IgG or IgM directed against the O2, O4 or O9 O-epitopes played an important role in disease prevention [24]. Consequently, the O-antigenic characteristics of S. Paratyphi A, S. Typhimurium and S. Enteritidis are hereafter referred to as O2, O4 and O9, rather than their full O-antigenic formulae.

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Fig 1. S. enterica group A1, B1 and D1 O-unit structures and the CDP-Sugar biosynthesis pathways.

(A) Square brackets denoted a single O-unit repeat. The arrows represented the linkages with the linkage types and the glycosyltransferase (GT) indicated. Note that the S. Paratyphi A, S. Enteritidis and S. Typhimurium all share a common trisaccharide backbone. The side-branch sugar of group A1, B1 and D1 was Par, Abe and Tyv, respectively, creating epitopes O2, O4 and O9. Note that the O-antigen galactose can be variably glucosylated, creating epitopes O:12 and O:1. Further, S. Typhimurium can variably O-acetylate its abequose in the C-2 position, creating epitope O:5. (B) Gene responsible for each glycosidic linkage synthesis was indicated adjacent to each arrow. The CDP-Sugars synthesis pathways are all initiated from glucose-1-phosphate. The genes involved in each reaction step of pathways were shown alongside the arrows. Abbreviations: Abe, abequose; Tyv, tyvelose; D-Man, D-mannose; D-Gal, D-galactose; L-Rha, L-rhamnose.

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The O-antigen gene cluster of S. Paratyphi A, S. Typhimurium and S. Enteritidis are all located between the galF and gnd gene in the chromosome [25,26] (S1 Fig). The main differences among these gene clusters are the regions responsible for synthesizing the 3,6-dideoxyhexosyl side-branch sugars, and the side-branch sugars are transferred from CDP-sugar precursors by glycosyltransferase WbaV [27,28]. The CDP-sugars synthesis pathways are all initiated from glucose-1-phosphate but diverge after DdhD. CDP-abequose synthase (Abe) and CDP-paratose synthase (Prt) reduce the keto group at C-4 to give either the galactose stereochemistry for CDP-Abe or the glucose stereochemistry for CDP-Par. CDP-Par 2-epimerase (Tyv) is responsible for the synthesis of CDP-tyvelose [27] (Fig 1B). Meanwhile, the Wzx is a multi-transmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varying degrees of preference [29,30].

Previously, we and others have shown that the immunodominant O-epitope of one S. enterica strain could be converted to another via chromosomally genetic manipulation [31–33]. For example, the O4 O-serotype in S. Typhimurium could be converted into O9 of S. Enteritidis by replacing the gene abe with prt-tyv_D1 without sacrificing its immunogenic properties [31,33]. Similarly, S. Typhimurium with abe-wzx_B1-wbaV_B1 gene replaced by prt-tyv_A1-wzx_A1-wbaV_A1 from S. Paratyphi A could convert its original O4 O-serotype into O2 and still retained an excellent immunogenic property [32]. So, we later come up with an interesting question. Could it be possible to simultaneously express immunodominant O2, O4 and O9 O-epitopes in one strain?

In this study, we explored the possibility of exhibiting O2, O4 and O9 immunodominant O-serotypes simultaneously in S. enterica serovars. The O2 & O4 double O-serotypes phenotype could be easily achieved in either S. Paratyphi A or S. Typhimurium background. So did the O4 & O9 double O-serotypes phenotype in S. Enteritidis or S. Typhimurium background. However, we encountered difficulties in chromosomally co-expressing O2 & O4 & O9 triple O-epitopes, which suggested S. enterica serovars exhibited different preferences over CDP-Par, CDP-Abe and CDP-Tyv precursors. Unexpectedly, increasing the pool of CDP-Par precursor and the synthesis of WbaV glycosyltransferase and Wzx flippase in S. Typhimurium by plasmids overexpression system could result in an O2 & O4 & O9 triple O-serotypes phenotype. A series of live attenuated S. enterica vaccine candidates were designed and constructed to evaluate their potential cross-immune responses and cross-protections against wild-type S. Paratyphi A, S. Typhimurium and S. Enteritidis. Our research highlights the strategy of manipulating bacteria surface polysaccharide epitopes to generate live attenuated vaccines offering multi-serotype protections.

Materials and methods

Ethics statement

All animal experiments were conducted in compliance with the Animal Welfare Act and regulations stated in the Guide for the Care and Use of Laboratory Animals, which was approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University (IACUC-20210525-02).

Bacteria, plasmids, and culture conditions

The bacteria and plasmids used in this study are listed in Table 1. E. coli, S. enterica serovars and their derivatives were aerobically grown at 37°C in Luria-Bertani (LB) broth or on LB agar. Allelic exchange in S. enterica serovars was achieved by sacB gene-based counter selection on LB agar plates that contained 10% sucrose with no sodium chloride added and incubated at 30°C [34]. When necessary, chloramphenicol was added at 25 μg/ml to select transconjugants. D-Alanine (D-Ala) (50 μg/ml) was added for the growth of Δalr ΔdadB strains [35]. Diaminopimelic acid (DAP) was added at 50 μg/ml for the growth of Δasd mutant χ7213 [36]. In vitro growth rates of Salmonella strains were determined by optical density measurements.

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Table 1. Bacterial strains and plasmids used in this study.

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Molecular and genetic procedures

Molecular biology techniques were performed following standard methods [37], and details regarding the primers used in this study are listed in the S1 Table. DNA concentration and purity were measured using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific), and DNA fragments were cyclized by Circular Polymerase Extension Cloning (CPEC) method [38].

Insertion or deletion mutant strains construction. As antibiotic resistance is not permitted in live vaccine strains, sacB gene-based sucrose counter-selectable suicide vectors were used to construct unmarked insertion or deletion mutations in S. enterica serovars [34]. Take the construction of L001 (SA-ddhc::abe) as an example; the upstream and downstream homologous regions were amplified from S. Paratyphi A using primer pairs P11/P12 and P15/P16. The abe gene was amplified from S. Typhimurium using primer pairs P13/P14 and the suicide vector backbone was amplified from pRE112 using primer pairs P1/P2. After purification, these three fragments were cyclized by CPEC method, resulting in a new suicide plasmid pHY005 (112-ddhc::prt_A1). The conjugational transfer of pHY005 to S. Paratyphi A was performed using the suicide vector donor strain χ7213 [39]. Transconjugants with the first homologous recombination event were selected on chloramphenicol agar without supplemental DAP. The positive clones were inoculated in fresh LB media without chloramphenicol addition. The second homologous recombination event, resulting in excision of the suicide vector from the S. Paratyphi A chromosome, was selected on 10% sucrose LB plates without sodium chloride and grown at 30°C. Successful gene insertion mutations were confirmed by PCR screening and DNA sequencing. Other insertion mutations are constructed following the same procedures. The insertion mutations constructed for this study are illustrated in S2 Fig, and the primer pairs used for each DNA fragment amplification were labeled accordingly. As for the crp and cya gene deletion mutations, the suicide plasmids 112-Dcrp and 112-Dcya were constructed previously [33]. The subsequent deletion mutation processes were similar to those described above for the insertion mutations. All successful gene deletion or insertion mutations were confirmed by DNA sequencing.

Recombinant plasmids construction. Briefly, prt, tyv, wzx and wbaV genes were cloned from S. Paratyphi A or S. Enteritidis using the same primer pairs P19/P20. To distinguish them from each other, we added an "A1" suffix subscript after each gene cloned from S. Paratyphi A, as S. Paratyphi A belongs to S. enterica serogroup A1. Similarly, a “D1” suffix subscript was added to each gene cloned from S. Enteritidis, as S. Enteritidis belongs to S. enterica serogroup D1. Next, the vector backbone was cloned from pSC101-asd and p15a-dadB using the same primer pairs P17/P18, as these two plasmids all contained a Ptrc promoter-multiple cloning site (MCS)-5ST1T2 terminator cassette. After purification, the DNA fragment prt_A1-tyv_A1-wzx_A1-wbaV_A1 and the linearized pSC101-asd vector were cyclized by CPEC method, resulting in pSC101-asd-O2. Similarly, the DNA fragment prt_D1-tyv_D1-wzx_D1-wbaV_D1 and the linearized p15a-dadB vector were cyclized by CPEC method, resulting in p15a-dadB-O9.

Phenotype characterization of mutant strains

Mutant strains phenotype evaluations included the LPS silver stain and western blot, slide agglutination test, P22 transduction studies, growth rates, motility test, and minimum inhibitory concentration (MIC) test of deoxycholate (DOC) and polymyxin B. These methods were all reported previously, and a complete description of all methods employed for these phenotype evaluations is provided in S1 Text.

Virulence determination and colonization in mice

Six-week-old female BALB/c mice were purchased from Dashuo Biotechnology Co., Ltd. (Chengdu, China). To determine the 50% lethality dose (LD₅₀), bacteria were grown to OD₆₀₀ of 0.8 to 0.9 and harvested by centrifugation at 3,452 × g at room temperature. The centrifuged S. Typhimurium, S. Enteritidis or their derivatives were resuspended and adjusted to the appropriate OD₆₀₀ value by buffered saline with gelatin (BSG) [40]. In contrast, the centrifuged S. Paratyphi A or its derivatives were resuspended and adjusted to the appropriate OD₆₀₀ value by 10% hog gastric mucin (Sigma). Six mice per group were infected orally with 20 μl of BSG or intraperitoneally injected with 500 μl 10% hog gastric mucin containing various doses of bacteria, ranging from 1 × 10⁴ CFU to 1 × 10⁸ CFU. Mice were monitored for mortality or signs of significant morbidity daily. The LD₅₀ was calculated using the method of Reed and Muench. To evaluate colonization, three mice per group were orally inoculated with 20 μl of BSG containing 1 × 10⁹ CFU bacteria. On days 4 and 8 post-inoculation, Peyer’s patches, spleen and liver samples were collected. Samples were homogenized, dilutions were plated onto MacConkey and LB agar to determine viable counts.

Vaccination and immune response measurement

Thirty mice per group were vaccinated orally on day 0 with 20 μl BSG containing approximately 1 × 10⁹ CFU vaccine strains and boosted on day 14 with the same dose. Blood samples and vaginal secretions were collected from randomly selected twelve mice in each group on day 28 after the booster immunization. On day 35, mice in each group were randomly subdivided into three small subgroups; mice in each subgroup were challenged orally with 5 × 10⁷ CFU of S. Typhimurium and S. Enteritidis or injected intraperitoneally with 1 × 10⁴ CFU of S. Paratyphi A (~100 times the LD₅₀) [40,41]. S. Paratyphi A, S. Typhimurium and S. Enteritidis LPS were purified as described previously [42] and used as coating antigens to measure the immune responses. IgM, IgG and IgA antibodies specific to S. Paratyphi A, S. Typhimurium and S. Enteritidis LPS in the serum or vaginal secretions were measured using the quantitative enzyme-linked immunosorbent assay (ELISA) as described previously [43]. Antibody concentrations were calculated based on absorbance values and the standard curve.

Serum bactericidal activity assay

The serum bactericidal assay (SBA) was performed as previously described with a few modifications [44]. Briefly, log-phase cultures of S. Paratyphi A, S. Typhimurium and S. Enteritidis were grown to an OD₆₀₀ of 0.6 in LB broth. After centrifugation and resuspension, the log-phase cultures were diluted in PBS to a concentration of approximately 1× 10⁴ CFU/ml. Sera samples from vaccinated mice were serially diluted from 1:100 to 128,000, and nonimmune sera were serially diluted from 1: 10 to 1,280. Optimal SBA results were achieved by combining 25 μl of active baby rabbit complement (BRC) (25% final concentration) with 15 μl of PBS, 50 μl of diluted mice pooled sera, and 10 μl of diluted bacteria (~350 CFU). In total, 10 μl of the mixture from each well was spread on LB agar plates after 60 min to assess the bactericidal activity. The spread LB agar plates were incubated overnight at 37°C, and the viable CFU were counted the next day. The negative control contained only bacteria and complement. The serum bactericidal antibody titer was defined as the reciprocal of the highest serum dilution that produced >50% killing in relation to the killing observed for the control wells. The titers were determined from the mean bacterial count from triplicate wells.

Statistical analysis

Data were analyzed using GraphPad Prism 5 software (Graph Software, San Diego, CA) by one-way or two-way ANOVA of variance followed by Tukey’s multiple-comparison post-test. Kaplan-Meier survival curve comparisons were calculated by comparing two groups at each time point through the log-rank (Mantel-Cox) test. The data were expressed as the means ± SD. P<0.05 was considered statistically significant.

Results

S. enterica mutants exhibiting double or triple immunodominant O-serotypes

The primary goal of this study is to try to exhibit O2, O4 and O9 O-serotypes simultaneously in one S. enterica serovar. To begin with, we explore the possibility of co-expressing O2 & O4 or O4 & O9 O-epitopes first. According to the O-antigen gene cluster analysis (S1 Fig), the major difference among S. Paratyphi A, S. Typhimurium and S. Enteritidis lie in the genes responsible for the synthesis of the side-branch sugar (Fig 1B), i.e., the abe, prt and tyv genes for CD-Abe, CD-Par and CDP-Tyv biosynthesis respectively. Consequently, we inserted the abe gene from S. Typhimurium between the ddhc and prt gene of S. Paratyphi A to test the potential of co-expressing O2 and O4 O-epitopes (S2A Fig). The results of bacterial agglutination (Table 2) and western blot (Fig 2A) showed that the SA-ddhc::abe mutant could react positively to O2 and O4 antisera, respectively, indicating the co-existence of O2 and O4 O-epitopes in the outer membrane of SA-ddhc::abe mutant. To be more persuasive, we inserted the prt_A1 gene from S. Paratyphi A similarly between the ddhc and abe gene of S. Typhimurium (S2B Fig) to see if these co-expressing phenomena still existed. Consistently, the bacterial agglutination (Table 2) and western blot results (Fig 2A) all indicated that the ST-ddhc::prt_A1 mutant exhibited O2 and O4 O-epitopes simultaneously. At the same time, the abe gene was inserted between the ddhc and prt_D1 gene of S. Enteritidis (S2C Fig) and the prt_D1-tvy_D1 gene was inserted between the ddhc and abe gene of S. Typhimurium (S2D Fig). Unsurprisingly, either SE-ddhc::abe or ST-ddhc::prt_D1-tyv_D1 could independently agglutinate with O4 and O9 antisera obviously (Table 2). However, we did not observe clear anti-O9 positive bands of SE-ddhc::abe or clear anti-O4 positive bands of ST-ddhc::prt_D1-tyv_D1 in western blot results (Fig 2B). Unfortunately, we failed to obtain a S. Paratyphi A, S. Typhimurium or S. Enteritidis mutant that could express O2, O4 and O9 O-epitopes simultaneously through chromosomally genetic modifications.

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Fig 2. Immunoblots analysis.

LPS was extracted from the S. enterica mutants exhibiting O2&O4 (A), O4&O9 (B) and O2&O4&O9 (C) O-serotypes and separated on a 12.5% (w/v) polyacrylamide gel by tricine-SDS-PAGE. Samples were then transferred to a nitrocellulose membrane for immunoblotting with anti-Par O2, anti-Abe O4 or anti-Tyv O9 antisera. The detected bands corresponded to LPS with different side-branch O-unit repeats.

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We have noticed that S. Enteritidis possess both functional prt and tyv genes and it turns out to be a dominant O9 O-serotype, while the tyv gene in S. Paratyphi A is frameshift and yet it turns out be a dominant O2 O-serotype. So, we speculate that when prt and tyv are co-expressed at a similar level, the CDP-Par will be mostly converted into CDP-Tyv, resulting in an O9 dominant O-serotype. Therefore, we hypothesize that it might be possible to express O2, O4 and O9 O-epitopes simultaneously by improving the CDP-Par substrate pool. Based on that assumption, we have built a dual-plasmid expression system. The ST-Δalr ΔdadB ΔrecF Δasd mutant was constructed deliberately to harbor pSC101-asd and p15a-dadB plasmids without antibiotic selection pressure [35]. The prt_A1-tyv_A1-wbaV_A1-wzx_A1 genes from S. Paratyphi A were cloned into pSC101-asd, resulting in pSC101-asd-O2, and the prt_D1-tyv_D1-wbaV_D1-wzx_D1 genes from S. Enteritidis were cloned into p15a-dadB, resulting in p15a-dadB-O9. As tyv_A1 possesses a loss-of-function mutation, when pSC101-asd-O2 and p15a-dadB-O9 were transferred into ST-Δalr ΔdadB ΔrecF Δasd, the prt gene were comparatively overexpressed. Except for anti-O9, we did not observe anti-O2 or anti-O4 positive bands of ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) in western blot results (Fig 2C). However, the bacterial agglutination assays had clearly shown that, in addition to O4 antiserum, ST-Δalr ΔdadB ΔrecF Δasd harboring pSC101-asd-O2 or p15a-dadB-O9 could agglutinate with O2 or O9 antiserum, respectively (Table 2). Most importantly, ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) could agglutinate with O2, O4 and O9 antisera independently, which indicated that we had successfully exhibited O2, O4 and O9 O-serotypes simultaneously in S. Typhimurium outer membrane.

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Table 2. Bacterial agglutination assays.

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Phenotype characterizations of S. enterica mutants

As we have achieved double or triple immunodominant O-epitopes co-expression in S. enterica mutants, we would like to know whether or not these O-serotypes’ co-existence would influence their phenotype characterizations. Firstly, the LPS profiles of these mutant strains were visualized by silver staining. Most mutants can synthesize a full length of LPS when compared with their wild-type parent strains, except for ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB), which exhibited a decrease in LPS synthesis (Fig 3). Next, the accessibilities of mutants’ O-antigen structure for Phage P22 were examined. The number of transductions obtained from double or triple O-serotypes exhibiting mutants was similar to that of their wild-type parent strains (Table 3), indicating that their O-antigen backbones were unaffected. Secondly, the growth rate of these mutant strains and their wild-type parent strains were all evaluated, and there was no significant difference between them (Fig 4).

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Fig 3. LPS profiles of S. enterica mutants.

S. enterica mutants exhibiting double or triple immunodominant O-serotypes were constructed by chromosomally gene insertion mutations (A) or plasmid overexpressing systems (B). The LPS extracted from these mutant strains was separated on a 12.5% (w/v) polyacrylamide gel by tricine-SDS-PAGE and visualized by the silver staining. Bands represented the LPS with different numbers of O-unit repeats.

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Fig 4. Growth curves.

In vitro growth rates of S. enterica and its derivatives were measured by optical density measurements as an OD₆₀₀ value at multiple time points.

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Table 3. Transduction efficiencies, MIC of DOC and Polymyxin B, swimming motility and virulence of wild type Salmonella and its derivatives.

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Meanwhile, their sensitivity to the bile salt DOC and the cationic antimicrobial peptide polymyxin B were tested. The DOC MICs had no difference among these strains, whereas the polymyxin B MICs for double or triple O-epitopes co-expressing mutant strains were twofold lower than their wild-type parent strains. All mutants retained similar swimming motility as their wild type parent strains (Table 3). Taken together, all these results indicated that double or triple O-epitopes co-expressing had a minor influence on the phenotype changes of our constructed S. enterica mutants.

Virulence and colonization of the S. enterica mutants in BALB/c mice

The LD₅₀ values of wild-type S. Typhimurium S100 and S. Enteritidis S246 were approximately 10⁵ CFU, whereas the LD₅₀ values of double or triple O-epitopes co-expressing mutant strains were approximately 10⁶ or 10⁷ CFU, showing around 10 times or 100 times attenuation (Table 3). Since S. Paratyphi A is a human host-restricted pathogen, its virulence attribute was evaluated in a lethal murine model that requires suspending the bacteria in 5–10% hog gastric mucin and then injecting the suspension intraperitoneally [45]. In this model, the LD₅₀ value of wild-type S. Paratyphi A was approximately 100 CFU, showing a highly virulent attribute.

Considering the phenotype characterizations and broad host adaption abilities, we select ST-ddhc::prt_A1, SE-ddhc::abe and ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) for further potential live attenuated vaccine candidates development. To guarantee their safety in the murine model, we further deleted the cya and crp global regulators of these mutant strains [46], resulting in L008 (ST-ddhc::prt_A1 Δcya Δcrp), L009 (SE-ddhc::abe Δcya Δcrp) and L083 [ST-Δalr ΔdadB ΔrecF Δcya Δcrp Δasd (pSC101-asd-O2, p15a-dadB-O9)], respectively. Again, their O-serotype phenotypes have been reconfirmed, and the deletion mutations of cya and crp genes did not affect the O-epitopes expression (S3 Fig) but could attenuate these candidates more than 1000 times (LD₅₀ > 5 ×10⁸ CFU). To be more briefly, these mutant strains will be hereafter referred to as L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9). The colonization of L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9) in murine Peyer’s patches, spleens, and livers was determined on days 4 and 8 after oral inoculation. All candidates displayed good colonization in Peyer’s patches, livers, and spleens. No deaths occurred during this period (Fig 5).

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Fig 5. Colonization of murine Peyer’s patches, livers and spleens by live attenuated S. enterica vaccines.

Colonization of mouse Peyer’s patches (A), livers (B) and spleens (C) 4 and 8 days post-inoculation were shown. The horizontal lines represent the means, and the error bars represent the standard deviation of the means.

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Immune responses induced by live attenuated S. enterica vaccines

To assess the immunogenicity of these vaccine candidates, mice were inoculated orally with approximately 10⁹ CFU of each strain on day 0 and boosted on day 14 with the same doses. Anti-S. Paratyphi A, anti-S. Enteritidis and anti-S. Typhimurium LPS serum antibodies were measured on day 28.

The IgG immune response are depicted in Fig 6. Mice vaccinated with L008 (O2&O4) and L083 (O2&O4&O9) mounted a significantly higher anti-S. Paratyphi A LPS immune responses than those of ST-Δcrp Δcya or SE-Δcrp Δcya. Similarly, mice vaccinated with SE-Δcrp Δcya, L009 (O4&O9) and L083 (O2&O4&O9) mounted a significantly higher anti-S. Enteritidis LPS immune responses than those of ST-Δcrp Δcya. Except for SE-Δcrp Δcya, mice vaccinated with other vaccine candidates could all mount a significantly higher anti-S. Typhimurium LPS responses. We did observe some cross-immunogenicity between ST-Δcrp Δcya and SE-Δcrp Δcya, but it had long been proved to be the common trisaccharide backbone and the conserved core oligosaccharide. So far, we did not obtain valid evidence showing that there were detectable cross-immune responses among immunodominant O2, O4 and O9 O-epitopes unless they were co-expressed simultaneously. L008 (O2&O4) could mount a significantly higher anti-S. Paratyphi A LPS immune response than those vaccinated with ST-Δcrp Δcya (O4), showing an improved cross-immune response against S. Paratyphi A. Consistently, L009 (O4&O9) could mount a significantly higher anti-S. Typhimurium LPS immune response than those vaccinated with SE-Δcrp Δcya (O9), showing an improved cross-immune response against S. Typhimurium. Most importantly, L083 (O2&O4&O9) could simultaneously mount a significantly higher anti-S. Paratyphi A and anti-S. Enteritidis LPS immune response than those vaccinated with ST-Δcrp Δcya (O4), showing a good sign of eliciting cross-protections against S. Paratyphi A and S. Enteritidis. Meanwhile, all vaccine candidates induced a significantly higher IgG2a response than IgG1 (S4 Fig), indicating a predominantly Th1-type response.

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Fig 6. Serum IgG antibody responses.

(A) Anti-S. Paratyphi A LPS serum IgG levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks (**, P<0.01; ***, P<0.001). (B) The anti-S. Enteritidis LPS serum IgG levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks (***, P<0.001). (C) Anti-S. Typhimurium LPS serum IgG levels. Responses that differed from the results in the SE-Δcya Δcrp group are noted by asterisks (**, P<0.01; ***, P<0.001). Antibody concentrations were calculated using a standard curve and all the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means calculated by GraphPad Prism software. These data are representative of at least two independent experiments.

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We also evaluated the serum IgM (S5 Fig) and vaginal washes IgA antibodies immune responses (S6 Fig). A similar trend of IgM and IgA immune responses has been observed compared to IgG antibodies. Unsurprisingly, L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9) could induce a significantly higher amount of cross-reactive IgM antibodies than their parent strains. However, the elicited IgM antibodies were even higher than their IgG counterpart, which is unexpected. A significantly higher level of secreted IgA antibodies against S. Paratyphi A and S. Enteritidis were observed in mice vaccinated by L083 (O2&O4&O9) when compared to its parent strain ST-Δcrp Δcya. However, L009 (O4&O9) could elicit a higher level of anti-S. Enteritidis LPS IgA compared to L083 (O2&O4&O9) and the level of anti-S. Paratyphi A LPS IgA was similar between L008 (O2&O4) and L083 (O2&O4&O9). Negative control groups did not mount a detectable immune response. All these ELISA results had shown that our vaccine candidates could induce promising cross-immune responses.

Antibody-dependent complement-mediated S. enterica killing

To evaluate the functional capacities of antibodies induced by our vaccine candidates, we performed the serum bactericidal assays (SBAs) using pooled serum from immunized mice or BSG control. Baby rabbit complement titration with serum samples from immunized mice had a significantly higher complement-mediated killing activity versus those from nonimmunized mice (Fig 7). The serum antibodies induced by ST-Δcrp Δcya (O4) or SE-Δcrp Δcya (O9) exhibited a high level of SBA activity against its homologous serotype strains. However, they had a limited SBA activity against heterologous serotype strains. On the contrary, the serum antibodies induced by L008 (O2&O4) and L009 (O4&O9) significantly improved SBA activity against heterologous serotype strains S. Paratyphi A and S. Typhimurium, respectively. More importantly, the bactericidal abilities induced by L083 (O2&O4&O9) covered all tested heterologous serotype strains, significantly improving SBA activity against S. Paratyphi A, S. Typhimurium and S. Typhimurium, simultaneously.

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Fig 7. Serum bactericidal activity.

Serum bactericidal assays (SBA) were performed with vaccinated mouse serum from the indicated groups against wild-type S. Paratyphi A (A), S. Enteritidis (B) and S. Typhimurium (C). Strains were grown in LB to log phase (OD₆₀₀, 0.4). The error bars represent the standard deviation of the mean titers calculated by GraphPad Prism software. The dashed lines indicate the detection limit of the assay. **, P<0.01; ***, P<0.001.

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Protective efficacy of live attenuated S. enterica vaccines against wild-type S. Typhimurium and S. Enteritidis

The immune protection against wild-type S. Paratyphi A, S. Typhimurium and S. Enteritidis were evaluated in the murine model (Fig 8). Unfortunately, most of the vaccinated mice succumbed to the 100 times LD₅₀ challenge from S. Paratyphi A. Only three mice, one out of twelve in L008 (O2&O4) and two out of twelve in L083 (O2&O4&O9) vaccinated group, survived in the end. However, mice vaccinated by L008 (O2&O4) and L083 (O2&O4&O9) could generally survive as long as 4 days, while the other vaccinated groups could barely survive a single day, showing a positive correlation of cross-protection. Meanwhile, mice vaccinated by ST-Δcrp Δcya (O4) or SE-Δcrp Δcya (O9) could survive a 100 times LD₅₀ challenge from S. Typhimurium or S. Enteritidis, respectively, indicating a high level of homologous protection. However, these mice succumbed to the heterogenous challenge. Intriguingly, mice vaccinated by L009 (O4&O9) could independently survive a 100 times LD₅₀ challenge from S. Typhimurium and S. Enteritidis, indicating a good cross-protection against heterogenous challenge. Similar results were also observed in mice vaccinated by L083 (O2&O4&O9). These mice could independently survive a 100 times LD₅₀ challenge from S. Typhimurium and S. Enteritidis. Taken together, our vaccine confidants possessed good abilities to elicit cross-immune responses and cross-protections.

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Fig 8. Survival curves after a challenge by wild-type virulent Salmonella.

Five weeks after primary immunization, nine to twelve mice from each group were challenged orally or intraperitoneally with approximately 100 times the LD₅₀ of the wild-type virulent S. Typhimurium (A), S. Enteritidis (B) and S. Paratyphi A (C). Curve comparisons were calculated using GraphPad Prism by comparing two groups by the log-rank (Mantel-Cox) test for all the marked groups versus the PBS group. ***, P<0.001. The numbers in brackets represented the number of mice alive at the end of the experiment/number of mice challenged.

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Discussion

Traditionally, Salmonella vaccine developments followed the strategies of targeting one particular serovar or serotype, leaving the cross-protection largely unexplored. However, recently, a bivalent outer membrane vesicle (GMMAs) approach targeting S. Enteritidis and S. Typhimurium [19] and a trivalent glycoconjugate approach targeting S. Enteritidis, S. Typhimurium, and S. Typhi [21] are showing the light of multi-valent strategies. In this study, we explored another way of improving the cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis by O-antigen O-epitopes rational design.

The antisera of patients who recovered from paratyphoid fever or iNTS diseases mainly target the O-antigen polysaccharides of S. enterica serovars, indicating the importance of anti-OAg antibodies in patients’ adaptive immune responses. The O-serotype specificity of S. Paratyphi A, S. Typhimurium and S. Enteritidis is determined by the 3,6-dideoxyhexosyl side-branch residues of the trisaccharide backbone (Figs 1 and S1) [22]. Therefore, our strategy was to exhibit double or triple immunodominant O-serotypes in one S. enterica serovar. In order to achieve that goal, we first explored the possibility of co-expression of two O-epitopes. As the results had shown, either inserting the abe gene in front of the prt_A1 gene in S. Paratyphi A (SA-ddhc::abe) or the prt_A1 gene in front of the abe gene in S. Typhimurium (ST-ddhc::prt_A1) (S2 Fig), we all get an obviously bacterial agglutination with O2 and O4 antisera (Table 2). Consistently, positive bands were observed in the western blot assays when using O2 and O4 antisera as the primary antibody (Fig 2A). These data all indicated that O2 and O4 could be simultaneously expressed in either S. Paratyphi A or S. Typhimurium background. Similarly, we inserted the abe gene in front of the prt_D1 gene in S. Enteritidis (SE-ddhc::abe), and the prt_D1 gene in front of the abe gene in S. Typhimurium (ST-ddhc::prt_D1-tyv_D1) to testify the possibility of O4 and O9 O-serotypes exhibition (S2 Fig). We observed a clear bacterial agglutination with O4 and O9 antisera in both SE-ddhc::abe and ST-ddhc::prt_D1-tyv_D1 (Table 2). However, the western blot assay did not show anti-O9 positive bands in SE-ddhc::abe sample or anti-O4 positive bands in ST-ddhc::prt_D1-tyv_D1 sample (Fig 2B). The possible explanation for this might be the unbalanced expression of O4 and O9 O-epitopes, as this phenomenon has already existed naturally in wild-type S. Enteritidis. In wild-type S. Enteritidis or S. Typhi, the CDP-Par and CDP-Tyv synthesize at a similar level. However, the immunodominant O-serotype of S. Enteritidis or S. Typhi is exclusively O9, with even undetectable bacterial agglutination to O2 antiserum. On the other hand, S. Paratyphi A exhibiting O2 O-serotype is due to the frameshift mutation in tyv gene [47], interrupting the synthesis of CDP-Tyv from CDP-Par, leaving the S. Paratyphi A with no choice but utilizing the CDP-Par as its substrate for side-branch sugar attachment. Lastly, we failed to express O2, O4 and O9 O-serotypes in either S. Paratyphi A, S. Typhimurium or S. Enteritidis strain by chromosomally genetic modification.

It seems that S. enterica serovars have a different preference for the CDP-Abe, CDP-Par and CDP-Tyv substrates when they are synthesized at a similarly level. At least, it is evident that S. enterica prefers CDP-Tyv rather than CDP-Par [28]. Therefore, we hypothesized that achieving an O2, O4 and O9 O-serotype phenotype might be possible by increasing the synthesis of CDP-Par. So, we built a dual-plasmid expression system in a S. Typhimurium mutant, i.e., ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd or p15a-dadB). As the WbaV glycosyltransferase and Wzx flippase might have influenced synthesizing and transferring of different side-branch O-units, we amplified the whole prt_A1-tyv_A1-wbaV_A1-wzx_A1 genes from S. Paratyphi A and inserted them into pSC101-asd vector, resulting in pSC101-asd-O2 in brief (S2 Fig). At the same time, we amplified the whole prt_D1-tyv_D1-wbaV_D1-wzx_D1 genes from S. Enteritidis and inserted them into p15a-dadB vector, resulting in p15a-dadB-O9 in brief. Predictably, ST-Δalr ΔdadB ΔrecF Δasd harboring both pSC101-asd-O2 and p15a-dadB-O9 would increase CDP-Par synthesis, as there are at least two copies of prt gene transcribing but with only one normal tyv gene. Meanwhile, the synthesis of CDP-Abe was unaffected. The bacterial agglutination assays demonstrated that ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) could agglutinate with O2, O4 and O9 antiserum individually, indicating that we had successfully achieved a triple immunodominant O-serotypes phenotype in S. Typhimurium background (Table 2). However, we did not receive a consistent result from western blot assays, which showed a predominantly anti-O9 positive band (Fig 2C). This inconsistency between the bacterial agglutination and western blot assay might indicate the amount or distribution of O2, O4 and O9 O-epitopes in ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) is most likely unequal or unbalanced. We could not explain clearly how these O-epitopes are arranged in the outer membrane, either homogeneously or heterogeneously attached to each of the O-antigen polysaccharides (S7 Fig), or merely mixed irregularity. It is also possible that new irrelevant epitopes are created and, in this case, a portion of the antibody response may not be functional.

Liu etc. had shown that the glycosyltransferase WbaV of S. enterica is in fact more effective in attaching the Tyv to the O-antigen common trisaccharide backbone than Par, and this inefficiency of low Par utilization could be primarily improved by overexpressing the wbaV gene [28]. Their findings were reconfirmed in this study. Meanwhile, Hong etc. had also shown that the Wzx flippases have a strong preference for their cognate substrate [29,30]. Furthermore, they demonstrated that the absence of the side-branch resides would dramatically decrease the efficiency of Wzx flippases [29]. However, no direct evidence had been put forward to illustrate the influence of different side-branch resides upon Wzx translocation efficiency. Our previous report demonstrated that the S. Typhimurium could tolerate well Tyv side-branch O-units but reject Par side-branch O-units [32], indicating that nonnative substrate does influence Wzx translocation efficiency. However, in this study, we further developed these theories by showing that S. Typhimurium did not exhibit severe rejection against CDP-Par if accompanied by CDP-Abe. Moreover, S. Typhimurium could utilize CDP-Par as its side-branch building block even in the presence of CDP-Tyv.

After successfully constructing a series of double or triple immunodominant O-serotype mutant strains, we would like to know their phenotype characterizations. The phage P22 tailspike protein recognizes and hydrolyzes the repetitive O-antigen polysaccharides of S. enterica serogroup A, B and D at the Rha-Gal (1→3)-glycosidic linkages during infections [48]. So, we first performed a P22 phage transduction assay to check the O-antigen integrality of our mutant strains. The transduction numbers of our constructed mutants were similar to their wild-type parent strains (Table 3), which indicated that our insertion mutations did not change the essential common trisaccharide backbone of O-antigen polysaccharides. Next, the LPS profile of each mutant strain was examined. It is obvious that ST-ddhc::prt_A1 and SE-ddhc::abe mutants could exhibit a full length of LPS (Fig 3A), while ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB) or ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd, p15a-dadB-O9) showed a decreased synthesis of LPS length (Fig 3B). Surprisingly, ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) exhibited a full length of LPS synthesis, indicating a higher level of CDP-Par synthesis might be the critical factor in achieving a triple O-serotype phenotype in a S. enterica strain. However, this assumption is not strictly demonstrated in this study. Meanwhile, we also conducted other phenotype evaluation assays (Table 3), such as the DOC and polymyxin B MICs, to roughly mimic the intestinal environment of the natural oral infection route and swimming abilities, an indicator of bacteria surface “wettability” [49]. After all these tests, we selected ST-ddhc::prt_A1, SE-ddhc::abe and ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd-O2, p15a-dadB-O9) for further cross-immunogenicity evaluation. To guarantee their safety in the murine model, the global regulator cya and crp were deleted in these mutant strains [50], resulting in a series of potential vaccine candidates. The vaccine candidates showed good colonization in Peyer’s patches, livers, and spleens (Fig 5).

We are primarily interested in whether or not our live attenuated vaccine candidates could elicit effective cross-immune responses. To evaluate that, we applied ELISA assays to detect the antibodies raised against the O-antigen polysaccharides of wild-type S. Paratyphi A, S. Typhimurium and S. Enteritidis (Fig 6). Consistent with the bacterial agglutination assays, L008 (O2&O4) could induce a significantly higher anti-O2 antibody response than ST-Δcrp Δcya (O4). Meanwhile, L009 (O4&O9) could induce a significantly higher anti-O4 antibody response than SE-Δcrp Δcya (O9). Most importantly, L083 (O2&O4&O9) could simultaneously induce significantly higher anti-O2, anti-O4 and anti-O9 antibody responses in mice when compared to the negative control, SE-Δcrp Δcya (O9) and ST-Δcrp Δcya (O4), respectively. These data showed that our vaccine candidates could elicit effective cross-immune responses. Although a high level of anti-S. Typhimurium, anti-S. Enteritidis and anti-S. Paratyphi A LPS serum antibodies were obtained, we would like to know whether or not these raised antibodies are indeed functional. The in vitro bactericidal data indicated that serum complement-mediated S. Paratyphi A, S. Typhimurium and S. Enteritidis killing depended upon anti-O2, anti-O4 and anti-O9 antibodies (Fig 7). Moreover, we challenged all vaccinated mice with wild-type virulent S. Paratyphi A, S. Typhimurium or S. Enteritidis strain (Fig 8). Compared with the negative control, ST-Δcrp Δcya (O4) and SE-Δcrp Δcya (O9) vaccinated mice could receive good homologous protection but failed to heterologous challenge. However, mice vaccinated by double or triple O-epitopes vaccine strains received a significantly higher protection rate even when challenged with heterologous wild-type virulent strains, showing adequate cross-immunity protection.

However, there are some limitations to this study. The S. Typhimurium and S. Enteritidis used in SBA assay are all animal isolates, which could not simply be equal to the human blood isolates. Other reports have shown that anti-LPS antibodies stimulate low complement-dependent killing against S. Enteritidis human blood isolates [51]. Furthermore, S. Typhimurium ST313, which dominates in sub-Saharan Africa, is highly serum resistant and expresses distinct transcriptional patterns that may aid in escaping this killing mechanism [52]. It is a good future research direction to illustrate how they escape this complement-dependent killing mechanism.

In summary, we expressed double or triple immunodominant O-epitopes in a S. Typhimurium or S. Enteritidis background strain and proved they are effective in inducing cross-immunity and cross-protection. Significantly, the strategies we present in this study are not limited to S. enterica O-antigen polysaccharides but have applicability for generating cross-protection for many other important human and animal pathogens. Notable examples include Shigella flexneri and many pathogenic Escherichia coli.

Supporting information

S1 Fig. The O-antigen gene cluster comparisons of S. Paratyphi A, S. Enteritidis and S. Typhimurium.

The O-antigen gene clusters are within the galF and gnd genes of S. Paratyphi A, S. Enteritidis and S. Typhimurium genome, which could be accessed through the genebank accession numbers NZ_CP019185.1, CP007361.1 and CP002614.1, respectively. The O-antigen gene clusters of groups A1 and D1 are highly homologous. The main differences between group B1 and group A1, D1 are the regions responsible for synthesizing the side-branch sugars. Note that the tyvA1 has a loss-of-function mutation due to ORF frameshift. Diagrams are drawn to scale.

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S2 Fig. Schematic representation of insertion mutations and plasmid constructions.

The abe gene from S. Typhimurium was inserted between the ddhc and prt_A1 gene of S. Paratyphi A. (B) The prt_A1 gene was inserted between the ddhc and abe gene of S. Typhimurium. (C) The abe gene was inserted between the ddhc and prt_D1 gene of S. Enteritidis. (D) The prt_D1-tyv_D1 genes were inserted between the ddhc and abe gene of S. Typhimurium. (E) The prt_A1-tyv_A1-wbaV_A1-wzx_A1 genes from S. Paratyphi A were cloned into pSC101-asd, resulting in pSC101-asd-O2. (F) The prt_D1-tyv_D1-wbaV_D1-wzx_D1 genes from S. Enteritidis were cloned into p15a-dadB, resulting in p15a-dadB-O9. Primer pairs used for each DNA fragment amplification were labeled accordingly.

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S3 Fig. The bacterial agglutination assays.

The agglutination assays were performed on glass slides and the used anti-Par O2, anti-Abe O4 and anti-Tyv O9 antiserum were indicated above. Positive or negative agglutination could be observed directly by the naked eye. Images were taken at 10 × 10 magnification.

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S4 Fig. Serum IgG2a and IgG1 responses.

Serum IgG2a and IgG1 responses against the LPS of S. Paratyphi A (A), S. Enteritidis (B) and S. Typhimurium (C) were determined by ELISA. A significantly higher level of IgG2a specific to the S. Paratypi A LPS compared to IgG1 was observed in L008 (O2&O4) and L083 (O2&O4&O9) (***, P<0.001). A significantly higher level of IgG2a specific to the S. Enteritis LPS compared to IgG1 was observed in SE-Δcrp Δcya (***, P<0.001). A significantly higher level of IgG2a specific to the S. Typhimurium LPS compared to IgG1 was observed in ST-Δcrp Δcya, L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9) (***, P<0.001). The antibody concentrations were calculated using a standard curve. All of the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means. These data are representative of at least two independent experiments.

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S5 Fig. Serum IgM responses.

(A) Anti-S. Paratyphi A LPS serum IgM levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks (***, P<0.001). (B) The anti-S. Enteritidis LPS serum IgM levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks (***, P<0.001). (C) Anti-S. Typhimurium LPS serum IgM levels. Responses that differed from the results in the SE-Δcya Δcrp group are noted by asterisks (***, P<0.001). Antibody concentrations were calculated using a standard curve and all the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means. These data are representative of at least two independent experiments.

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S6 Fig. IgA antibody responses in mice vaginal secretions.

(A) Anti-S. Paratyphi A LPS serum IgA levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks (*, P<0.05; **, P<0.01). (B) The anti-S. Enteritidis LPS serum IgA levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks (***, P<0.001). (C) Anti-S. Typhimurium LPS serum IgA levels. Responses that differed from the results in the SE-Δcya Δcrp group are noted by asterisks (**, P<0.01). Antibody concentrations were calculated using a standard curve and all the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means calculated by GraphPad Prism software. These data are representative of at least two independent experiments.

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S7 Fig. A schematic diagram of S. Typhimurium outer membrane O-antigen epitopes arrangement.

(A) The O2, O4 and O9 O-epitopes are homogeneously attached to each one of the O-antigen polysaccharides. (B) The O2, O4 and O9 O-epitopes are heterogeneously attached to each one of the O-antigen polysaccharides. Note that the number of each attached O-epitope does not represent the real case.

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S1 Text. Supplementary Methods.

Methods for LPS preparation and western blotting, the bacterial slide agglutination test, P22 transduction studies, motility test and minimum inhibitory concentration (MIC) test are described in supporting information supplementary methods.

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S1 Table. Primers used in this work.

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Acknowledgments

We thank Qingqe Kong (Southwest University, Beibei, Chongqing Province, China) for providing the anti-Par O2, anti-Abe O4 and anti-Tyv O9 antiserum. We thank Haoju Wang (Southwest University, Beibei, Chongqing Province, China) for critically reading and improving the manuscript.

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Figures

Abstract

Author summary

Introduction

Materials and methods

Ethics statement

Bacteria, plasmids, and culture conditions

Molecular and genetic procedures

Phenotype characterization of mutant strains

Virulence determination and colonization in mice

Vaccination and immune response measurement

Serum bactericidal activity assay

Statistical analysis

Results

S. enterica mutants exhibiting double or triple immunodominant O-serotypes

Phenotype characterizations of S. enterica mutants

Virulence and colonization of the S. enterica mutants in BALB/c mice

Immune responses induced by live attenuated S. enterica vaccines

Antibody-dependent complement-mediated S. enterica killing

Protective efficacy of live attenuated S. enterica vaccines against wild-type S. Typhimurium and S. Enteritidis

Discussion

Supporting information

S1 Fig. The O-antigen gene cluster comparisons of S. Paratyphi A, S. Enteritidis and S. Typhimurium.

S2 Fig. Schematic representation of insertion mutations and plasmid constructions.

S3 Fig. The bacterial agglutination assays.

S4 Fig. Serum IgG2a and IgG1 responses.

S5 Fig. Serum IgM responses.

S6 Fig. IgA antibody responses in mice vaginal secretions.

S7 Fig. A schematic diagram of S. Typhimurium outer membrane O-antigen epitopes arrangement.

S1 Text. Supplementary Methods.

S1 Table. Primers used in this work.

Acknowledgments

References