Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge

Vaccine 35 (2017) 2862–2869

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge Pei Li a,c,1, Qing Liu b,⇑,1, Chun Huang a, Xinxin Zhao a, Kenneth L. Roland c, Qingke Kong a,c,⇑ a

Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China Department of Bioengineering, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China c Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-5401, USA b

a r t i c l e

i n f o

Article history: Received 14 June 2016 Received in revised form 14 March 2017 Accepted 3 April 2017 Available online 12 April 2017 Keywords: Colanic acid O-antigen Mannose manB Live attenuated Salmonella vaccine Cross-immunity

a b s t r a c t Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Dpmi, DrfbK or DcpsG) and a double mutation (DrfbK DcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Dcya Dcrp vaccine strain. The Dpmi, DrfbK and DcpsG DrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Dcrp Dcya DcpsG DrfbK) and its parent S738 (Dcrp Dcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Salmonella enterica is an intracellular pathogen that infects both humans and a wide range of animals, with notable economic consequences [1–3], which can be categorized into typhoidal and nontyphoidal serovars [4,5]. Typhoidal Salmonella are restricted to human hosts, and include Salmonella serovars Typhi, Paratyphi A and Paratyphi B, which cause typhoid and paratyphoid enteric fevers. Many non-typhoidal Salmonella enterica serovars (NTS) are

⇑ Corresponding authors at: Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China (Q. Kong). E-mail addresses: [email protected] (Q. Liu), [email protected] (Q. Kong). 1 These authors contributed equally to this research. http://dx.doi.org/10.1016/j.vaccine.2017.04.002 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.

characterized as ‘‘generalists” because they have a broad host range, infecting both animals and humans [4,5]. NTS strains, which generally cause a self-limiting gastroenteritis in immunocompetent individuals, result in an estimated 94 million cases infections and 155,000 deaths worldwide each year [1,2]. S. Typhimurium (serogroup B), S. Enteritidis (serogroup D), S. Choleraesuis (serogroup C) and S. Newport (serogroup C) account for more than 95% of all NTS infections worldwide [6–10]. Salmonella infections are typically treated with antibiotics. The emergence of multipledrug resistance in clinical isolates poses severe threats to public health [11–13]. Control of Salmonella in food animals is primarily achieved via vaccination. However, because of the serological diversity of S. enterica, these vaccines are typically not cross protective against the major NTS serovars. The various Salmonella serovars possess

2863

P. Li et al. / Vaccine 35 (2017) 2862–2869 Table 1 Bacterial strains and plasmids used in this study. Strains or plasmids

Description

Source

S. Typhimurium S100 S089 S113 S114 S116 S738 S119 S120 S121 S122

Clinical isolate from duck Dpmi-2 DcpsG3 DrfbK4 DcpsG3 DrfbK4 Dcrp-24 Dcya-25 Dcrp-24 Dcya-25 Dpmi-2 Dcrp-24 Dcya-25 DcpsG3 Dcrp-24 Dcya-25 DrfbK4 Dcrp-24 Dcya-25 DcpsG3 DrfbK4

[29] This This This This [58] This This This This

Other Salmonella serovars and E. coli strains S246 S. Enteritidis clinical isolate from pig S340 S. Choleraesuis clinical isolate from chicken v7232 E. coli endA1 hsdR17 (rK
, mK+) glnV44 thi-1 recA1 gyrA relA1 D(lacZYA-argF)U169 kpir deoR (80dlac D(lacZ)M15) v7213 E. coli thi-1 thr-1 leuB6 glnV44 fhuA21 lacY1 recA1 RP4-2-Tc::Mu kpir DasdA4 Dzhf-2::Tn10

[29] [29] [59] [59]

Plasmids pYA4278 pSS16 pSS44 pSS45 pYA4518 pSS47 pSS48

[60] This This This [61] This This

sacB mobRP4 R6 K ori Cm+ Dpmi-2 construction DcpsG3 construction DrfbK4 construction p15a ori Cm+ p15a-rfbK p15a-cpsG

a number of immunologically related cross-reactive outer membrane protein (OMP) antigens, which are typically masked by highly immunogenic, but antigenically variable structural elements, primarily the O-antigen polysaccharide component of LPS [14,15]. While these OMPs exhibit some micro-heterogeneity, they share many conserved antigenic determinants [16]. Previous reports have demonstrated that down-regulation of LPS or reversible synthesis of LPS enhances immune responses to OMPs [17–21]. Reversible synthesis of LPS can be achieved by deleting genes to block synthesis of dNDP-sugar precursors required for O-antigen synthesis, i.e., the O-antigen structure is truncated due to the unavailability of dNDP sugar precursor, which could be restored by supplying the dNDP sugar in the growth media [18,19,22]. For example, S. Typhimurium Dpmi (manA) mutants produce a truncated LPS in the absence of mannose and exhibits the full LPS pattern when free mannose is provided during bacterial growth [18,19]. Hence, the production of O-antigen is reversible depending on the availability of mannose. S. Typhimurium pmi mutants are attenuated and immunogenic, inducing antibodies that crossreact with OMPs from other Salmonella serovars [18,19]. ManA (type I phosphomannose isomerase) catalyses the reversible interconversion of D-fructose-6-phosphate to D-mannose 6-phosphate (Man-6-P), the first step in GDP-mannose synthesis. The next step is the conversion of Man-6-P to D-mannose-1-phosphate (Man-1P) by phosphomannomutase (PMM), encoded by manB [14,23]. In S. Typhimurium, there are two copies of manB. One copy is designated rfbK (manBOAg), located in the O-antigen gene cluster [24,25] and the other is cpsG (manBCA), located in the gene cluster responsible for synthesis of colanic acid (CA) [14,24]. Sequence alignment analysis shows that these two genes exhibit about 40% and 20% identity in nucleotide sequence and amino acid level, respectively. CA is a surface exopolysaccharide produced by many enteric bacteria that serves as a scaffold for biofilm formation and protects cells from adverse physicochemical and environmental conditions [26–28]. Deletion of the entire colanic acid gene cluster does not attenuate S. Typhimurium virulence, but leads to increased synthesis of heterologous antigens [27]. In addition, regulated LPS truncation can enhance induction of cross-reactive immune response against conserved OMPs of enteric bacteria [15,20,29,30], which

study study study study study study study study

study study study study study

was further demonstrated by our recent research in which both O-antigen polysaccharide and enterobacterial common antigen (ECA) polysaccharide were synthesized only in the presence of exogenously supplied arabinose in the media via regulation of rfbB gene, a key gene for synthesis of O-antigen and ECA [31]. It was speculated that the gradual loss of both CA and O-antigen polysaccharides in vivo would contribute to exposing the OMPs to the host immune system and enhance cross-protective immunity. In this study, Dpmi and DrfbK (manBOAg) DcpsG (manBCA) double mutants were compared for their in vitro and in vivo phenotypes. The results showed that the double DrfbK (manBOAg) DcpsG (manBCA) mutant exhibited an attenuated, mannose-dependent reversibly rough phenotype that induced enhanced serum antibody responses cross-reactive with OMPs from heterologous Salmonella strains. 2. Materials and methods 2.1. Bacterial strains, plasmids, media, and growth conditions The bacterial strains and plasmids used in this study were listed in Table 1. All the mutant strains were derived from the virulent wild-type strain S100. Bacteria were grown at 37 °C in LuriaBertani (LB) medium or on LB agar plates [32] with or without mannose (1%) as needed. sacB-based counterselection was used for allelic exchange to construct Salmonella mutants [33]. Chloramphenicol was added at a final concentration of 25 lg/ml when necessary. 2.2. Construction of plasmids and mutant strains The primers used in this study were listed in Supplementary Table 1. DNA manipulations were carried out using standard techniques [34]. Transformation of E. coli and S. Typhimurium was performed by electroporation. To construct complementation plasmids, two DNA fragments of approximately 1400-bp were amplified using the primer pairs of rfbK-F-BamHI/rfbK-R-PstI and cpsG-F-BamHI/cpsG-R-PstI. After

2864

P. Li et al. / Vaccine 35 (2017) 2862–2869

simultaneously digested by BamHI and PstI, the PCR products were ligated into pYA4518 to generate pSS47 and pSS48, respectively. The resulting plasmids were sequenced to confirm the DNA sequence of the cpsG and rfbK. Suicide vector technology was used to generate precise deletion mutations. Two primer pairs, DcpsG-1F/DcpsG-1R and DcpsG-2F/ DcpsG-2R, were used to amplify approximately 400-bp DNA fragments upstream and downstream of the cpsG gene. These two fragments were fused by PCR using primers DcpsG-1F and DcpsG-2R. The fused PCR product was ligated into pYA4278 to generate plasmid pSS44. The same strategy was applied to construct plasmids pSS16 and pSS45 for deletion of pmi and rfbK genes, respectively. The mutations were independently introduced into S. Typhimurium S100 by allelic exchange using pSS16, pSS44 and pSS45, resulting in S089, S113 and S114, respectively. Similarly, the rfbK mutation was introduced into S113 to yield S116 (DcpsG3 DrfbK4). The crp and cya mutations were also introduced into S089, S113, S114 and S116, resulting in S119, S120, S121 and S122, respectively.

complied with the Guide for the Care and Use of Laboratory Animals. Six-week-old, female BALB/c mice were purchased from Dashuo Biotechnology Co., Ltd. (Chengdu, China). For the 50% lethal dose (LD50) determinations, static overnight LB cultures were diluted 1:100 into fresh LB containing 1% mannose and harvested by centrifugation at 3452g at room temperature when the OD600 reached 0.8 to 0.9. Cells were washed once in PBS and normalized to the required inoculum density in buffered saline with gelatin (BSG) [36]. Groups of six mice were infected orally with 20 ll BSG containing S. Typhimurium doses ranging from 1  104 CFU to 1  109 CFU. Animals were observed for 4 weeks after infection. The LD50 for each strain was calculated using the method of Reed and Muench [37]. To evaluate colonization, mice were orally inoculated with 20 ll BSG containing 1  109 CFU. On days 4 and 8 post-inoculation, Peyer’s patches, spleen and liver samples were collected from three mice per group. Samples were homogenized and dilutions were plated onto MacConkey and LB agar to determine viable counts. Twenty colonies from each animal were randomly selected to confirm the mutant genotype by PCR.

2.3. LPS preparation and silver staining 2.7. Immunization and measurement of immune response 2 ml of overnight cultures were pelleted by centrifugation. The bacterial pellets were boiled about 10 min in 100 ll 0.0625 M TrisHCl buffer (pH 6.8) containing 2% (w/v) SDS, 0.05% (v/v) bmercaptoethanol and 0.01% bromophenol blue. 10 ll of boiled sample supernatant was added into 90 ll of the 0.0625 M TrisHCl buffer (pH 6.8) containing 10% glycerol, and 0.01% bromophenol blue. For every 100 ll of solution, 1 ll proteinase K (20 mg/ml) was added and inoculated at 37 °C for approximately 1 h. 10 ll of each digested sample was separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver staining as described by Hitchcock and Brown [35]. 2.4. Motility and polymyxin B killing assays The motility assay was performed on LB plates solidified with 0.3% agar. When required, 1% mannose was added. The plates were allowed to dry at room temperature for approximately 2 h before use. Six ll of freshly grown bacteria (approximately 1  106 CFU) was spotted onto the middle of the plates and incubated at 37 °C for 6 h. The halo diameter of each colony resulting from bacterial motility was measured. Overnight cultures were diluted 1:100 into fresh LB media and continued to grow until the OD600 reached 0.8. Then, samples were diluted 100-fold into 1 ml of LB media with or without 1 lg/ml polymyxin B. Mannose (1%) was added to some samples, as indicated. The mixture was incubated statically at 37 °C for 1 h. Cells were serially diluted and plated onto LB agar plates to determine the number of colony forming units (CFU). The survival ratio was calculated by comparing the number of colonies obtained after polymyxin B treatment with the number of colonies treated to the same conditions without polymyxin B. The assays were performed in triplicate.

Mouse immunizations were performed as described previously [38]. Mice were inoculated orally with approximately 109 CFU of each strain on days 0 and 35. Blood and vaginal washes sample were collected at biweekly intervals. Mice were challenged orally on day 63 with 1  109 CFU of S. Typhimurium (10,000 times LD50) or with 1  107 CFU of S. Choleraesuis or S. Enteritidis at (50 times LD50). Salmonella OMPs were purified as described previously [39]. S. Typhimurium LPS was purchased from Sigma (St. Louis, MO, USA). A quantitative enzyme-linked immunosorbent assay (ELISA) was used to determine antibody levels in the serum and the vaginal washes as described [40] with the following modifications. Microtiter plates were coated with OMPs or LPS. The capture antibody, unlabelled goat anti-mouse IgG or IgA (H + L) (BD Pharmingen, San Diego, CA) at 1 lg/ml in PBS, was added to extra uncoated wells to generate the standard curve. The plates were incubated overnight at 4 °C, followed by blocking with PBS containing 10% FBS for 1 h at room temperature. A 100 ll diluted serum or vaginal washes sample was added to individual wells in triplicate, and the purified mouse IgG or IgA standard (for the standard curve quantification, BD Pharmingen, San Diego, CA, USA) was added to capture antibody-coated wells and 2-fold serially diluted starting at 0.5 lg/ml. The plates were incubated for 1 h at 37 °C and then treated with biotinylated goat anti-mouse IgG or IgA (Southern Biotechnology Associates, Birmingham, AL, USA). Subsequent steps for colour development were described previously [40]. Absorbance was recorded at 405 nm using an iMarkTM Microplate Reader (Bio-Rad, Hercules, CA, USA). The ELISA standard curve was drawn using software Curve Expert (Hyams DG, Starkville, MS, USA). Serum and vaginal secretion antibody levels were quantified based on absorbance values and the standard curve.

2.5. Attachment and invasion assay 2.8. Statistical analysis The human intestinal cell line INT407 (ATCC strain CCL-6) was used to perform bacterial attachment and invasion assays as described previously [15]. Both assays were repeated three times in triplicate. 2.6. Virulence determination and colonization in mice All animal research was conducted in compliance with the Animal Welfare Act, and manipulations related to animal experiments

For antibody responses to OMPs or LPS antigens analysis, data were analysed using one-way ANOVA of variance followed by Tukey’s multiple-comparison posttest, GraphPad Prism 5 software package (Graph Software, San Diego, CA) [41]. Kaplan-Meier survival Curve comparisons were calculated by comparing two groups each time through the log-rank (Mantel-Cox) test. The data were expressed as the means ± SEM. P < 0.05 was considered as significant difference.

2865

P. Li et al. / Vaccine 35 (2017) 2862–2869

Fig. 1. LPS profiles of mutant strains. (A) LPS profiles of S. Typhimurium S100 and its derivatives. (B) Effect of extracellular mannose on O-antigen synthesis. LPS patterns of strain S116 (DcpsG DrfbK) were grown in nutrient broth (NB) with various concentrations of mannose, ranging from 10% to 0% (w/v) in 2-fold dilution increments. The last lane showed the LPS pattern of wild-type S. Typhimurium S100.

Table 2 Polymyxin B sensitivity, swimming motility and virulence of wild type S. Typhimurium and its derivatives. Strain

1% Mannose

Survival ratio of polymyxin B killinga

Swimming Motilityb (mm)

Virulence (LD50)c

S100


+

0.33 0.562

38.4 ± 9.9 45.3 ± 11.6

NDd 8.57  104

Dpmi-2


+

0.037* 0.495

21.1 ± 5.1 35.7 ± 6.6

ND 1.44  106

DcpsG3


+

0.27 0.451

26.2 ± 5.2 33.2 ± 7.3

ND 3.06  106

DrfbK4


+

0.016* 0.536

25.3 ± 4.0 30.3 ± 8.1

ND 1.23  107

DcpsG3 DrfbK4


+

0.006* 0.225

9.4 ± 1.1y 27.1 ± 4.9

ND >109

a Polymyxin B killing assays were performed and the survival ratio calculated as described in Materials and Methods. Significant differences between mutant strains and their corresponding wild type strains were indicated as ‘‘*”, using GraphPad Prism. P < 0.05. b Average diameter (mm) of swimming colonies were measured after 6 h incubation. Significant differences was indicated as ‘‘y”, S116 (DcpsG3 DrfbK4) versus S100, using GraphPad Prism. P < 0.05. c Virulence expressed as the LD50 value (CFU) for the murine model of typhoid. d ND: not determined.

3. Results 3.1. In vitro characterization of mutant strains There are two copies of manB in Salmonella, rfbK (manBOAg) located in the O-antigen gene cluster and cpsG (manBCA) in the CA gene cluster [14,24]. However, it is unknown whether either of the genes can complement the other. The chromosomal copy of cpsG was examined to complement a DrfbK mutation. As CA is highly expressed only under stress conditions [42–45], It was anticipated that cpsG would be poorly expressed when grown in LB in the laboratory and therefore may not be able to fully complement the DrfbK mutation. As expected, the LPS pattern of the DrfbK mutant was incomplete, although some O-antigen was detected (Fig. 1A, lane 3). The DcpsG mutation had no detectable effect on O-antigen synthesis (Fig. 1A, lane 2). The DrfbK mutant was transformed with either pSS45 (rfbK) or pSS48 (cpsG). Both plasmids were able to fully restore full length LPS synthesis to the DrfbK strain (Fig. 1A, lane 4 and 5). Similar results were observed when pSS45 or pSS48 were introduced into the DcpsG DrfbK double mutant strain (Fig. 1A, lane 7 and 8). These results suggested that the products of both genes carried out the same function. Moreover, some O-antigen was produced in the DrfbK mutant, indicating that cpsG from the CA operon could naturally complement

rfbK, albeit to a lesser extent (Fig. 1A, lane 3). Deletion of pmi or rfbK plus cpsG blocked the synthesis of LPS O-antigen in nutrient broth, a carbohydrate-free medium (Fig. 1A and data not shown). To determine whether O-antigen could be restored by mannose, cells were grown in nutrient broth containing various concentrations of mannose. The results showed that the addition of mannose restored full length O-antigen synthesis in this strain (Fig. 1B). Loss of O-antigen in Salmonella can increase sensitivity to cationic antimicrobial peptides [20,21]. Mutants were evaluated for sensitivity to polymyxin B. When grown in the absence of mannose, strains S089 (Dpmi), S114 (DrfbK) and S116 (DcpsG DrfbK) were all more sensitive than the wild type (Table 2). Growth in mannose restored polymyxin B resistance for all mutants. Strains S089 (Dpmi), S113 (DcpsG) and S114 (DrfbK) exhibited minor motility defects compared to wild-type Salmonella, and S116 (DcpsG DrfbK) was significantly less motile than the wildtype (Table 2). Addition of mannose to the media partially restored motility. Plasmid complementation completely restored the motility of each mutant to wild-type levels (data not shown). 3.2. Attachment and invasion When grown in mannose, strains S114 (DrfbK) and S116 (DcpsG DrfbK) were significantly better able to adhere to INT407 cells than

2866

P. Li et al. / Vaccine 35 (2017) 2862–2869

Fig. 2. Attachment and invasion assays in INT407 cells. INT407 cells were seeded at 5  105 cells/ml in and allowed to grow approximately 24 h to a confluent monolayer. The mutant strains were added to each well at a multiplicity of infection (MOI) of 10:1. All mutants were cultured with 1% mannose before the assay. Error bars represented standard errors of the means, calculated using GraphPad Prism. Significant differences between strains is indicated as P < 0.05.

wild type (Fig. 2). Strain S114 (DrfbK) invaded INT407 cells significantly better than wild type (P < 0.05). The 2-fold increase in invasion by strain S116 (DcpsG DrfbK) was not significant. The Dpmi and DcpsG mutants were statistically indistinguishable from wild type. 3.3. Virulence and colonization by mutant strains in BALB/c mice The wild-type S100 had an LD50 of 8.57  104 CFU. The LD50s of S089 (Dpmi) and S113 (DcpsG) were increased 35-fold, and the LD50 of S114 (DrfbK) was 140-fold higher than wild type (Table 2). The DcpsG DrfbK mutant S116 was highly attenuated, with an LD50 > 1000-fold higher than wild type, indicating that S116 (DcpsG DrfbK) was more attenuated than S089 (Dpmi) in this model. Colonization of murine Peyer’s patches, spleen and liver was determined on days 4 and 8 after oral inoculation. There were no significant differences among the strains (Fig. 3). 3.4. Immunogenicity and protective efficacy of live attenuated vaccine strains Anti-S. Typhimurium-OMPs serum IgG was detected 4 and 8 weeks after the primary immunization (Fig. 4A). Mice immunized with strain S122 (Dcrp Dcya DcpsG DrfbK) mounted a

stronger anti-OMP serum IgG response after 8 weeks than those immunized with any of the other strains, including the parent for this study, strain S738 strain (Dcrp Dcya) (Fig. 4A). Anti-OMP responses to heterologous strains S. Choleraesuis and S. Enteritidis were also examined. In each case, all mutant derivatives of strain S738 elicited higher levels of OMP-specific IgG than S738 (P < 0.05). Serum IgG responses against OMPs derived from E. coli O157 showed the same trend, with all of the mutant strains outperforming S738 (P < 0.05). There was no significant difference in the levels of anti-S. Typhimurium LPS IgG among the strains (Fig. 4B). By 8 weeks, the anti-S. Typhimurium OMP mucosal IgA levels elicited by S738 strain (Dcrp Dcya) and S120 strain (Dcrp Dcya DcpsG) were significantly higher than the others (Fig. 4C). The mucosal IgA responses against heterologous OMPs were similar for all strains. Negative control groups (BSG) did not mount a detectable immune response. Upon lethal challenge with wild-type virulent S. Typhimurium S100, complete protection was observed in all immunized mice (Fig. 5A), whereas approximately 50% protection was obtained in all immunized mice challenged with wild-type virulent S. Choleraesuis or S. Enteritidis strains (Fig. 5B and C).

4. Discussion Immune responses elicited by smooth Salmonella are directed primarily against the structurally hyper-variable O-antigen domain, posing a challenge for designing a cross-reactive vaccine against multiple Salmonella serovars [46,47]. Many conserved OMPs, including iron regulated outer membrane proteins and lipoproteins, induce cross-protective immunity [48–50]. Regulated LPS synthesis can enhance production of cross-reactive anti-OMP antibodies against heterologous enteric bacteria [20,21]. Regulated synthesis of enterobacterial common antigen, a major Salmonella surface polysaccharide, also enhances cross-immune responses against enteric bacteria [31]. The aim of this study was to engineer a strain with regulated synthesis of O-antigen and CA polysaccharides to enhance crossimmunity against heterologous Salmonella serovars and other enteric bacteria. The results demonstrated the necessity of deleting both rfbK and cpsG to achieve complete shutoff of O-antigen synthesis in the absence of mannose, due to partial complementation of DrfbK with chromosomal cpsG (Fig. 1A). The DcpsG DrfbK mutant adhered and invaded as well or better than wild type (Fig. 2) and colonized deeper tissues such as spleen

Fig. 3. Colonization of murine Peyer’s patches, spleens and livers by live attenuated S. Typhimurium vaccine strains. All mutants were derived from the S738 (Dcrp Dcya) parental strain, as indicated. Vaccine strains were grown in LB broth containing 1% mannose. Colonization of Peyer’s patches (A), liver (B) and spleen (C) at 4 and 8 days postinoculation were shown. The horizontal lines represented the means, and the error bars represented standard errors of the means calculated using GraphPad Prism. P value of <0.05 was considered as significant difference.

P. Li et al. / Vaccine 35 (2017) 2862–2869

2867

Fig. 4. Serum IgG and mucosal IgA responses against purified outer membrane proteins (OMPs) and S. Typhimurium LPS OMPs purified from different enteric bacteria were used for coating the ELISA plate, the concentration of antibodies was calculated based on the standard curve of commercial antibodies, All concentrations of the measured samples were within the range of the standard curve. Error bars represented the standard errors of the means calculated using GraphPad Prism. (A) Anti-OMPs IgG antibody levels in sera from mice orally immunized with live attenuated Salmonella strains after 8 weeks. (B) Anti-S. Typhimurium LPS IgG antibody levels in sera from mice orally immunized with the indicated live attenuated Salmonella for 4 and 8 weeks. (C) Anti-OMPs IgA antibody levels in vaginal washes from mice orally immunized with live attenuated Salmonella for 8 weeks.

Fig. 5. Survival curves after oral challenge with wild-type virulent Salmonella Female BALB/c mice were immunized with the indicated vaccine strains. Nine weeks after the second immunization, mice were challenged with wild-type virulent S. Typhimurium at 10,000 times the LD50 (A), wild-type virulent S. Choleraesuis at 100 times the LD50, (B) or wild-type virulent S. Enteritidis at 50 times the LD50, (C). Curve comparisons were calculated by comparing two groups each time through the log-rank (Mantel-Cox) test, using GraphPad Prism. P < 0.05, for all marked group versus BSG group.

and liver as well as the parent strain (Fig. 3). In addition, the DcpsG DrfbK double mutation represented an improvement on Dpmi because the Dpmi mutation was only partially attenuated (Table 2)

[19], while the DcpsG DrfbK mutant was completely attenuated (Table 2). Furthermore, a Dpmi mutant grown in LB can produce O-antigen when provided with as little as 0.2% mannose [51], while

2868

P. Li et al. / Vaccine 35 (2017) 2862–2869

the strain S116 (DcpsG DrfbK) mutant required 1% mannose to produce full length O-antigen (Fig. 1B). The LPS profile of S738 was the same as S100 (data not shown), which indicated that, after invading and colonizing the host tissue, the S122 (Dcrp Dcya DcpsG DrfbK) mutant could be promptly truncated to expose the conserved outer membrane proteins to the host immune system. The strains with deletions in cpsG, rfbK or both induced higher levels of anti-OMPs serum IgG titers than those produced by S738 (Dcrp Dcya) (Fig. 4A), consistent with our previous observations using mutants with truncated LPS [20,31]. The profiles of OMPs from the mutants did not exhibit obvious differences when grown in LB media (data not shown), which also was consistent with previous observations that the OMPs isolated from Salmonella mutants with altered length of LPS resulting from mutations including DwaaC12, DwaaF15, DwaaG42, DrfaH49, DwaaI43, DwaaJ44, DwaaL46, DwbaP45 and Dwzy-48 showed only minor alterations [29]. This enhancement of cross-reactivity may be largely due to enhanced exposure of outer membrane proteins that were accessible to the host immune system rather than altered components of OMPs from the mutants. There were no significant differences in anti-S. Typhimurium LPS IgG levels among the different vaccine strains, but trend was gradually decreased as mutations were introduced into the smooth parent strain S738 (Dcrp Dcya) (Fig. 4B), indicating that Salmonella LPS synthesis was gradually shut off after bacteria invaded the host, in accordance with the results obtained from the mutants with arabinose-regulated synthesis LPS [20,21,31]. After the boost, mice vaccinated with S738 (Dcrp Dcya) or S120 (DcpsG Dcrp Dcya) elicited significantly higher anti-S. Typhimurium OMPs mucosal IgA responses than mice vaccinated with other strains. Salmonella LPS contributes to the activation of dendritic cells and intestinal IgA induced by dendritic cells protects against mucosal penetration by microbes [52–55]. From this, it is inferred that in the early stages of Salmonella infection, intact LPS plays an important role in induction of mucosal immune responses. If so, it is possible that the LPS in strains with regulated O-antigen synthesis may have a deficiency in their ability to stimulate dendritic cells, posing a requirement to systemically investigate the outcome of mucosal immunity induced by Salmonella mutants with diverse length of LPS. Mice vaccinated with S120 (Dcrp Dcya DcpsG), S121 (Dcrp Dcya DrfbK) or S122 (Dcrp Dcya DcpsG DrfbK) were completely protected against challenge with wild-type S100 (Fig. 5A). However, only 50% protection was observed in mice challenged with S. Choleraesuis or S. Enteritidis while high cross-reaction against heterologous OMPs were observed (Fig. 5B and C). These intermediate levels of protection could be due, at least in part, to the fact that some abundant Salmonella OMPs are highly immunogenic but not protective, such as OmpA or OmpC porins, which are conserved and abundantly present on the surface of Salmonella serovars [49,56,57], or to an insufficient mucosal response. Future studies will focus on reducing the induction of immune responses to non-protective OMPs and on exposing and displaying crossprotective surface antigens while achieving a stronger mucosal response.

Conflict of interest The authors have no conflicts of interest.

Acknowledgements This work was supported by grants 31570928 and 31472179 from the National Natural Science Foundation of China, and by

the National Institutes of R01AI112680).

Health

(NIH

R21AI100199

and

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2017.04. 002.

References [1] Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O’Brien SJ, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis 2010;50:882–9. [2] Crump JA, Mintz ED. Global trends in typhoid and paratyphoid fever. Clin Infect Dis 2010;50:241–6. [3] Dougan G, John V, Palmer S, Mastroeni P. Immunity to salmonellosis. Immunol Rev 2011;240:196–210. [4] Tennant SM, Diallo S, Levy H, Livio S, Sow SO, Tapia M, et al. Identification by PCR of non-typhoidal Salmonella enterica serovars associated with invasive infections among febrile patients in Mali. PLoS Neglect Trop D 2010;4:e621. [5] Levy H, Diallo S, Tennant SM, Livio S, Sow SO, Tapia M, et al. PCR method to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella Isolates from the blood of patients with clinical enteric fever. J Clin Microbiol 2008;46:1861–6. [6] Fuche FJ, Sow O, Simon R, Tennant SM. Salmonella Serogroup C: current status of vaccines and why they are needed. Clin Vaccine Immuno: CVI 2016;23:737–45. [7] Gal-Mor O, Boyle EC, Grassl GA. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front Microbiol 2014;5:391. [8] Hoelzer K, Moreno Switt AI, Wiedmann M. Animal contact as a source of human non-typhoidal salmonellosis. Vet Res 2011;42:34. [9] Mughini-Gras L, Enserink R, Friesema I, Heck M, van Duynhoven Y, van Pelt W. Risk factors for human salmonellosis originating from pigs, cattle, broiler chickens and egg laying hens: a combined case-control and source attribution analysis. PLoS ONE 2014;9:e87933. [10] Galanis E, Lo Fo Wong D, Patrick ME, Binsztein N, Cieslik A, Chalermchaikit T, et al. Web-based surveillance and global Salmonella distribution, 2000–2002. Emerg Infect Dis 2006;12:381–9. [11] Threlfall EJ. Antimicrobial drug resistance in Salmonella: problems and perspectives in food-and water-borne infections. FEMS Microbiol Rev 2002;26:141–8. [12] Tadesse DA, Singh A, Zhao S, Bartholomew M, Womack N, Ayers S, et al. Antimicrobial resistance in Salmonella in the United States: 1948–1995. AAC. 2016:AAC. 02536–15. [13] Ricke SC, Rivera Calo J, Kaldhone P, Ricke S, Donaldson J, Phillips C. Salmonella control in food production: current issues and perspectives in the United States. Food Safety: Emerging Issues, Technologies and Systems; 2015. p. 107. [14] Stevenson G, Lee S, Romana L, Reeves P. The cps gene cluster of Salmonella strain LT2 includes a second mannose pathway: sequence of two genes and relationship to genes in the rfb gene cluster. Mol Gen Genet 1991;227. [15] Kong Q, Yang J, Liu Q, Alamuri P, Roland KL, Curtiss 3rd R. Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar Typhimurium. Infect Immun 2011;79:4227–39. [16] Malik M, Butchaiah G, Bansal M, Siddiqui M, Bakshi C, Singh R. Antigenic relationships within the genus Salmonella as revealed by anti-Salmonella enteritidis monoclonal antibodies. Vet Res Commun 2002;26:179–88. [17] Nagy G, Danino V, Dobrindt U, Pallen M, Chaudhuri R, Emödy L, et al. Downregulation of key virulence factors makes the Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate. Infect Immun 2006;74:5914–25. [18] Curtiss III R, Zhang X, Wanda S-Y, HoYoung K, Konjufca V, Li Y, et al. Induction of host immune responses using Salmonella-vectored vaccines. Virulence mechanisms of bacterial pathogens; 2006. p. 297–313. [19] Collins L, Attridge S, Hackett J. Mutations at rfc or pmi attenuate Salmonella Typhimurium virulence for mice. Infect Immun 1991;59:1079–85. [20] Kong Q, Liu Q, Roland KL, Curtiss 3rd R. Regulated delayed expression of rfaH in an attenuated Salmonella enterica serovar Typhimurium vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen. Infect Immun 2009;77:5572–82. [21] Kong Q, Liu Q, Jansen AM, Curtiss 3rd R. Regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Vaccine 2010;28:6094–103. [22] Hone D, Morona R, Attridge S, Hackett J. Contruction of defined galE mutants of Salmonella for use as vaccines. J Infect Dis 1987;156:167–74. [23] Samuel G, Reeves P. Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly. Carbohydr Res 2003;338:2503–19.

P. Li et al. / Vaccine 35 (2017) 2862–2869 [24] Jensen SO, Reeves PR. Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica. Microbiology 2001;147:599–610. [25] Frick DN, Townsend BD, Bessman MJ. A novel GDP-mannose mannosyl hydrolase shares homology with the MutT family of enzymes. J Biol Chem 1995;270:24086–91. [26] Flemming H-C, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010;8:623–33. [27] Wang S, Shi H, Li Y, Shi Z, Zhang X, Baek CH, et al. A colanic acid operon deletion mutation enhances induction of early antibody responses by live attenuated Salmonella vaccine strains. Infect Immun 2013;81:3148–62. [28] Flemming H-C, Neu TR, Wozniak DJ. The EPS matrix: the ‘‘house of biofilm cells”. J Bacteriol 2007;189:7945–7. [29] Liu Q, Liu Q, Zhao X, Liu T, Yi J, Liang K, et al. Immunogenicity and crossprotective efficacy induced by outer membrane proteins from Salmonella Typhimurium mutants with truncated LPS in mice. Int J Mol Sci 2016;17:416. [30] Liu Q, Liu Q, Yi J, Liang K, Liu T, Roland KL, et al. Outer membrane vesicles derived from Salmonella Typhimurium mutants with truncated LPS induce cross-protective immune responses against infection of Salmonella enterica serovars in the mouse model. Int J Med Mic 2016;306:697–706. [31] Huang C, Liu Q, Luo Y, Li P, Liu Q, Kong Q. Regulated delayed synthesis of lipopolysaccharide and enterobacterial common antigen of Salmonella Typhimurium enhances immunogenicity and cross-protective efficacy against heterologous Salmonella challenge. Vaccine 2016;34:4285–92. [32] Bertani G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 1951;62:293. [33] Blomfield I, Vaughn V, Rest R, Eisenstein B. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol 1991;5:1447–57. [34] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989. [35] Hitchcock PJ, Brown TM. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 1983;154:269–77. [36] Curtiss R. Chromosomal aberrations associated with mutations to bacteriophage resistance in Escherichia coli. J Bacteriol 1965;89:28–40. [37] Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938;27:493–7. [38] Wang S, Li Y, Scarpellini G, Kong W, Shi H, Baek C-H, et al. Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity. Infect Immun 2010;78:3969–80. [39] Kang HY, Srinivasan J, Curtiss R. Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar Typhimurium vaccine. Infect Immun 2002;70:1739–49. [40] Li Y, Wang S, Xin W, Scarpellini G, Shi Z, Gunn B, et al. A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Infect Immun 2008;76:5238–46. [41] Motulsky H. Prism 5 statistics guide. GraphPad Software. 2007;2007:1–26. [42] Chen J, Lee SM, Mao Y. Protective effect of exopolysaccharide colanic acid of Escherichia coli O157: H7 to osmotic and oxidative stress. Int J Food Microbiol 2004;93:281–6. [43] Ophir T, Gutnick DL. A role for exopolysaccharides in the protection of microorganisms from desiccation. Appl Environ Microbiol 1994;60:740–5.

2869

[44] Navasa N, Rodriguez-Aparicio L, Martinez-Blanco H, Arcos M, Ferrero MA. Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92. Appl Microbiol Biot 2009;82:721–9. [45] Sailer FC, Meberg BM, Young KD. Β-Lactam induction of colanic acid gene expression in Escherichia coli. FEMS Microbiol Lett 2003;226:245–9. [46] Grimont PA, Weill F-X. Antigenic formulae of the Salmonella serovars. WHO collaborating centre for reference and research on Salmonella. vol. 9; 2007. [47] Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA. Invasive nontyphoidal Salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 2012;379:2489–99. [48] Yang Y, Wan C, Xu H, Wei H. Identification and characterization of OmpL as a potential vaccine candidate for immune-protection against salmonellosis in mice. Vaccine 2013;31:2930–6. [49] Barat S, Willer Y, Rizos K, Claudi B, Mazé A, Schemmer AK, et al. Immunity to intracellular Salmonella depends on surface-associated antigens. PLoS Pathog 2012;8:e1002966. [50] Confer AW, Suckow MA, Montelongo M, Dabo SM, Miloscio LJ, Gillespie AJ, et al. Intranasal vaccination of rabbits with Pasteurella multocida A: 3 outer membranes that express iron-regulated proteins. Am J Vet Res 2001;62:697–703. [51] Li Y, Wang S, Scarpellini G, Gunn B, Xin W, Wanda SY, et al. Evaluation of new generation Salmonella enterica serovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA. P Natl Acad Sci USA 2009;106:593–8. [52] Neutra MR, Pringault E, Kraehenbuhl J-P. Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annu Rev Immunol 1996;14:275–300. [53] Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 2004;303:1662–5. [54] Lamm ME. Interaction of antigens and antibodies at mucosal surfaces. Annu Rev Microbiol 1997;51:311–40. [55] Zenk SF, Jantsch J, Hensel M. Role of Salmonella enterica lipopolysaccharide in activation of dendritic cell functions and bacterial containment. J Immunol 2009;183:2697–707. [56] Toobak H, Rasooli I, Talei D, Jahangiri A, Owlia P, Astaneh SDA. Immune response variations to Salmonella enterica serovar Typhi recombinant porin proteins in mice. Biologicals 2013;41:224–30. [57] Cho Y, Park S, Barate AK, Truong QL, Han JH, Jung CH, et al. Proteomic analysis of outer membrane proteins in Salmonella enterica Enteritidis. J Microbiol Biotechn 2015;25:288–95. [58] Liu X, Liu Q, Xiao K, Li P, Liu Q, Zhao X, et al. Attenuated Salmonella Typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus. Vet Microbiol 2016;186:189–98. [59] Roland K, Curtiss III R, Sizemore D. Construction and evaluation of a Dcya Dcrp Salmonella Typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian Dis 1999:429–41. [60] Edwards RA, Keller LH, Schifferli DM. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 1998;207:149–57. [61] Zhang X, Wanda S-Y, Brenneman K, Kong W, Zhang X, Roland K, et al. Improving Salmonella vector with rec mutation to stabilize the DNA cargoes. BMC Microbiol 2011;11:31.