Abstract

The attenuation and immunoenhancing effects of rpoS and phoPSalmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS–phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP–rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD50 of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP–rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP–rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.

Introduction

The alternative sigma factor σs (rpoS) of Salmonella is involved in general stress resistance, survival under starvation conditions, and virulence in mice (Fang et al., 1992; Coynault et al., 1996). rpoS mutations in Salmonella strains have various attenuating effects on the growth rate of deep lymphoid organs and on systemic infection and bacteremia in animals and humans (Fang et al., 1992; Norel et al., 1992; Kowarz et al., 1994; Coynault et al., 1996). rpoS mutations also reduce the ability of Salmonella enterica serovar strain Typhimurium (Salmonella typhimurium) to colonize Peyer's patches of infected mice (Coynault et al., 1996; Nickerson & Curtiss, 1997). In addition, rpoS mutants of Salmonella enterica serovar strain Typhi (Salmonella typhi) appear to be less cytotoxic for macrophages than wild-type S. typhi (Khan et al., 1998).

The phoP locus is a two-component regulatory system that controls the expression of genes necessary for the virulence and survival of Salmonella strains within host macrophages (Hohmann et al., 1996b). This locus regulates multiple unlinked phoP-activated and phoP-repressed genes: both classes of gene have been documented to encode virulence determinants in S. typhimurium (Behlau & Miller, 1993; Belden & Miller, 1994). phoP mutants of Salmonella are markedly attenuated for virulence in mice, survive poorly within cultured murine macrophages, and are effective vaccines in the mouse model (Galan & Curtiss, 1989; Miller et al., 1989).

Many previous reports document that either phoP or rpoS mutants of Salmonella have potential as oral live vaccine candidates, eliciting protective immune responses in mice (Galan & Curtiss, 1989; Miller et al., 1989; Fang et al., 1992; Kowarz et al., 1994; Coynault et al., 1996; Nickerson & Curtiss, 1997). However, all of those studies were performed using S. typhimurium. Experimentation with S. typhi has been much more limited than experimentation with S. typhimurium due to the lack of a reliable preclinical animal model to allow immunological assessment of typhoid vaccine candidates. Recently, other groups demonstrated that strong immune responses can be elicited in mice immunized intranasally with attenuated S. typhi live vaccines (Barry et al., 1996; Galen et al., 1997), and bacterial characterizations of S. typhi in vivo could therefore be performed directly using a nasally infected mouse model. Thus, the bacterial characterizations performed using S. typhimurium to evaluate Typhoid vaccines need to be reconfirmed using the nasal-infected mouse model and S. typhi.

The phoP and rpoS genes are known to be important factors in determining the virulence of Salmonella. Oral live vaccine candidates (Ty800 and Ty21a) have been developed with these genes mutated in S. typhi and used as oral live vaccines worldwide (Robbe-Saule et al., 1995; Hohmann et al., 1996a). However, these mutants need to be characterized further using the nasal infected mouse model and S. typhi. In particular, a comparison between phoP and rpoS mutants has not been performed. Three S. typhi deletion mutants (rpoS, phoP, and rpoS–phoP double mutant) were constructed and these mutants were characterized for invasiveness, virulence, and protective immune response compared with wild-type Ty2.

Materials and methods

Bacterial strains and culture conditions

The Salmonella strains used in this study are listed in Table 1. The highly virulent S. typhi strain Ty2 (ATCC 19430) was used as the parent for the genetically modified strains. The construction of defined deletion mutants or null mutants of S. typhi Ty2 was performed using the allelic exchange method (Kang et al., 2002). The resulting mutants were designated LF1021 (phoP deletion), LF1036 (rpoS deletion), and LF1037 (phoP rpoS double deletion).

Table 1

Bacterial strains and plasmids

Strain or plasmid Relevant phenotype Reference 
χ7213 (E. coliΔasd Kang et al. (2002) 
S. typhi Ty2 Wild type, ATCC19430 ATCC 
ELF256 χ7213/pLL6.12 This work 
ELF265 χ7213/pWL2.1 This work 
LF1021 S. typhi Ty2/phoP::km This work 
LF1036 S. typhi Ty2/ΔrpoS This work 
LF1037 S. typhi Ty2/phoP::km, ΔrpoS, double mutant This work 
pBSL86 Source of Km gene/Hind III Alexeyev et al. (1995) 
pDMS197 Suicide plasmid of Salmonella Kang et al. (2002) 
pLL6.12 pDMS197/ contains flanking regions of phoP and Km genes This work 
pWL2.1 pDMS197/contains flanking regions of rpoS This work 
Strain or plasmid Relevant phenotype Reference 
χ7213 (E. coliΔasd Kang et al. (2002) 
S. typhi Ty2 Wild type, ATCC19430 ATCC 
ELF256 χ7213/pLL6.12 This work 
ELF265 χ7213/pWL2.1 This work 
LF1021 S. typhi Ty2/phoP::km This work 
LF1036 S. typhi Ty2/ΔrpoS This work 
LF1037 S. typhi Ty2/phoP::km, ΔrpoS, double mutant This work 
pBSL86 Source of Km gene/Hind III Alexeyev et al. (1995) 
pDMS197 Suicide plasmid of Salmonella Kang et al. (2002) 
pLL6.12 pDMS197/ contains flanking regions of phoP and Km genes This work 
pWL2.1 pDMS197/contains flanking regions of rpoS This work 

Bacterial strains were grown overnight at 37°C in 10 mL Luria–Bertani (LB) broth in rollers. A 0.1 mL overnight culture was used to inoculate 10 mL LB broth, and bacteria were grown at 37°C for 4 h in a roller. When required, antibiotics were added to the culture medium at the following concentrations: ampicillin, 50 µg mL−1; chloramphenicol, 12 µg mL−1; and tetracycline, 15 µg mL−1. Diaminopimelic acid was added for the growth of Asd-strains. The number of CFU was determined by plating serial 10-fold dilutions on LB plates.

The human colon carcinoma cell line HT-29 was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and was cultured in RPMI-1640 medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1 mM penicillin–streptomycin (Sigma) at 37°C in a 5% CO2 atmosphere.

Construction of S. typhi Ty2 mutants by conventional allelic exchange

The plasmids and bacterial strains are listed in Table 1. Escherichia coliχ7213 and pDMS197 were kindly provided by Roy Curtiss III (Kang et al., 2002). An S. typhi Ty2 phoP::Km mutant (LF1021) was constructed by conjugation between the wild-type strain S. typhi Ty2 and E. coliχ7213, which had plasmid pLL6.12 inserted into the regions flanking the phoP locus. To integrate the selective maker into the phoP locus of S. typhi, the kanamycin cassette gene was ligated into the Hind III site between the upstream and downstream flanking regions of pLL6.12. Strains containing a single-crossover plasmid insertion (S. typhi Ty2 with phoP replaced by pLL6.12, Fig. 1) were isolated on plates containing tetracycline and kanamycin. Loss of the suicide vector after the second recombination between homologous regions was selected on LB containing 10% sucrose using the sacB-based sucrose sensitivity counter-selection system. The presence of the phoP::Km allele in LF1021 was confirmed by PCR using primers 5′-CGAGCTCCGTCATCGCCTGACGAC-3′ and 5′-GCTCTAGACACCGATTATAACGGATGC-3′.

Figure 1

Construction of mutants by allelic exchange. (a) Genetic organization of recombinant constructs used to make a phoP::Km mutation and a defined rpoS deletion. (b) Analysis of PCR products amplified from whole-cell lysates of the wild type and mutant strains. The phoP allele was amplified from selected clones, yielding a 4.28-kb product compared with a 3.75-kb product from the wild type (lanes 3 and 2). The rpoS allele yielded a 3.07-kb product compared with a 4.22-kb product from the wild type (lanes 5 and 4). The phoP–rpoS double-mutant Salmonella typhi strain was amplified from selected clones using primer sets for phoP and rpoS allele, yielding 4.28 and 3.07-kb products, respectively (lane 6 and 7).

Figure 1

Construction of mutants by allelic exchange. (a) Genetic organization of recombinant constructs used to make a phoP::Km mutation and a defined rpoS deletion. (b) Analysis of PCR products amplified from whole-cell lysates of the wild type and mutant strains. The phoP allele was amplified from selected clones, yielding a 4.28-kb product compared with a 3.75-kb product from the wild type (lanes 3 and 2). The rpoS allele yielded a 3.07-kb product compared with a 4.22-kb product from the wild type (lanes 5 and 4). The phoP–rpoS double-mutant Salmonella typhi strain was amplified from selected clones using primer sets for phoP and rpoS allele, yielding 4.28 and 3.07-kb products, respectively (lane 6 and 7).

A defined deletion rpoS mutant (LF1036) in S. typhi Ty2 was constructed by allelic exchange as described above, with conjugation between S. typhi Ty2 and E. coliχ7213 containing pWL2.1 inserted into flanking regions near the rpoS locus. Mutants were selected on LB containing 10% sucrose. The deletion of rpoS in LF1036 was confirmed by PCR amplification and comparison with wild-type S. typhi Ty2 using primers 5′-CGAGCTCCAGCGAGGAAGTGAATGC-3′ and 5′-GCTCTAGACGCTATCATTGTGACGGC-3′.

An S. typhi Ty2ΔrpoS phoP::Km double mutant (LF1037) was constructed by conjugation by allelic exchange between E. coliχ7213 and LF1021. The presence of the ΔrpoS phoP::Km double mutation in LF1037 was confirmed by PCR with primer sets for phoP and rpoS.

Tissue culture invasion assay

Invasion assays were performed using gentamicin protection assays as described previously (Tsolis et al., 1995). In brief, HT-29 cells were seeded at c. 4 × 105 cells well−1 in 24-well plates and the invasion assay was performed the following day. Approximately 1 × 107 CFU bacteria were added to HT-29 cells and incubated for 1 h at 37°C in 5% CO2 to allow invasion. Each well was washed three times with sterile phosphate-buffered saline (PBS, pH 7.4) to remove extracellular bacteria, and a medium containing 0.1 mg mL−1 gentamicin was added for 1 h. After three washes with PBS, cells were lysed by addition of 1 mL 1% Triton X-100 (Sigma). Tenfold serial dilutions of cell lysates in PBS were plated onto LB agar to count the intracellular bacteria.

Animal maintenance

Three independent animal studies were performed in this study: assessment of virulence by intraperitoneal inoculation (eight mice per group); estimation of serological immune responses and protective effects by two rounds of intranasal immunization (five mice per group); and proliferation assay (10 mice per group). Four-week-old specific-pathogen-free BALB/c female mice (Orient Co. Ltd, Seoul, Korea) were used in this study. Animals were housed in BSL grade 2 animal rooms at 22±2°C under a 12 h light and dark cycle. Food and water were provided ad libitum. All animal experimentation was performed according to the guidelines for the care and use of laboratory animals approved by IACUC of Seoul National University.

Assessment of virulence by intraperitoneal inoculation of mice

Female BALB/c mice (eight mice per group) were injected intraperitoneally (i.p.) with 10-fold dilutions of the different serovar Typhi strains (grown in LB broth, washed, and resuspended in sterile PBS) mixed with 5% (w/v) hog gastric mucin in a final volume of 0.5 mL. Mice mortality was monitored for 7 days after inoculation (Powell et al., 1980), and the LD50 was determined using the origin program (Origin 5.0 Pro, Northampton, MA)

Immunization, estimation of serum antibody response, and assessment of protective ability against wild-type challenge

Salmonella typhi mutant strains and wild-type Ty2 were cultured in LB broth and washed three times with sterile PBS. Mice were intranasally immunized with 2 × 109 CFU of S. typhi. Booster doses (2 × 109 CFU) were given 4 weeks after the first immunization. Mice were bled from the retro-orbital sinus before immunization (day 0) and every 2 weeks during the immunization schedule (days 14, 28, 42, and 56), and sera were stored at −70°C until tested.

Serum antibody titers against Salmonella lipopolysaccharide were measured by an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with S. enterica serovar Typhi lipopolysaccharide (Difco, Detroit, MI) at 1 µg well−1 in carbonate buffer. Salmonella-specific IgG, IgG1, IgG2a, IgG2b, and IgA were detected with peroxidase-conjugated antibodies (Zymed, San Francisco, CA) diluted 1 : 10 000 in 5% skim milk in PBS containing 0.05% Tween 20. One hundred microliters of o-phenylenediamine dihydrochloride (Sigma) was added and the absorbance was measured at OD450 nm. Linear regression curves were plotted for each serum sample, and IgG titers were calculated as the inverse of the serum dilution that produces an OD of 0.5 above the value for the blank (ELISA units mL−1).

To evaluate the protective efficacy, five mice in each group were intraperitoneally challenged with a superlethal dose of wild-type Ty2 (1 × 103 CFU in 5% hog gastric mucin) at 8 weeks after immunization, and the survival rate was evaluated for the following 7 days.

Proliferation assay

Five mice in each group were necropsied at 4-week intervals after the first immunization. Each mouse was intranasally inoculated with 2.0 × 109 CFU of each mutant at 0 and 4 weeks. Control mice received the same volume of PBS. The spleens were removed aseptically and placed individually into Petri dishes containing 3 mL RPMI 1640 medium. Single-cell suspensions were prepared as described previously (Park et al., 2005). The proliferation responses of spleen cells to mitogens were determined using a commercial cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany) as described previously (Park et al., 2005). Fifty microliters of ConA (2.5 µg mL−1; Sigma, St Louis, MO) and lipopolysaccharide (5 µg mL−1, derived from E. coli; Sigma) were used as mitogens.

Statistical analysis

Significant differences between the experimental and control groups were determined using Duncan's Multiple Range Test (sas version 8.2, SAS Institute Inc., Cary, NC). Values of P<0.05 were considered to be significant.

Results and discussion

Typhoid vaccines and murine intranasal immunization model

Oral administration of live attenuated Salmonella vaccines is beneficial because it stimulates all immune responses, including mucosal, humoral, and cellular immunities (Medina & Guzman, 2001; Kang et al., 2002). However, a significant obstacle to the development of attenuated S. enterica serovar Typhi vaccines is the lack of a small-animal model in which to assess reliably the immunogenicity of candidate vaccine strains following mucosal inoculation (Gonzalez et al., 1994; Medina & Guzman, 2001). Because of the narrow host restriction of S. enterica serovar Typhi, oral inoculation of mice with potential S. enterica serovar Typhi vaccine strains typically resulted in minimal or no immune response. Therefore, most work on the development of typhoid vaccines has used S. enterica serovar Typhimurium, which causes an invasive infection in mice that resembles typhoid in humans (Cardenas & Clements, 1992; Curtiss et al., 1994). Recently, there have been several reports that attenuated S. enterica serovar Typhi administered intranasally to mice was strongly immunogenic, particularly in eliciting a serum IgG response (Galen et al., 1997; Pasetti et al., 2000; Pickett et al., 2000). Subsequently, studies have been undertaken to better understand the success of this preclinical model. The main contribution of this report is the direct comparison of phoP and rpoS mutants of Typhi strains in vitro and in vivo, with an assessment of both serologic and cell-mediated immune responses. As expected based on earlier reports (Galen et al., 1997; Pasetti et al., 2000; Pickett et al., 2000), serovar Typhi was found in this study to be highly immunogenic when given intranasally (Fig. 4).

Figure 4

Protection against wild-type Ty2 challenge following intranasal immunization with Salmonella typhi mutants. Groups of five mice were immunized twice with LF1021 (−□-) or LF1036 (−•-) or LF1037 (−○-) or nonimmunized (−▪-). After intranasal immunization at 0 and 4 weeks, mice were challenged intraperitoneally with 1 × 103 CFU of wild-type Ty2 at 8 weeks. The graph shows the number of animals surviving this challenge over 7 days.

Figure 4

Protection against wild-type Ty2 challenge following intranasal immunization with Salmonella typhi mutants. Groups of five mice were immunized twice with LF1021 (−□-) or LF1036 (−•-) or LF1037 (−○-) or nonimmunized (−▪-). After intranasal immunization at 0 and 4 weeks, mice were challenged intraperitoneally with 1 × 103 CFU of wild-type Ty2 at 8 weeks. The graph shows the number of animals surviving this challenge over 7 days.

Construction of S. typhi mutants by allelic exchange

Using bacterial allelic exchange mediated by the sacB-based recombinant suicide system (Gay et al., 1985), three serovar Typhi mutant strains were obtained. Mutants were screened by selecting clones that were able to grow only in specific antibiotic-supplemented agar and thus contained disruptions of the phoP and rpoS genes. Successful gene deletion was confirmed by PCR amplification. The mutant phoP allele was amplified from selected clones, yielding a 4.28-kb product compared with a 3.75-kb product from the wild type (Fig. 1b, lanes 3 and 2, respectively), while the mutant rpoS allele yielded a 3.07-kb product compared with a 4.22-kb product from the wild type (Fig. 1b, lanes 5 and 4). For the phoP–rpoS double-mutant S. typhi strain, primer sets for both phoP and rpoS alleles were used, yielding the 4.28- and 3.07-kb products, respectively (Fig. 1b, lanes 6 and 7).

Previous methods for construction of mutants in Salmonella, especially S. typhimurium, used P22 bacteriophage-mediated transduction and a gene fusion protocol. However, these methods do not adapt to wild-type strains such as S. typhi because P22 is not able to infect S. typhi. To construct defined deletions and/or null mutants in S. typhi, a bacterial allelic exchange mediated by a sacB-based recombinant suicide system has been developed (Gay et al., 1985). Such suicide vectors replicate in narrow host ranges, and bacterial strains that contain suicide plasmid vectors are selected by environmental stresses, such as higher temperatures (Hamilton et al., 1989), or antibiotics, especially streptomycin (Skorupski & Taylor, 1996), and fusaric acid (Maloy & Nunn, 1981). At the present time, allelic exchange methods are the only way to introduce selectable markers, such as genes for antibiotic resistance, or reporter genes such as lacZYA or GFP into S. typhi.

Virulence of mutant strains

The cellular invasive ability of Ty2 and S. typhi mutants (phoP, rpoS and phoP–rpoS) was examined in the HT-29 cell line. As shown in Fig. 2, the three S. typhi mutants were significantly less invasive for HT-29 cells than wild-type Ty2, especially the phoP and phoP–rpoS mutants (Fig. 2).

Figure 2

Tissue culture invasion assay. HT-29 cells were seeded at 4 × 105 cells well−1 in 24-well plates and coincubated with 1 × 107 CFU of each Salmonella typhi mutant for 1 h to allow invasion. After washing with gentamicin and PBS, the cells were lysed by 1% Triton X-100. Tenfold serial dilutions of lysates in PBS were plated onto LB agar to count the intracellular bacteria. The assay was performed three times. Bars are expressed as mean log 10 CFU of live bacteria±SD. Means labeled with the same letter are not significantly different (P<0.05).

Figure 2

Tissue culture invasion assay. HT-29 cells were seeded at 4 × 105 cells well−1 in 24-well plates and coincubated with 1 × 107 CFU of each Salmonella typhi mutant for 1 h to allow invasion. After washing with gentamicin and PBS, the cells were lysed by 1% Triton X-100. Tenfold serial dilutions of lysates in PBS were plated onto LB agar to count the intracellular bacteria. The assay was performed three times. Bars are expressed as mean log 10 CFU of live bacteria±SD. Means labeled with the same letter are not significantly different (P<0.05).

The degree of attenuation of the three mutants was evaluated using the murine hog mucin challenge assay. LD50 values were determined and compared with those of the wild-type strain Ty2 (Table 2). The LD50 of phoP and rpoS mutants were found to differ by <3 log units (Table 2). In particular, the LD50 of the phoP–rpoS double mutant showed much greater attenuation than that of the single mutants.

Table 2

Virulence of bacterial strains in mice

Bacterial strain LD50 in eight mice (log 10 CFU of bacteria) 
Wild-type Ty2 1.28 
LF1021 4.57 
LF1036 4.49 
LF1037 7.58 
Bacterial strain LD50 in eight mice (log 10 CFU of bacteria) 
Wild-type Ty2 1.28 
LF1021 4.57 
LF1036 4.49 
LF1037 7.58 

The LD50 was determined using origin program (Origin 5.0 Pro, Northampton, MA). Each group contained eight mice. Mortality was recorded over 7 days.

Previously, it has been reported that mutations in phoP and rpoS have some similar phenotypes, suggesting a possible relationship between PhoP and RpoS. Inactivation of either the phoP or rpoS genes results in Salmonella strains that are highly attenuated for virulence in mice (Miller et al., 1989; Fang et al., 1992; Lee et al., 1995). In this study, phoP and rpoS mutant strains showed similar results with respect to virulence (Table 2). Moreover, the phoP mutant strain was much less invasive to HT-29 cells than the rpoS mutant strain, while the phoP–rpoS mutant did not differ from the phoP single mutant. These results indicate that phoP may control the expression of the rpoS gene (Table 2 and Fig. 2), as substantiated by previous reports that PhoP controls the expression of the RpoS-regulated spv virulence genes and rpoS gene (Heithoff et al., 1997; Tu et al., 2006). However, the phoP single mutant did not show the same degree of attenuation as phoP–rpoS mutants when mutant strains were intraperitoneally inoculated into mice (Table 2). It is therefore possible that another rpoS-mediated virulence gene that is not regulated by phoP might be involved in the virulence of S. typhi.

Comparisons on eliciting immune responses

After two nasal immunizations, all S. typhi mutants induced high titers of serum IgG against lipopolysaccharide of S. typhi at 8 weeks in the immunization schedule (Fig. 3a and b). For all mutants, the serum titer of IgG was more than 1000-fold higher than in control mice. However, there was no significant difference in antibody titer between the groups administered with the different S. typhi mutants. In addition, the 450 nm wave-length absorption value of IgG2 considerably increased for all S. typhi mutants (Fig. 3d and e), while that of IgG1 and IgA did not increase at 8 weeks after immunization (Fig. 3c and f).

Figure 3

Measurement of serum antibody titers against Salmonella lipopolysaccharide by ELISA. Optical densities of Salmonella-specific IgG (a), IgG1 (c), IgG2a (d), IgG2b (e), and IgA (f) were measured at 2-week intervals. Mice were intranasally immunized with LF1021 (−□-), LF1036 (−•-), LF1037 (−○-), and nonimmunized (−▪-) at 0 and 4 weeks. In (b), IgG titers were calculated as the inverse of the serum dilution that produces an OD450 nm of 0.5 above the value for the blank (ELISA units per milliliter) at 8 weeks.

Figure 3

Measurement of serum antibody titers against Salmonella lipopolysaccharide by ELISA. Optical densities of Salmonella-specific IgG (a), IgG1 (c), IgG2a (d), IgG2b (e), and IgA (f) were measured at 2-week intervals. Mice were intranasally immunized with LF1021 (−□-), LF1036 (−•-), LF1037 (−○-), and nonimmunized (−▪-) at 0 and 4 weeks. In (b), IgG titers were calculated as the inverse of the serum dilution that produces an OD450 nm of 0.5 above the value for the blank (ELISA units per milliliter) at 8 weeks.

The protective effects of mutant S. typhi strains against wild-type Ty2 challenge was also evaluated. After two nasal immunizations with each S. typhi mutant at 4-week intervals, mice were intraperitoneally challenged with 1 × 103 CFU of wild-type Ty2 at 8 weeks. More than 60% of mice that were immunized with S. typhi mutant strains survived. After the challenge, only one and two mice in each group (n=5) died during 7 days in the mice immunized with single mutants. All mice given the phoP–rpoS mutant survived for 7 days, while those without immunization (administration of PBS only) died within 1 day after challenge (Fig. 4).

Proliferation assay

Splenocytes from mice immunized with live S. typhi mutants showed significantly increased proliferative responses following in vitro stimulation with ConA and lipopolysaccharide (Fig. 5). At 8 weeks after immunization, ConA-stimulated splenocytes displayed a higher proliferative response than lipopolysaccharide-stimulated cells. There were no significant differences in the magnitude of the proliferative response to either ConA or lipopolysaccharide stimulation between the groups immunized with different mutant S. typhi strains.

Figure 5

Proliferation of spleen cells, stimulated by Con A or lipopolysaccharide at 4 and 8 weeks. Five mice from each group were necropsied at 4 weeks, and five were necropsied at 8 weeks. Each mouse was intranasally inoculated with 2.0 × 109 CFU of each mutant at 0 and 4 weeks. Control mice received the same volume of PBS (the white bar). Results are expressed as mean±SD. a,bMeans labeled with the same letter are not significantly different (P<0.05).

Figure 5

Proliferation of spleen cells, stimulated by Con A or lipopolysaccharide at 4 and 8 weeks. Five mice from each group were necropsied at 4 weeks, and five were necropsied at 8 weeks. Each mouse was intranasally inoculated with 2.0 × 109 CFU of each mutant at 0 and 4 weeks. Control mice received the same volume of PBS (the white bar). Results are expressed as mean±SD. a,bMeans labeled with the same letter are not significantly different (P<0.05).

Lymphocyte proliferation responses to mitogens are widely used to assess T- and B-cell function (Mosmann & Coffman, 1989; Muotiala & Makela, 1990; Gill et al., 1992). Mice exposed to various external antigens exhibit enhanced T- and B-cell functions as indicated by elevated proliferation responses to the T-cell mitogen ConA, and the B-cell mitogen lipopolysaccharide. In the present study, both ConA and lipopolysaccharide effectively stimulated and induced proliferation in the splenocytes of mice immunized with phoP, rpoS, and phoP–rpoS mutants. Moreover, ConA-stimulated splenocytes were found to exhibit a higher proliferative response than those stimulated with lipopolysaccharide (Fig. 5). The results of this study indicate that immunization by nasal inoculation with phoP, rpoS, and phoP–rpoS mutants increased the function of T-cells in mice, and that this enhanced T-cell function might play an important role in eliciting a protective response against a wild-type Ty2 challenge.

In addition, as shown in Fig. 3, immunization with phoP, rpoS, and phoP–rpoS mutants increased the serum antibody titers of IgG, IgG2a, and IgG2b, but not of IgA and IgG1. T-cells are the main effectors and regulators of cell-mediated immunity and are subdivided into two functional types: Th1 and Th2. Th1 cells produce IL-2, IFN-γ, and tumor necrosis factor and promote class switching to IgG2a, while Th2 cells produce IL-4, IL-5, and IL-10 and promote class switching to IgG1 and IgA (Mosmann & Coffman, 1989; Kantele, 1990; Spellberg & Edwards, 2001). Previous reports have suggested that cell-mediated responses are important mediators of the protection conferred by S. enterica serovar Typhi strain Ty21a and other attenuated S. enterica serovar Typhi live oral vaccines (Wang et al., 2001).

Conclusion

This study shows that phoP and rpoS play an important role in the attenuation and cellular invasiveness of S. typhi, and confirms that the role of these genes is similar to that of genes in S. typhimurium. Both phoP and rpoS mutants were effectively attenuated and elicited protective immune responses in mice immunized by nasal inoculation. Furthermore, the phoP–rpoS double mutant was found to have lower virulence than the single mutants but to elicit the same protective immunity. The phoP–rpoS double mutant of S. typhi is therefore a promising candidate for an attenuated typhoid vaccine.

Acknowledgements

This work was supported in part by a grant from the Korea Food & Drug Administration, and a grant from the Brain Korea 21 Project. Additional support was also provided by Korea Research Foundation grants (KRF-005-E00077) and the Research Institute of Veterinary Science, College of Veterinary Medicine, Seoul National University.

References

Alexeyev
M.F.
(
1995
)
Three kanamycin resistance gene cassettes with different polylinkers
.
Biotechniques
 
18
:
52
,
54
,
56
.
Barry
E.M.
Gomez-Duarte
O.
Chatfield
S.
Rappuoli
R.
Pizza
M.
Losonsky
G.
Galen
J.
Levine
M.M.
(
1996
)
Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908
.
Infect Immun
 
64
:
4172
4181
.
Behlau
I.
Miller
S.I.
(
1993
)
A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells
.
J Bacteriol
 
175
:
4475
4484
.
Belden
W.J.
Miller
S.I.
(
1994
)
Further characterization of the PhoP regulon: identification of new PhoP-activated virulence loci
.
Infect Immun
 
62
:
5095
5101
.
Cardenas
L.
Clements
J.D.
(
1992
)
Oral immunization using live attenuated Salmonella spp. as carriers of foreign antigens
.
Clin Microbiol Rev
 
5
:
328
342
.
Coynault
C.
Robbe-Saule
V.
Norel
F.
(
1996
)
Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon
.
Mol Microbiol
 
22
:
149
160
.
Curtiss
R.
III
Kelly
S.M.
Tinge
S.A.
Tacket
C.O.
Levine
M.M.
Srinivasan
J.
Koopman
M.
(
1994
)
Recombinant Salmonella vectors in vaccine development
.
Dev Biol Stand
 
82
:
23
33
.
Fang
F.C.
Libby
S.J.
Buchmeier
N.A.
Loewen
P.C.
Switala
J.
Harwood
J.
Guiney
D.G.
(
1992
)
The alternative sigma factor katF (rpoS) regulates Salmonella virulence
.
Proc Natl Acad Sci USA
 
89
:
11978
11982
.
Galan
J.E.
Curtiss
R.
III
(
1989
)
Virulence and vaccine potential of phoP mutants of Salmonella typhimurium
.
Microb Pathog
 
6
:
433
443
.
Galen
J.E.
Gomez-Duarte
O.G.
Losonsky
G.A.
Halpern
J.L.
Lauderbaugh
C.S.
Kaintuck
S.
Reymann
M.K.
Levine
M.M.
(
1997
)
A murine model of intranasal immunization to assess the immunogenicity of attenuated Salmonella typhi live vector vaccines in stimulating serum antibody responses to expressed foreign antigens
.
Vaccine
 
15
:
700
708
.
Gay
P.
Le Coq
D.
Steinmetz
M.
Berkelman
T.
Kado
C.I.
(
1985
)
Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria
.
J Bacteriol
 
164
:
918
921
.
Gill
H.S.
Watson
D.L.
Brandon
M.R.
(
1992
)
In vivo inhibition by a monoclonal antibody to CD4+T cells of humoral and cellular immunity in sheep
.
Immunology
 
77
:
38
42
.
Gonzalez
C.
Hone
D.
Noriega
F.R.
et al
. (
1994
)
Salmonella typhi vaccine strain CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum: strain construction and safety and immunogenicity in humans
.
J Infect Dis
 
169
:
927
931
.
Hamilton
C.M.
Aldea
M.
Washburn
B.K.
Babitzke
P.
Kushner
S.R.
(
1989
)
New method for generating deletions and gene replacements in Escherichia coli
.
J Bacteriol
 
171
:
4617
4622
.
Heithoff
D.M.
Conner
C.P.
Hanna
P.C.
Julio
S.M.
Hentschel
U.
Mahan
M.J.
(
1997
)
Bacterial infection as assessed by in vivo gene expression
.
Proc Natl Acad Sci USA
 
94
:
934
939
.
Hohmann
E.L.
Oletta
C.A.
Killeen
K.P.
Miller
S.I.
(
1996a
)
phoP/phoQ-deleted Salmonella typhi (Ty800) is a safe and immunogenic single-dose typhoid fever vaccine in volunteers
.
J Infect Dis
 
173
:
1408
1414
.
Hohmann
E.L.
Oletta
C.A.
Miller
S.I.
(
1996b
)
Evaluation of a phoP/phoQ-deleted, aroA-deleted live oral Salmonella typhi vaccine strain in human volunteers
.
Vaccine
 
14
:
19
24
.
Kang
H.Y.
Dozois
C.M.
Tinge
S.A.
Lee
T.H.
Curtiss
R.
III
(
2002
)
Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors
.
J Bacteriol
 
184
:
307
312
.
Kantele
A.
(
1990
)
Antibody-secreting cells in the evaluation of the immunogenicity of an oral vaccine
.
Vaccine
 
8
:
321
326
.
Khan
A.Q.
Zhao
L.
Hirose
K.
Miyake
M.
Li
T.
Hashimoto
Y.
Kawamura
Y.
Ezaki
T.
(
1998
)
Salmonella typhi rpoS mutant is less cytotoxic than the parent strain but survives inside resting THP-1 macrophages
.
FEMS Microbiol Lett
 
161
:
201
208
.
Kowarz
L.
Coynault
C.
Robbe-Saule
V.
Norel
F.
(
1994
)
The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes
.
J Bacteriol
 
176
:
6852
6860
.
Lee
I.S.
Lin
J.
Hall
H.K.
Bearson
B.
Foster
J.W.
(
1995
)
The stationary-phase sigma factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium
.
Mol Microbiol
 
17
:
155
167
.
Maloy
S.R.
Nunn
W.D.
(
1981
)
Selection for loss of tetracycline resistance by Escherichiacoli
.
J Bacteriol
 
145
:
1110
1111
.
Medina
E.
Guzman
C.A.
(
2001
)
Use of live bacterial vaccine vectors for antigen delivery: potential and limitations
.
Vaccine
 
19
:
1573
1580
.
Miller
S.I.
Kukral
A.M.
Mekalanos
J.J.
(
1989
)
A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence
.
Proc Natl Acad Sci USA
 
86
:
5054
5058
.
Mosmann
T.R.
Coffman
R.L.
(
1989
)
TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties
.
Annu Rev Immunol
 
7
:
145
173
.
Muotiala
A.
Makela
P.H.
(
1990
)
The role of IFN-gamma in murine Salmonella typhimurium infection
.
Microb Pathog
 
8
:
135
141
.
Nickerson
C.A.
Curtiss
R.
III
(
1997
)
Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection
.
Infect Immun
 
65
:
1814
1823
.
Norel
F.
Robbe-Saule
V.
Popoff
M.Y.
Coynault
C.
(
1992
)
The putative sigma factor KatF (RpoS) is required for the transcription of the Salmonella typhimurium virulence gene spvB in Escherichia coli
.
FEMS Microbiol Lett
 
78
:
271
276
.
Park
J.H.
Lee
Y.
Moon
E.
Seok
S.H.
Cho
S.A.
Baek
M.W.
Lee
H.Y.
Kim
D.J.
Park
J.H.
(
2005
)
Immunoenhancing effects of a new probiotic strain, Lactobacillus fermentum PL9005
.
J Food Prot
 
68
:
571
576
.
Pasetti
M.F.
Pickett
T.E.
Levine
M.M.
Sztein
M.B.
(
2000
)
A comparison of immunogenicity and in vivo distribution of Salmonella enterica serovar Typhi and Typhimurium live vector vaccines delivered by mucosal routes in the murine model
.
Vaccine
 
18
:
3208
3213
.
Pickett
T.E.
Pasetti
M.F.
Galen
J.E.
Sztein
M.B.
Levine
M.M.
(
2000
)
In vivo characterization of the murine intranasal model for assessing the immunogenicity of attenuated Salmonella enterica serovar Typhi strains as live mucosal vaccines and as live vectors
.
Infect Immun
 
68
:
205
213
.
Powell
C.J.
Jr
DeSett
C.R.
Lowenthal
J.P.
Berman
S.
(
1980
)
The effect of adding iron to mucin on the enhancement of virulence for mice of Salmonella typhi strain TY 2
.
J Biol Stand
 
8
:
79
85
.
Robbe-Saule
V.
Coynault
C.
Norel
F.
(
1995
)
The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses
.
FEMS Microbiol Lett
 
126
:
171
176
.
Skorupski
K.
Taylor
R.K.
(
1996
)
Positive selection vectors for allelic exchange
.
Gene
 
169
:
47
52
.
Spellberg
B.
Edwards
J.E.
Jr
(
2001
)
Type 1/Type 2 immunity in infectious diseases
.
Clin Infect Dis
 
32
:
76
102
.
Tsolis
R.M.
Baumler
A.J.
Heffron
F.
(
1995
)
Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774 macrophages
.
Infect Immun
 
63
:
1739
1744
.
Tu
X.
Latifi
T.
Bougdour
A.
Gottesman
S.
Groisman
E.A.
(
2006
)
The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica
.
Proc Natl Acad Sci USA
 
103
:
13503
13508
.
Wang
J.Y.
Pasetti
M.F.
Noriega
F.R.
Anderson
R.J.
Wasserman
S.S.
Galen
J.E.
Sztein
M.B.
Levine
M.M.
(
2001
)
Construction, genotypic and phenotypic characterization, and immunogenicity of attenuated DeltaguaBA Salmonella enterica serovar Typhi strain CVD 915
.
Infect Immun
 
69
:
4734
4741
.

Author notes

Editor: Johannes Kusters