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Infection and Immunity, January 2003, p. 30-39, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.30-39.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Oral Immunization with ATP-Dependent Protease-Deficient Mutants Protects Mice against Subsequent Oral Challenge with Virulent Salmonella enterica Serovar Typhimurium

Hidenori Matsui,1,2* Masato Suzuki,3 Yasunori Isshiki,2,{dagger} Chie Kodama,1 Masahiro Eguchi,1,2 Yuji Kikuchi,1,2 Kenji Motokawa,4 Akiko Takaya,3 Toshifumi Tomoyasu,3 and Tomoko Yamamoto3

Laboratory of Immunoregulation, Department of Infection Control and Immunology, Kitasato Institute for Life Sciences, Kitasato University, Minato-ku, Tokyo 108-8641,1 Center for Basic Research, The Kitasato Institute, Minato-ku, Tokyo 108-8642,2 Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522,3 Division of Research and Development, Research Center for Biologicals, The Kitasato Institute, Kitamoto, Saitama 364-0026, Japan4

Received 29 April 2002/ Returned for modification 15 July 2002/ Accepted 15 October 2002


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ABSTRACT
 
We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 108 CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 103 to 104 CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.


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INTRODUCTION
 
In a mouse infection model, Salmonella enterica serovar Typhimurium can reside and proliferate within phagocytes in deeper tissues (35, 47) and it can follow up to induce macrophage death by a possible necrosis (19). This model has been studied as a model of typhoid fever infection in humans caused by S. enterica serovars Typhi and Paratyphi that remains a significant problem in developing countries (46). Three vaccines against typhoid fever are presently in use. Two are injected, and one is orally administered. The two injected vaccines consist of killed whole bacteria and capsular material (Vi antigen) (51). The one live oral vaccine strain, serovar Typhi Ty21a, is an attenuated strain of mutations in galE (a gene in the galactose utilization pathway that affects O-antigen production) (10) and rpoS ({sigma}S) (48, 49). Generally, an oral vaccine is cheaper, easier, and safer to administer than an injected vaccine. In human field trials, the oral vaccination with serovar Typhi Ty21a proved to be safe and effective, but its efficacy varied considerably from trial to trial (9, 51). To develop more promising vaccine strains, researchers have carried out human trials to test various serovar Typhi vaccine candidate strains that harbor precise mutations in specific genes (29). The following four candidate strains show promise for use in serovar Typhi live oral vaccines: CVD 908-htrA (mutations in aroC-aroD [aromatic amino acid biosynthesis] and htrA [a stress-regulated periplasmic protease]), Ty800 (mutations in phoP-phoQ), CVD 909 (CVD 908 with constitutively expressed Vi polysaccharide antigen), and {chi}4073 (mutations in cya-crp and cdt [a gene involved in dissemination of Salmonella from gut-associated lymphoid tissue to deep organs]) (29).

In the mouse model, at least three distinct genetic loci (ity, lps, and xid) affect the ability of the animal to successfully resist systemic infections by serovar Typhimurium. The itys (designating susceptible response) allele of BALB/c or C57BL/6 mice and the lpsd (designating defective response) allele of C3H/HeJ mice are associated with increased susceptibility to infection (30, 62) and decreased susceptibility to endotoxin lethality (44), respectively. The ity phenotype is linked to Nramp1 (61), which encodes a macrophage-specific phosphoglycoprotein that is required by the phagosomal membrane during phagocytosis (15, 61). The increased susceptibility to infection is associated with a nonconservative amino acid substitution at position 169 (glycine to aspartic acid, Nramp1Asp169) (61). In addition, the lack of the xid genotype of CBA/N mice is associated with decreased antibody production and increased susceptibility to infection (45). We have reported that gamma interferon (IFN-{gamma}) or tumor necrosis factor alpha (TNF-{alpha}) depletion with a monoclonal antibody is found to sensitize mice to infection (19). Moreover, T and B lymphocytes have no detectable role in suppressing or enabling systemic infection by serovar Typhimurium within 5 days after oral inoculation (20). The confocal laser scanning microscopy analysis of immunostained sections has revealed that the host cells associated with the serovar Typhimurium infection are macrophages in the liver (47) and spleen (35). In contrast, it has also been reported that a number of different mutations introduce the attenuation of virulence into serovar Typhimurium, such as aroA (aromatic amino acid biosynthesis) (23), ompR (a positive regulator of the expression of tripeptide permease and outer membrane protein genes) (8), purA (adenylosuccinate synthetase) (37), rpoE ({sigma}E) (25), rpoS (11), slyA (a gene required for survival within macrophages) (4), htrB (a gene involved in the acylation of the lipid A portion of the lipopolysaccharide) (56), dam (DNA adenine methylase) (22), and hns (a bacterial DNA binding protein) (21), not to mention cya-crp (7), htrA (5), and phoP (13). The aroA and crp/cya mutants were equally attenuated and produced vigorous mucosal, humoral, and cellular immune responses on oral inoculation (53). The phoP mutant failed to grow or persist and was not immunosuppressive, but it gave 100% protection against challenge with wild-type serovar Typhimurium (10).

It was recently shown that the stress-induced protease ClpP was required for virulence of the facultative intracellular pathogen Listeria monocytogenes. Following high infectious doses of the mutant with a Clp deletion, a sufficient amount of listeriolysin O was produced to induce specific immunity against L. monocytogenes (12). In Escherichia coli, ClpP and ClpX have been demonstrated to be involved in a function of proteolytic activity and an ATPase activity, respectively. In serovar Typhimurium, the ATP-dependent protease ClpXP had a role in the regulation of flagellum synthesis (58). The mutations with a deletion of ClpXP exhibited overproduction of the flagellar proteins (58) and could persist in BALB/c mice for long periods of time without causing an overwhelming systemic infection (64). Lon is an ATP-dependent protease that is known to be a major contributor to proteolysis in E. coli. The mutant of serovar Typhimurium with a disruption of Lon was able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified Salmonella pathogenicity island 1 proteins, SipA, SipC, and SipD (57). Remarkably little is known about how the ClpXP- or Lon-deficient mutant of serovar Typhimurium lost its virulence in mice. However, we found that the serovar Typhimurium ClpXP- or Lon-deficient mutant was immunogenic for mice and that immunization of mice with each strain induced protection against challenge by the virulent strain. To the best of our knowledge, this is the first paper to establish that a single oral immunization of the ClpXP- or Lon-deficient serovar Typhimurium strongly elicits a protective immunity against Salmonella infection.


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MATERIALS AND METHODS
 
Bacterial strains, media, and growth conditions. The relevant bacterial strains used are listed in Table 1. To construct the fliA, fliC, fljB, and flhD disruption mutants, P22 phages were propagated on KK1108 (26), KK1110 (26), KK2504 (serovar Typhimurium LT2 fljB::Tn10), and KK1107 (26) media to transduce fliA::MudI, fliC::MudI, fljB::Tn10, and flhD::MudI, respectively. The lysates were used for infection of the serovar Typhimurium SR-11 {chi}3306 and CS2007, and the transducts were selected by tetracycline (Tet) or ampicillin (Amp) resistance. Bacteria were grown at 37°C in Luria-Bertani (LB) broth or LB agar (Difco Laboratories, Detroit, Mich.) (34). When necessary, the antibiotics were supplemented with nalidixic acid (Nal; 25 µg/ml), chloramphenicol (Chl; 30 µg/ml), Tet (15 µg/ml), kanamycin (Kan; 40 µg/ml), and Amp (100 µg/ml).


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  		TABLE 1. Serovar Typhimurium SR-11 strains

Immunization and subsequent challenge of virulent serovar Typhimurium. Five-week-old female BALB/c mice (Charles River Japan, Yokohama, Japan), which are sensitive to infection by serovar Typhimurium because of the itys mutation (55), were orally inoculated with 5 x 108 CFU of exponential-growth-phase salmonellae concentrated in 60-µl (CS2022) or 20-µl (other strains) doses mixed with phosphate-buffered saline (PBS), pH 7.4, containing 0.01% (wt/vol) gelatin, as described previously (18, 36). The inoculated mice were harvested, and the spleen, mesenteric lymph nodes, Peyer's patches, and cecum were homogenized, diluted in PBS containing 0.01% (wt/vol) gelatin, and plated to facilitate the enumeration of CFU.

To determine the salmonella clearance, immune and naive (unimmunized) mice were orally infected with {chi}3456 in large doses of 5 x 108 CFU. Recovery (number of CFU) of infected bacteria colonized in the spleen, mesenteric lymph nodes, Peyer's patches, or cecum was determined 5 days later. In the same tissue sample, mixed bacteria were distinguished as belonging to the avirulent strain (CS2007 or CS2022) or the virulent strain ({chi}3456) on LB agar plates containing Nal or Tet.

Immunological sampling. For assays of serum and mucosal antibodies, mouse samples were prepared according to the method described by Ryan et al. (50). Briefly, serum was prepared from blood by centrifugation. Bile was collected from the gallbladder. Intestines were washed with 3 ml of solution A (0.1 mg of soybean trypsin inhibitor [Sigma, St. Louis, Mo.]/ml, freshly prepared 2 mM phenylmethylsulfonyl fluoride [Sigma], 50 mM EDTA [Wako, Osaka, Japan], and 0.1% bovine serum albumin [BSA, Fraction V; Sigma] in PBS), and the supernatant was pooled after centrifugation for 15 min at 12,000 x g. The prepared samples of serum, bile, and intestinal washes were divided into aliquots and stored at -70°C for subsequent analysis.

Preparation of FliC and FljB proteins. Strains CS2031 and CS2034 were used for the preparation of FliB and FliC proteins, respectively. The bacterial cells were removed by centrifugation from the overnight shaking culture of each strain in LB broth (3 liters each) at 37°C. The supernatant containing the secreted protein components was filtrated (0.22-µm pore size; Millipore, Bedford, Mass.) and precipitated by 50% (wt/wt) ammonium sulfate. The precipitants were suspended in 80 ml of solution B (20 mM Tris-HCl, 5 mM MgSO4, pH 8.0), desalted by gel filtration using Bio-GelR A-5m (Bio-Rad, Hercules, Calif.) equilibrated with solution C (10 mM KPO4, 150 mM NaCl, and 5 mM MgSO4, pH 7.0), and reprecipitated by ultracentrifugation (105,000 x g for 1 h). To prepare the monomeric flagella, we dissolved the obtained pellets in solution C and added the mixture to 0.1 M HCl (50 µl/ml of suspension). The resulting mixture stood for 30 min at room temperature, and we then adjusted the pH of the solution to 8.5 by adding 0.1 M NaCO3 (1). The flagellar proteins were obtained in the supernatant fraction after ultracentrifugation (105,000 x g for 1 h) and were stored at -70°C until use.

Quantitation of IgG and IgA antibodies. We used the enzyme-linked immunosorbent assay (ELISA) to detect antibodies to lipopolysaccharide (LPS) according to the method described by Ryan et al. (50), as follows: each well of 96-well immune plates (no. 475094; Nalge Nunc, Rochester, N.Y.) was coated with 100 µl of antigen solution (2 µg of smooth Westphal LPS from serovar Typhimurium [Sigma] or 0.2 µg of purified FliC or FljB in 0.175 M borate-buffered saline [BBS], pH 8.3). After overnight incubation at 4°C, plate contents were incubated with 100 µl of PBS-T (PBS containing 0.05% Tween 20 [Sigma]) including 1% BSA (Fraction V; Sigma). After 20 min at 37°C, plates were washed with PBS-T. We added 100 µl of serial threefold dilution of each mouse sample in 1% BSA in PBS-T to individual wells. The initial dilution ratio of each mouse sample was previously determined (1:10 to 1:104). After incubation for 1 h at 37°C, plates were washed and 100 µl of goat anti-mouse horseradish peroxidase (HRPO) conjugate immunoglobulin G (IgG) heavy- and light-chain antibody; goat anti-mouse HRPO conjugate IgG1, IgG2a, IgG2b, or IgG3 heavy-chain specific antibody; or goat anti-mouse HRPO conjugate IgA alpha-chain specific antibody (Bethyl, Montgomery, Tex.) diluted in PBS-T was added to each well. The optimum dilution ratio of each second antibody was previously determined (1:10,000 or 1:2,000 for IgG or IgA, respectively). After 45 min of incubation at 37°C, plates were washed with PBS-T, and 100 µl of tetramethylbenzidine peroxidase enzyme immunoassay substrate solution (Bio-Rad) was prepared and added according to the manufacturer's instructions. After 20 min at 37°C, 50 µl of 2 M H2SO4 was added to each well and plates were read at 450 nm in a kinetic microplate reader (Molecular Devices, Sunnyvale, Calif.). ELISA to detect total IgA was carried out according to the above protocol except that each well of the plates was coated with 100 µl of BBS containing 0.1 µg of goat anti-mouse IgA alpha chain (Bethyl). To compute the antibody concentration using obtained data, we generated a calibration curve using different dilutions of mouse IgG or IgA standard (chromatographically purified mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgA [Zymed, South San Francisco, Calif.]; starting dilution, 1.0 µg/ml) in every immune plate. Each well of the plates was previously coated with 100 µl of BBS containing 0.1 µg of affinity-purified goat anti-mouse IgG heavy and light chain or affinity-purified rabbit anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (Zymed).

Quantitative RT-PCR. Five spleens from mice in the same experimental group were combined, and then total RNA was isolated from the spleens with ISOGEN (Wako, Osaka, Japan). Reverse transcriptase PCR (RT-PCR) was carried out using the one-step RNA PCR kit (avian myeloblastosis virus) (Takara, Otsu, Japan) according to the manufacturer's instructions with IFN-{gamma} (5'-TACTGCCACGGCACAGTCATTGAA-3', 5'-GCAGCGACTCCTTTTCCGCTTCCT-3')-, interleukin-4 (IL-4) (5'-ACGGAGATGGATGTGCCAAACGTC-3', 5'-CGAGTAATCCATTTGCATGATGC-3')-, inducible nitric oxide synthase (iNOS) (5'-TTTCTCTTCAAAGTCAAATCCTACCA-3', 5'-TGTGTCTGCAGATGTGCTGAAAC-3')-, and hypoxanthine phosphoribosyltransferase (5'-GTAATGATCAGTCAACGGGGGAC-3', 5'-CCAGCAAGCTTGCAACCTTAACCA-3')-specific primers. The amount of amplified cDNA was determined by agarose gel electrophoresis. All cytokine and iNOS values were normalized to corresponding hypoxanthine phosphoribosyltransferase values by using NIH Image version 1.6.1.

Statistics. Significant differences between the means plus or minus standard deviations (SD) of different groups were examined using a two-tailed Student's t test. A P of <0.05 was regarded as statistically significant.


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RESULTS
 
Phenotype of the serovar Typhimurium ClpXP- or Lon-deficient mutant. It was recently demonstrated that the ATP-dependent ClpXP protease of serovar Typhimurium regulates flagellum synthesis. Bacterial cells with the clpP gene deleted showed a "hyperflagellate" phenotype (58). On the other hand, in E. coli, bacterial cells of a Lon-deficient mutant strain caused an appearance of filamentous shape, since the Lon protease had an ability to degrade the SulA protein, which was an inhibitor of cell division (24, 43, 54). In addition, Lon protease could also degrade RcsA protein, which was a positive regulator of the transcription of cps genes involved in colanic acid synthesis. Therefore, the stabilization of RcsA resulted in the overproduction of capsular polysaccharide and the formation of a wet glistening (mucoid) colony (59). CS2022 (the Lon-deficient mutant strain of serovar Typhimurium) also exhibited the long-filament phenotype, but in this case the colonies were not the mucoidal phenotype (data not shown). The bacterial cells of CS2022, with volumes up to 50-fold those of {chi}3306 (the virulent strain) or CS2007 (the ClpXP-deficient mutant strain of serovar Typhimurium) calculated by a ratio of the optical density at 600 nm to the number of CFU, had growth rates equal to each other's under the culturing condition of being shaken at 37°C in LB broth. The doubling time of each strain was 24 min (data not shown).

Virulence of the serovar Typhimurium ClpXP- or Lon-deficient mutant in BALB/c mice. We previously reported that CS2007 continuously resided in the spleen or liver of BALB/c mice, with more than 104 CFU monitored for up to 35 days after a single intraperitoneal administration with 102 CFU of salmonellae (64). In the present study, young (5-week-old) mice were orally inoculated with 5 x 108 CFU of salmonellae. CS2007 or CS2022 was found in the spleen at week 1 in amounts of about 1.6 x 104 CFU or 3.7 x 103 CFU, respectively. After week 1, the number of bacteria decreased by stages in every tissue sample. Both mutant strains were cleared from the spleen by week 12 after oral inoculation; however, about 103 CFU of bacteria was still recovered from the cecum at week 12 after oral inoculation (Fig. 1). A 50% lethal dose (LD50) in female BALB/c mice for CS2007 or CS2022 in the oral inoculation could not be estimated because mice had not died after oral inoculation with the maximum dose (109 CFU) of either mutant strain. In contrast, an LD50 for the parent strain {chi}3306 (3 x 105 CFU) under the same conditions was determined by Gulig and Curtiss (17).



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  		FIG. 1. Colonization of serovar Typhimurium CS2007 and CS2022 in BALB/c mice. At weeks 1, 2, 3, 4, 8, and 12 after a single oral inoculation of bacteria with a dose of 5 x 108 CFU, recoveries of salmonellae from the spleen, mesenteric lymph nodes (MLN), Peyer's patches (PP), and cecum were measured. Results were combined from two experiments with five mice in each group. Data represent the means ± SD (n = 10). ND, not detected.

If a hyperflagellation or filamentous structure of bacterial cells directly leads to the attenuation of virulence of CS2007 or CS2022, we considered the possibility that CS2007 or CS2022 might restore virulence spontaneously during persistent infection by the elimination or suppression of the synthesis of hyperflagellation or filamentous structure of bacterial cells.

The flagellar operons are divided into three classes with respect to their transcription hierarchy (27). Class 3 contains three operons, including a filament formation. Salmonella has two genes (fliC and fljB), at different locations on the chromosome, that code for the antigenically distinct flagellar types H1 (phase 1) (27) and H2 (phase 2 [FljB]) (33). The expression of the class 3 operons requires the class 3 operon-specific sigma factor FliA. The fliA gene is included in class 2, and it positively regulated expression by activator proteins, FlhD and FlhC, which are encoded by the flhD class 1 operon lying at the top of the transcription hierarchy (28, 31). We constructed each class-specific flagellum-defective mutant strain with or without the ClpXP-deficient background, as shown in Table 1. We inoculated them into mice orally in order to study the effect of flagella on the virulence of salmonellae. As shown in Fig. 2, the virulence levels of splenic infection among flagellum-defective mutant strains (CS2055, CS2061, and CS2085), which were constructed from {chi}3306, were much the same. CS2055, CS2061, and CS2085 were still virulent strains, since there were no discrepancies in the numbers of splenic CFU between mutant strains (CS2055, CS2061, and CS2085) and {chi}3306-inoculated mice. Similarly, the virulence levels of splenic infection were the same among the other flagellar mutant strains with the ClpXP-deficient background (CS2056, CS2062, and CS2086), and it appeared that there were also no discrepancies in the numbers of splenic CFU between CS2056-, CS2062-, or CS2086- and CS2007-inoculated mice. Therefore, when mice are orally inoculated, the flagellar structures do not affect the virulence of CS2007 that enables splenic infection.



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  		FIG. 2. Effects of flagella on the virulence of serovar Typhimurium. Mice were orally inoculated with 5 x 108 CFU of serovar Typhimurium {chi}3306, CS2007, CS2055, CS2056, CS2061, CS2064, CS2085, or CS2086. Five days later, recoveries of salmonellae from the spleen were measured. Data represent the means ± SD (n = 5 to 9). P > 0.5 ({chi}3306 versus CS2055, CS2061, or CS2085; and CS2007 versus CS2056, CS2062, or CS2086).

Recalling Fig. 1, we noted that there was a certain number of salmonellae of each vaccine strain from every tissue except for the spleen at week 12 after oral inoculation. Therefore, we continued to monitor the colonization level of CS2007 or CS2022 in every tissue for another 12 weeks. In 9 out of 10 mice we found that no bacterium originated from CS2007 or CS2022 in any tissue by week 24 after oral inoculation. The exception was one mouse inoculated with CS2022 in which we found 40 CFU in only the cecum. We chose eight colonies of recovered CS2022 and compared the properties of salmonellae in each colony with regard to the bacterial cell shape, UV sensitivity, growth rate in LB medium at 37 and 28 or 43°C, and ratio of the optical density to the number of CFU with those of CS2022 or {chi}3306. We eventually selected two colonies whose salmonellae still had the lon-deficient genotype and found that their properties were close to those of {chi}3306 but not those of CS2022 (data not shown). Therefore, we concluded that at least a quarter of the recovered CS2022 from the cecum showed the normal cell division. We designated this as revertant CS2022 (CS2022R), and to examine whether CS2022R was the virulent strain or not, we inoculated mice orally with 5 x 108 CFU of {chi}3306 (virulent strain), CS2022, or CS2022R. As shown in Fig. 3, we measured the recovery of bacterial cells in the spleen, mesenteric lymph nodes, and Peyer's patches of mice 5 days after oral inoculation. As expected, CS2022 was greatly attenuated for splenic infection (log10 CFU/spleen: 3.06 ± 0.51) compared with {chi}3306 (log10 CFU/spleen: 6.24 ± 0.50, P = 0.001), whereas CS2022R was still attenuated for splenic infection (log10 CFU/spleen: 4.04 ± 0.84) compared with {chi}3306 (P = 0.015). CS2022R appeared to be more capable of splenic infection than CS2022, but the difference was not significant (P = 0.1). The recovery of CS2022R from mesenteric lymph nodes or Peyer's patches seemed to be less effective than that of CS2022. These data indicate that CS2022 did not restore virulence during persistent infection within the mouse.



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  		FIG. 3. Effects of filamentous structure of bacterial cells on the virulence of serovar Typhimurium. Mice were orally inoculated with 5 x 108 CFU of serovar Typhimurium {chi}3306, CS2022, or CS2022R. Five days later, recoveries of salmonellae from the spleen, mesenteric lymph nodes (MLN), and Peyer's patches (PP) were measured. Data represent the means ± SD (n = 5). *, P = 0.001; **, P < 0.015; ***, P = 0.15; #, P = 0.015, ##, P = 0.02; and ###, P < 0.05 compared with each tissue of {chi}3306.

Our test results, taken together, indicate that the serovar Typhimurium ClpXP- or Lon-deficient mutant was an avirulent strain; however, the hyperflagellation or filamentous phenotype of the mutation did not attenuate the virulence of salmonellae.

Immune responses induced by oral immunization with the serovar Typhimurium ClpXP- or Lon-deficient strain. We used ELISA to detect the presence of antibodies in various fluids of mice. Through the use of calibration curves with mouse IgG or mouse myeloma IgA as the standard antibody, this method allowed us to measure the amount of serovar Typhimurium LPS-specific IgG and secretory IgA (S-IgA) antibodies or total S-IgA antibodies in mouse samples. As shown in Fig. 4, a certain increase in the serovar Typhimurium LPS-specific IgG and S-IgA antibodies was observed at week 4 after a single oral inoculation of CS2007 or CS2022 in the serum or bile, respectively. Serum or bile samples were subsequently taken at weeks 8 and 12 after oral inoculation. Although a certain quantity of antibodies was continuously detected from each sample for up to 12 weeks after inoculation, it seemed that the increases in antibodies from both tissue samples of CS2022-inoculated mice were larger than those of CS2007-inoculated mice at each time point after inoculation, although there were no significant differences between the CS2007- and CS2022-inoculated mice at most time points. In addition, we precisely analyzed the antibody response at week 4. A certain amount of S-IgA was detected in intestinal washes following both CS2007 and CS2022 immunization, with no significant difference between them (Fig. 5). CS2007 exhibits overproduction of the flagellar protein and shows a fourfold increase in the rate of transcription of the fliC gene encoding the flagellar filament (58). Therefore, the production of flagellar protein-specific antibodies might be induced by the CS2007 immunization. However, there was no difference in the induced amount of either anti-FliC or anti-FljB IgG between the CS2007 and CS2022 immunizations (data not shown).



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  		FIG. 4. Changes in the amount of serovar Typhimurium LPS-specific IgG in serum and local S-IgA in bile after a single oral inoculation of serovar Typhimurium CS2007 or CS2022 at a dose of 5 x 108 CFU. Mice were harvested at weeks 1, 2, 3, 4, 8, and 12 after inoculation with 5 x 108 CFU, and an ELISA was carried out to detect the serovar Typhimurium LPS-specific IgG in micrograms per milliliter of serum (A) and S-IgA in micrograms per gallbladder (B). Shown are the combined results from three experiments. Data represent the means ± SD (n = 5 to 15). *, P = 0.8.



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  		FIG. 5. Intestinal S-IgA responses to the serovar Typhimurium LPS at week 4 after oral immunization. LPS-specific S-IgA contents are given in micrograms per intestine (A) or in the percentage of total S-IgA (B). Data represent the means ± SD (n = 5).

The serovar Typhimurium ClpXP- or Lon-deficient strain induces a significant protective immune response against the virulent strain. The immune or naive (unimmunized) mice were orally challenged or infected with 5 x 108 CFU of {chi}3456 (the virulent strain) at week 4 after immunization. This challenge dose was greater than 1,500 LD50s. All naive and/or infected mice died by 7 days postinfection. In contrast, immune and/or challenged mice with CS2007 or CS2022 did not become sick, and all these mice survived for 2 weeks after the challenge. Therefore, we analyzed the number of bacterial cells in each type of tissue 5 days after the challenge. As shown in Fig. 6, neither {chi}3456 nor CS2007 or CS2022 was detected in the spleen, mesenteric lymph nodes, or Peyer's patches of mice 5 days after the challenge. It is noteworthy that the salmonellae were completely eliminated from the cecum of the CS2007- or CS2022-immunized mice. This result suggests that a single oral immunization of CS2007 or CS2022 should be effective in protecting against the colonization of wild-type serovar Typhimurium within the intestinal tract.



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  		FIG. 6. Protection against the virulent serovar Typhimurium strain. Mice were orally immunized with 5 x 108 CFU of serovar Typhimurium CS2007 or CS2022. Four weeks later, the mice were challenged orally with 5 x 108 CFU of serovar Typhimurium {chi}3456. After an additional 5 days, recoveries of two strains ({chi}3456 and CS2007 or CS2022) were distinguishably measured from the spleen, mesenteric lymph nodes (MLN), Peyer's patches (PP), and cecum of challenged immune mice. Naive (unimmunized) mice were also infected with the virulent strain ({chi}3456) as controls. Shown are the combined results from two experiments with five mice from each group. Data represent the means ± SD (n = 20 [naive mice] or 10 [immune mice]). ND, not detected.

Challenge serovar Typhimurium may boost protective immunity. The above results showed that oral vaccination with CS2007 or CS2022 induced protective immunity against Salmonella infection in mice. We considered both vaccine strains to have induced sufficient amounts of S-IgA on the mucosal surfaces to protect against the colonization of wild-type serovar Typhimurium after a single oral inoculation. We found it interesting that CS2007 or CS2022 persistently resided in each tissue before the challenge (Fig. 1) but that each vaccine strain was completely cleared from each sampled tissue of mouse within 5 days after the challenge. To examine how salmonellae of vaccine strains were cleared in immune mice after the challenge, we compared the antibody amount with and without the challenge. We hypothesized that the clearance of CS2007 or CS2022 after the challenge was the result of the stimulated humoral and/or cell-mediated immunities caused by the challenge. As shown in Fig. 7A, a fivefold boost in the amount of LPS-specific IgG in serum was found in the CS2007- or CS2022-immunized mice 5 days after the challenge. In contrast, significant increases in the amount of LPS-specific S-IgA in bile did not occur in either CS2007- or CS2022-immunized mice (Fig. 7B). To demonstrate the consequence of introducing Th1 and Th2 cells on serum antibody responses, we measured the Salmonella LPS-specific IgG subclass responses. In mice, IFN-{gamma} (Th1-type cytokine) or IL-4 (Th2-type cytokine) provides essential signals for antibody isotype switching to IgG2a and IgG3 or to IgE and IgG1 (6). As shown in Fig. 8, the amount of IgG2a included the majority of IgG subclasses in serum with or without the challenge of CS2007- or CS2022-immunized mice. In contrast, IgG1 was not elicited by an immunization with CS2007 or CS2022 and the subsequent challenge with the virulent strain. In addition, we employed a quantitative RT-PCR to assess levels of the expression of cytokine and iNOS genes in the spleen homogenates of mice that were naive (uninoculated), vaccinated without challenge, or vaccinated with challenge (Fig. 9). It is generally thought that IFN-{gamma} activates macrophages to promote the intracellular killing of microbes by iNOS production. The produced iNOS leads to the production of large amounts of nitric oxide, which plays a role in host defense in mice (3). Interestingly, the expression of IFN-{gamma} and iNOS mRNAs was significantly increased by the immunization and strongly stimulated by the consequent challenge. On the other hand, the expression of IL-4 was not activated by the immunization, and it was hardly stimulated by the challenge. These results suggest that both Th1-and Th2-type responses were induced by the immunization of every vaccine strain. However, the Th1-type responses might be activated immediately after the challenge of a virulent strain, and this quick Th1-type response might be adequate for the clearance of persistent infection with each vaccine strain in mice.



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  		FIG. 7. Booster immunization by the serovar Typhimurium challenge. Immune mice were orally challenged with the virulent strain of serovar Typhimurium. Mice were harvested 5 days after the challenge, as described in the Fig. 6 legend. An ELISA was carried out to detect the serovar Typhimurium LPS-specific IgG in serum (A) and S-IgA in bile (B). Data represent the means ± SD (n = 5 to 10).



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  		FIG. 8. IgG subclass antibody titers. The serum IgG subclass anti-serovar Typhimurium LPS titers in Fig. 7 are shown. Data represent the means ± SD (n = 5). *, P > 0.2 compared with each IgG2a sample of no-challenge mice.



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  		FIG. 9. Expression of IL-4, IFN-{gamma}, and iNOS mRNAs. Immune mice were orally challenged with the virulent strain of serovar Typhimurium. Mice were harvested 5 days after the challenge, as described in the Fig. 6 legend. Total RNA was prepared from the mixed spleens of mice from the same experimental group. Quantitative RT-PCR was carried out as described in Materials and Methods. The amplified cDNA was detected on the agarose gel electrophoresis (A). All cytokine and iNOS values were normalized to corresponding hypoxanthine phosphoribosyltransferase (HPRT) values (B). ND, not detected.


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DISCUSSION
 
In this study, we demonstrated that serovar Typhimurium with a deletion of the ATP-dependent protease ClpXP or Lon persistently resided in mice after oral infection and that, as the result of this persistence, these mice developed strong protective immunity. Ultimately, immune mice were completely protected against infection with virulent serovar Typhimurium. In vitro, CS2007 (clpP::Chlr) was unable to grow or survive within peritoneal macrophages of BALB/c mice because the bacteria were too sensitive to the intracellular environment compared with the wild-type strain (64). In contrast, CS2022 (lon::Chlr) showed a dramatic increase in the ability of the bacteria to invade Intestine-407 cells, in the secretion of invasion-associated proteins, and in the expression of invasion genes encoded on Salmonella pathogenicity island 1 (57). However, CS2022 could not survive or proliferate within murine macrophages because of the enhanced susceptibility to the oxygen-dependent, killing mechanism-associated respiratory burst and the low phagosomal pH (A. Takaya et al., submitted for publication). In E. coli, little is known about the state of ClpXP or Lon in vivo; however, ClpXP is necessary for the degradation of {lambda}O protein, Phd (the P1 addiction protein), mutant Mu repressors, RpoS, and some proteins tagged by the carboxyl-terminal SsrA system, and the Lon protease can degrade {lambda}N protein, SulA, RcsA, and CcdA (the F factor addiction system protein) (14). SulA is a member of the SOS regulon and can inhibit septation by binding to FtsZ, a key cell division protein (32). We gained the revertant of CS2022 from the cecum of 1 mouse out of 10 mice tested at week 24 after oral vaccination. Two distinct possibilities as to the nature of a revertant of CS2022 (CS2022R) were considered. First, a suppressor may represent a revertant that reduces the sulA expression. Second, a dominant point mutation of sulA or ftsZ may represent a revertant that reduces the binding of SulA and FtsZ. While that was happening, CS2022R as well as CS2022 did not have mucoid colonies, suggesting that the stabilization of RcsA was not induced by the Lon protease disruption or that the stabilized RcsA was not able to act as a positive regulator of colanic acid synthesis in both strains. It thus appears unlikely that the inability to synthesize colanic acid allows for restoration of the virulence of CS2022. Incidentally, in serovar Typhi, a capsular polysaccharide (Vi antigen) synthesis is involved in a Lon-independent manner (63). The point is that an attenuation of virulence of CS2007 or CS2022 was clearly independent of its hyperflagellation or filamentous phenotype, respectively (Fig. 2 and 3). Therefore, even if CS2007 or CS2022 loses the ability to synthesize flagella or gains the ability of normal bacterial cell division in a host, respectively, the revertant strain would not restore the virulence.

It seems that CS2022 maintained the infection in mice longer than did CS2007 (Fig. 1). In other words, it was not easy to clear the bacteria of CS2022, and it was somewhat easier to clear the bacteria of CS2007 in mice. This could be due to the cell volume of CS2022 being approximately 50-fold larger than that of the wild-type strain. Therefore, immunization with CS2022 might result in more serovar Typhimurium LPS-specific IgG production in serum and S-IgA production in bile than would immunization with CS2007 (Fig. 4). However, there was no difference between CS2007 and CS2022 in the amounts of the intestinal washes containing S-IgA (Fig. 5) and the stimulated IgG in serum after the challenge (Fig. 7) in the immune mice. Nevertheless, they both resulted in a complete prevention against orally challenged virulent serovar Typhimurium and colonized CS2007 or CS2022 in every tissue by 5 days after the challenge (Fig. 6). We believe that the S-IgA on the mucosal surfaces induced by vaccination mainly prevented the infection by the virulent strain and that simultaneously the cell-mediated immunity stimulated by challenge mainly eradicated the colonization of a residual vaccine strain in each tissue (Fig. 8 and 9). In other words, both CS2007 and CS2022 could elicit the humoral and possible cell-mediated immunities after a single oral vaccination. We hope to develop an effective vaccine strain that will be effective with a single oral administration. Both CS2007 and CS2022 fully satisfy this requirement. Actually, we have observed that both CS2007- and CS2022-vaccinated mice hold a certain amount of serovar Typhimurium LPS-specific antibodies for longer than 6 months after the immunization (data not shown). Even so, we evaluated the booster effect in the amount of LPS-specific IgG by an additional oral immunization of CS2007 or CS2022 to immune mice at week 4 after the primary immunization. A 5- or 13-fold boost in the amount of LPS-specific IgG in serum was given in the CS2007- or CS2022-immunized mice 5 days after the additional immunization of CS2007 or CS2022, respectively. When we analyzed the number of bacterial cells in each tissue sample with or without the additional inoculation, a small amount of each vaccine strain (<103 CFU) was detected in each sample with or without the additional inoculation. We conclude that it is likely that the clearance of vaccine strains might be slower than that of wild-type bacteria (data not shown).

Salmonella given via the oral route initially attaches to enterocytes and M cells in Peyer's patches on the surface of gut-associated lymphoid tissue and then invades mucosa, prior to colonizing deeper tissues such as the spleen and liver (16). There were two strong rationales for pursuing the strategy of a live oral Salmonella vaccine. One was the perceived desirability of inducing S-IgA, which would presumably block entry of the organisms into the Peyer's patches; the other was the superior protective efficacy of live versus killed vaccines in some mouse strains, with concomitant induction of cell-mediated immunity as evidenced by delayed-hypersensitivity reactions by live but not killed Salmonella (9). Michetti et al. showed that secretion of anti-serovar Typhimurium S-IgA alone was able to block the oral challenge with the wild-type serovar Typhimurium, presumably by immune exclusion at the mucosal surface. It did not protect against the intraperitoneal challenge and did not possess complement-fixing or bactericidal activity in vitro (40). For that matter, the present results demonstrated that providing sufficient amounts of anti-Salmonella S-IgA from mucus in the intestine 4 weeks after a single oral immunization of CS2007 or CS2022 could prevent the spread of bacteria from mucosa while allowing entry into Peyer's patches through M cells.

Recently, Mittrucker et al. analyzed the role of B cells protecting against serovar Typhimurium by using B-cell-deficient Igµ-/- mouse mutants of susceptible background (C57BL/6). Their results showed that B cells were required for efficient protection against oral infection with virulent serovar Typhimurium, indicating that B cells had an essential role in host defense. In contrast, after systemic infection, Igµ-/- mice were cleared of attenuated serovar Typhimurium, but vaccine-induced protection against systemic infection with virulent serovar Typhimurium involved both B-cell-dependent and -independent effector mechanisms (42). In a related study, MacSorley and Jenkins also demonstrated that CD28+ CD4+ T cells were sufficient for the clearance of avirulent serovar Typhimurium in naive mice, whereas CD4+ T cells and specific antibodies were required for protection from virulent serovar Typhimurium in immune mice (39). In contrast, as Mittrucker et al. pointed out, primary immunization with an attenuated strain probably induced Salmonella-specific T cells, activation of macrophages, and other effector mechanisms capable of controlling subsequent infection with virulent serovar Typhimurium in the absence of antibodies (42). Moreover, the response to serovar Typhimurium involves both T- and B-cell-mediated immunity, and mechanisms mediated by both lymphocyte populations are probably important for the control of primary infection and protection against secondary infection (41). In our study, although the anti-serovar Typhimurium LPS IgG was not detected at week 2 (Fig. 1), the oral immunization of CS2007 or CS2022 could partly but not completely protect mice against the challenge with virulent serovar Typhimurium (data not shown). These data suggest that the Salmonella-specific T cells induced by the immunization can protect the host from infection in some degree but that specific antibodies are required for complete protection from virulent serovar Typhimurium. However, we did not examine the T-cell-dependent mechanisms accurately with regard to the induction of protective and sustained humoral responses in this study. We are presently studying these mechanisms using B-cell-deficient mice.

It has been confirmed that IFN-{gamma} and TNF-{alpha} are essential components of the host response for inhibiting systemic infection by serovar Typhimurium in the early phase of disease, before specific immunity has been stimulated in mice (19). There is a delicate balance between virulence/attenuation and inflammation within the host. VanCott et al. hypothesized that T cells were essential for distinguishing the immune mechanisms elicited by the two mutant strains (serovar Typhimurium with a disruption of PhoP or AroA) in vivo. Interestingly, in C57BL/6 (itys) mice, IFN-{gamma} was not required for clearance of a phoP deletion mutant strain of serovar Typhimurium, whereas alteration of the IFN-{gamma}-dependent clearance was required for infection with the wild-type strain. Oral administration of the phoP-deficient serovar Typhimurium promoted potent innate immune responses of macrophages in Peyer's patches and spleens with high levels of reactive nitrogen and oxygen intermediates. In contrast, administration of an aroA mutant strain elicited stronger specific antibody and Th-cell responses, wherein Th1-type cells were required for clearance (60). In our experiments, we observed significant increases of the serovar Typhimurium LPS-specific IgG or S-IgA 2 weeks after the three-time oral vaccination of the mutant strain with a disruption of phoP or aroA at 10-day intervals in serum or bile, cecal homogenate, and lung washes, but the serovar Typhimurium LPS-specific S-IgA was not detected in intestinal washes. However, we did not observe the serovar Typhimurium LPS-specific antibodies in any tissue sample after a single oral vaccination of the mutant strain with a disruption of phoP or aroA (unpublished data). In the present study, CS2007 and CS2022 could elicit sufficient amounts of antibodies to protect mice against serovar Typhimurium after a single oral vaccination.

Brown and Hormaeche studied the antibody response to salmonellae in mice and humans using immunoblots and ELISA. The data indicated that sera from mice infected intravenously with an aroA mutant strain of serovar Typhimurium recognized up to 45 different bands on immunoblots at the height of the response, including outer membrane proteins, a putative heat shock protein, flagella, and lipoprotein (2). Interestingly, immunization with the flagellar filament protein, FliC, showed the protection of naive C57BL/6 mice against oral challenge with lethal serovar Typhimurium, since FliC was the major target of CD4+ T cells during primary and secondary infection (38). On the contrary, a recent report showed that Salmonella flagellin did not efficiently activate systemic or secreted antibody responses after oral or nasal administration to mice (52). In our study, the amount of serum flagellin (FliC or FljB)-specific IgG elicited by the immunization of CS2007 (exhibiting hyperflagellation) was small and equal to that elicited by the immunization with CS2022. However, the ratios of anti-FliC or anti-FljB IgG to anti-LPS IgG were different for the two cases (data not shown). These findings support the possibility that CS2007 exhibited overproduction of the flagellar proteins in mice and consequently that the flagellar proteins produced worked as protective antigens. Similarly, the mechanisms by which B cells and antibodies contribute to protection against serovar Typhimurium remain unclear.

The ideal vaccine strain should be avirulent for immune hosts. After that, strong protection against pathogens is an additional requirement. Serovar Typhimurium CS2007 and CS2022 can completely satisfy the latter point and can satisfy the former point in part. ClpXP- or Lon-deficient Salmonella gives rise to unique and potentially excellent chances to develop a live vaccine strain of Salmonella. It remains important to continue to identify the role of stress proteins in the pathogenesis of intracellular bacteria.


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ACKNOWLEDGMENTS
 
This work was supported in part by grants-in-aid for scientific research 13670288, 13670327, and 13470058 to H.M., Y.K., and T.Y., respectively, from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Immunoregulation, Department of Infection Control and Immunology, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Phone and fax: 81-3-5791-6267.E-mail: hmatsui{at}lisci.kitasato-u.ac.jp. Back

Editor: B. B. Finlay

{dagger} Present address: Department of Microbiology, School of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan. Back


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Infection and Immunity, January 2003, p. 30-39, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.30-39.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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