Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium

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 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) 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.

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 × 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.

FIG. 2.

Effects of flagella on the virulence of serovar Typhimurium. Mice were orally inoculated with 5 × 10⁸ CFU of serovar Typhimurium χ3306, 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 (χ3306 versus CS2055, CS2061, or CS2085; and CS2007 versus CS2056, CS2062, or CS2086).

FIG. 3.

Effects of filamentous structure of bacterial cells on the virulence of serovar Typhimurium. Mice were orally inoculated with 5 × 10⁸ CFU of serovar Typhimurium χ3306, 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 χ3306.

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 × 10⁸ CFU. Mice were harvested at weeks 1, 2, 3, 4, 8, and 12 after inoculation with 5 × 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.

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).

FIG. 6.

Protection against the virulent serovar Typhimurium strain. Mice were orally immunized with 5 × 10⁸ CFU of serovar Typhimurium CS2007 or CS2022. Four weeks later, the mice were challenged orally with 5 × 10⁸ CFU of serovar Typhimurium χ3456. After an additional 5 days, recoveries of two strains (χ3456 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 (χ3456) 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.

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).

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.

FIG. 9.

Expression of IL-4, IFN-γ, 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.