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Infection and Immunity, July 2004, p. 4297-4301, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4297-4301.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Oral Immunization with an rfaH Mutant Elicits Protection against Salmonellosis in Mice

Gábor Nagy,¹ Ulrich Dobrindt,² Jörg Hacker,² and Levente Emödy^1*

Institute of Medical Microbiology and Immunology, University of Pécs, 7624 Pécs, Hungary,¹ Institut für Molekulare Infektionsbiologie, Universität Würzburg, 97070 Würzburg, Germany²

Received 4 October 2003/ Returned for modification 1 December 2003/ Accepted 23 March 2004

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Loss of the transcriptional antiterminator RfaH results in virulence attenuation (>10⁴-fold increase in 50% lethal dose) of the archetypal Salmonella enterica serovar Typhimurium strain SL1344 by both orogastric and intraperitoneal routes of infection in BALB/c mice. Oral immunization with the mutant efficiently protects mice against a subsequent oral infection with the wild-type strain. Interestingly, in vitro immunoreactivity is not confined to strain SL1344; rather, it is directed also towards other serovars of S. enterica and even Salmonella bongori strains.

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Salmonella enterica has over 2,300 serotypes, which were formerly designated as distinct species but recently, however, have been termed serovariants. Salmonella can cause diseases in humans and in a wide variety of animals. Development of a vaccine against salmonellosis has long been of high concern (13). There have been several vaccination strategies against serovar Typhi; however, none of them is optimal in all aspects. Typhoid fever can be induced experimentally only by oral infection of humans or higher primates. Salmonella enterica serovar Typhimurium infection, however, causes a typhoid fever-like disease in certain mutant strains of mice. Intestinal and extraintestinal lesions in this model highly resemble those observed in humans suffering from typhoid fever; therefore, this model has been widely used to mimic infection caused by Salmonella enterica serovar Typhi in humans (27). Design of several new live attenuated vaccine strains of serotype Typhi was based on experiments using the mouse model of typhoid fever.

Regulatory protein RfaH is a transcriptional antiterminator (1) that reduces the polarity of long operons encoding cell components involved in the virulence of Escherichia coli (2). Originally, RfaH was discovered as a regulator of lipopolysaccharide (LPS) synthesis in S. enterica (17) and E. coli (5). Later RfaH was shown to be essential for the expression of other cell components encoded on long operons in E. coli. RfaH-affected operons include those encoding the F plasmid (26), different capsules (4, 23, 30), and hemin uptake receptor (18), as well as the toxins alpha-hemolysin (3, 16) and cytotoxic necrotizing factor 1 (CNF-1) (15). A global regulatory role of RfaH in the virulence of uropathogenic E. coli has recently been shown (19). As rfaH seems to be conserved among various enterobacteria, its role in the regulation of virulence of various gram-negative pathogens has been suggested.

Bacterial strains and culture conditions. SL1344 is a fully mouse-virulent, invasive strain that has been described previously (8, 12, 32). Inactivation of rfaH in strain SL1344 was performed using the Red recombinase method explained elsewhere (6) with primers S-rfaH-cm1 and S-rfaH-cm2 (S-rfaH-cm1, 5'-ATG CAA TCC TGG TAT TTA CTG TAC TGC AAA CGC GGG CAA CTT CAG CGT GCT CAG GAA CAC CTC GTG TAG GCT GGA GCT GCT T-3'; S-rfaH-cm2, 5'-TTG CGA AAA CCG GTG TTT TTT ACG CTC TGC TTC ACT TCT TTA TTG AGT AAA TTA AGC ATA TGA ATA TCC TCC TTA GTT CCT A-3'; italics indicate sequences complementary to the cat gene). The mutant strain in which rfaH was inactivated through disruption by a cat cassette has been termed SL1344-R1. Complementation of the mutant with rfaH was performed principally as described by Diederich et al. (7). The gene rfaH_SL1344 together with its promoter region was amplified using primer pair rfaH-1 (5'-CAC GCA AAG TGC GGT CAG C-3') and rfaH-rev-X (5'-TTA TCT AGA CGC CGT ATC TGT TGC CTC GCG ATC T-3'; the restriction site for XbaI is indicated in italics) and was introduced into the chromosomal attB site by use of the integrase system (7), giving rise to the trans-complemented strain SL1344-R2. Wild-type Salmonella strains were obtained from Zoltán Péterfi (Department of Medical Microbiology and Immunology, University of Pécs) and from the strain collection of the Institut für Molekulare Infektionsbiologie, University of Würzburg. Bacteria were grown routinely in Luria-Bertani (LB) broth or on LB broth plates. When appropriate, media were supplemented with the following concentrations of antibiotics: 100 µg of ampicillin/ml, 30 µg of chloramphenicol/ml, 20 µg of tetracycline/ml, and 30 µg of kanamycin/ml, respectively.

Loss of RfaH results in virulence attenuation of strain SL1344. Animal experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals in a laboratory accredited by the Hungarian government (decree no. XXVII, 1998) and according to the subsequent regulation (government order no. 243/1998). Six- to eight-week-old female BALB/c mice (Charles River, Budapest, Hungary) were used in all cases. Bacteria grown in LB broth were washed and resuspended in phosphate-buffered saline for the inoculum. Orogastric infections were performed using a sterile gavage without prior neutralization of gastric acid (9). Intraperitoneal injection was carried out by direct puncture through the abdominal wall with a 25-gauge needle (10).

Four groups of five BALB/c mice each were infected orally with 5 x 10² to 5 x 10⁵ CFU of wild-type strain SL1344. In this experimental setup, a 50% lethal dose (LD₅₀) of 2.1 x 10³ CFU was calculated (data not shown) by the method of Reed and Muench (24). To assess virulence attenuation of SL1344-R1, groups of 10 mice (five mice in each of two independent experiments) were infected with 5 x 10⁵ CFU (200 LD₅₀) and groups of five mice were infected with 5 x 10⁷ CFU (2 x 10⁴ LD₅₀) of either the wild-type strain SL1344 or its isogenic rfaH mutant (SL1344-R1). Infectious lethality rates are summarized in Table 1. Loss of RfaH in the mutant strain resulted in abolishment of virulence of strain SL1344 in oral infection of BALB/c mice. trans-complementation of the mutant strain with rfaH (in strain SL1344-R2) completely restored its virulence.

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TABLE 1. Death rates elicited by S. enterica serovar Typhimurium SL1344 and its isogenic rfaH mutant after oral infection

Wild-type strain SL1344 is extremely virulent when administered parenterally to mice. Determination of an accurate LD₅₀ in the case of intraperitoneal infection is not possible, but the dose was reported to be under 13 CFU (28). To investigate whether RfaH plays a role in parenteral virulence as well, groups of four mice were challenged intraperitoneally with 10², 10⁴, and 10⁶ CFU of the rfaH mutant strain SL1344-R1 and, as a control, groups of four mice were infected with 10² CFU of either the wild-type strain or the complemented mutant strain SL1344-R2. Seventy-five percent of mice died between postinfection days 5 and 11 in the group infected with the highest dose (10⁶ CFU) of the mutant strain, while all mice survived in the other two groups. The surviving mice exhibited signs of disease (scruffiness and lethargy) between days 5 and 10, which completely ceased afterwards. On the other hand, all four mice challenged with 10² CFU of the wild-type strain SL1344 or the trans-complemented mutant SL1344-R2 died on days 5 and 6 postinfection. Our study shows for the first time that the presence of an intact rfaH gene is obligatory for full virulence of Salmonella. Inactivation of rfaH in a highly invasive strain severely attenuates mouse virulence in both orogastric and parenteral models of infection.

Vaccination with SL1344-R1 elicits protective immunity. Mice were immunized via the orogastric route with SL1344-R1 (5 x 10⁷ CFU) three times at 2-week intervals. Test blood samples were obtained by retrobulbar puncture with heparinized capillary tubes in the 5th week (days 36 to 37) after the first immunization (day 0). On day 42 (2 weeks after the last booster immunization) groups of four immunized mice were orally challenged with 5 x 10³ CFU (2 LD₅₀) or 5 x 10⁷ CFU (2 x 10⁴ LD₅₀) of wild-type strain SL1344. Postinfection survival rates of vaccinated and naïve mice are compared in Fig. 1A (5 x 10³ CFU) and B (5 x 10⁷ CFU). While 50 and 100% of control mice died after challenge with the wild-type strain, respectively, immunization fully protected mice regardless of the challenge dose. In the case of the higher challenge dose (Fig. 1B), the difference in survival was statistically significant (P = 0.0067) as calculated by the log rank test with GraphPad Prism software.

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FIG. 1. Survival of BALB/c mice immunized with attenuated S. enterica serovar Typhimurium strain SL1344-R1 (see text) following a subsequent challenge with the parental wild-type strain. Survival of immunized mice is represented with dashed lines, while that of control mice is shown with solid lines. Groups of four mice were challenged orally with 5 x 103 (A) or 5 x 107 (B) CFU of wild-type strain SL1344 on day 42 (2 weeks after the last booster immunization). Groups of six mice were challenged orally with 5 x 103 (C) or 5 x 107 (D) CFU of wild-type strain SL1344 on day 84 (8 weeks after the last booster immunization).

In order to prove that protection of mice was due to specific immunity and not to a nonspecific cellular immune response mediated by the presence of the vaccine strain in the host organs, the same experiment with a longer time interval between the last booster immunization and the challenge was repeated. In a pilot study, SL1344-R1 (with a dose corresponding to that used for the vaccination experiments) was shown to be cleared from the organs (liver, spleen, and Peyer's patches) of mice within 2 weeks postinfection (data not shown). Groups of six mice vaccinated as described above were orally challenged with 5 x 10³ CFU (2 LD₅₀) or 5 x 10⁷ CFU (2 x 10⁴ LD₅₀) of wild-type strain SL1344 on day 84 (6 weeks after the vaccine strain had been cleared from the immunized mice). Immunization significantly increased survival rates (depicted in Fig. 1C and D) with both the lower (P = 0.0387) and the higher (P = 0.0024) challenge doses.

Test sera of immunized mice (from three independent vaccination experiments) were collected into three pools: pools 1, 2, and 3 were obtained from sera of 12, 8, and 9 mice, respectively. Control pools were made by mixing sera of corresponding numbers of age-matched naïve mice. Immunoreactivities of sera obtained from immunized mice were tested on different serovars of S. enterica as well as Salmonella bongori strains in vitro. Ninety-six-well plates were coated overnight with 0.1 ml of bacterial suspensions (10⁹ CFU/ml), and immunoreactivities of pooled sera (diluted 1:50 in phosphate-buffered saline containing 0.25% bovine serum albumin) were determined in an enzyme-linked immunosorbent assay (ELISA) by standard protocols. Sera of immunized mice (in comparison to those of naïve mice) exhibited high reactivity to Salmonella strains (Fig. 2). Interestingly, sera of immunized mice showed in vitro immunoreactivity to not only the isogenic virulent strain but also other serovars of S. enterica as well as to S. bongori.

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FIG. 2. Immunoreactivities of pooled sera obtained from control mice (plain bars) and from mice immunized with S. enterica serovar Typhimurium strain SL1344-R1 (striped bars) to different Salmonella strains. Pools were obtained by mixing sera of 12 (pool 1, white background), 8 (pool 2, gray background), and 9 (pool 3, black background) mice. v.; serovariant of S. enterica. Serotypes of S. enterica strains are shown in parentheses. OD600, optical density at 600 nm.

Isotype determination of the Salmonella-specific antibodies (with isotype-specific anti-mouse immunoglobulins [Sigma-Aldrich, Budapest, Hungary]) revealed that the low background immunoreactivity seen with the control sera originated from immunoglobulins of the M (IgM) isotype. On the other hand, the vast majority of specific antibodies raised during the immunization process were of the IgG isotype, belonging mainly to the IgG1 and IgG2a subclasses (Fig. 3), indicating a systemic Th1-directed immune response in vaccinated mice.

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FIG. 3. Isotypes of reactive immunoglobulins to S. enterica SL1344 raised upon immunization of BALB/c mice with an isogenic rfaH mutant. Three pools of sera obtained from control mice (plain bars) and three pools of sera obtained from immunized mice (striped bars) were tested by ELISA. Pools were obtained by mixing sera of 12 (pool 1, white background), 8 (pool 2, gray background), and 9 (pool 3, black background) mice. OD600, optical density at 600 nm.

Beside the fact that the rfaH mutant is attenuated, evidence has been shown that oral vaccination with this attenuated strain elicits protective immunity against a subsequent challenge with wild-type strain SL1344. Interestingly, high in vitro immunoreactivity can be detected not only to the isogenic wild-type strain but also to different serovar Typhimurium strains and to heterologous serovariants of S. enterica. Since the mutant strain used for vaccination exhibits a rough phenotype (see below), immunity is not based on antibodies raised against O antigens. Cross-immunity comprises S. bongori strains as well, suggesting that a conserved antigen(s) shared among members of the genus Salmonella serves as an immunotarget on the surface of salmonellae. The major immunogenic molecules on the surface of S. enterica are the LPS and flagella, antigenic variants of which serve as bases for classification into serogroups. Cross-immunity among strains belonging to different serogroups is normally not elicited or is at a very low level (11, 29). Humoral protection against Salmonella is characterized by the bulk of antibodies being directed against the O determinant. Therefore, we suggest that using a vaccine strain with a rough LPS phenotype allows the immune system to raise higher titers of antibodies against those antigens that otherwise possess minor immunogenicity. Cross-immunity between different serovars mediated by the LP fimbriae has been recently shown elsewhere (21). Another study has demonstrated that outer membrane proteins from S. enterica serovar Typhimurium are capable of conferring protection against a lethal challenge with homologous bacteria (31). Developing an immunization strategy by exploitation of a conserved Salmonella surface antigen to elicit protection against multiple serogroups is of high importance, due to the huge number of serovars capable of colonizing farm animals with a consequent danger of infecting humans.

Loss of RfaH results in a rough phenotype and serum sensitivity. LPS originated from strain SL1344, and its derivatives were purified by the procedure of Hitchcock and Brown (10). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on a 12.5% polyacrylamide gel according to the method of Laemmli (14). Gels were fixed overnight in a solution of 7% acetic acid and 25% 2-propanol and were silver stained as described by Nelson et al. (20). A deep rough phenotype could be seen in the case of the rfaH mutant (SL1344-R1), in contrast to the smooth LPS structures obtained from the wild-type strain (SL1344) or the trans-complemented mutant (SL1344-R2) (data not shown). In agreement with our observation on strain SL1344, loss of RfaH results in a rough phenotype of different Salmonella strains as well (17, 25). Smooth LPS is an important virulence factor of Salmonella that plays a role in resistance to the complement-mediated lytic effect of serum. Serum resistance of strain SL1344 and its derivatives was compared in sera originating from humans, mice, and guinea pigs by a previously described procedure (19). Briefly, bacteria grown in LB medium were washed in saline and diluted to 10⁶ CFU/ml. One-hundred-microliter aliquots of bacterial suspensions were mixed with an equal volume of human serum and incubated at 37°C for 4 h in microtiter plates. Samples were taken at 0-, 0.5-, 1-, 2-, 3-, and 4-h time points. Viable cell counts were determined by plating aliquots onto LB broth plates and incubating them overnight at 37°C. Wild-type strain SL1344 proved to be resistant against the bactericidal effect of sera of all three investigated species (Fig. 4). On the other hand, the resistance of its isogenic rfaH mutant was dependent on the origin of the serum. Though the mutant strain was able to survive in mouse serum, it exhibited high susceptibility to the killing effect of human serum, and its resistance was intermediate in serum obtained from guinea pigs (Fig. 4). Complementation of the mutant with rfaH restored resistance to the lytic effect of sera originating from all three investigated species. Others have also reported that rough Salmonella mutants exhibit different levels of susceptibility towards sera of human and mouse origins (12). As mouse serum is not active against S. enterica serovar Typhimurium (22), attenuation of the rfaH mutant in the mouse model cannot to be explained by sensitivity to the killing effect of serum. The smooth phenotype, nevertheless, may be involved in other virulence properties, such as modifying host cell functions upon release of LPS from bacterial cells in intracellular compartments (9).

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FIG. 4. Serum resistance of wild-type strain SL1344 (black diamonds), its isogenic rfaH mutant SL1344-R1 (gray squares), and trans-complemented strain SL1344-R2 (white circles). Bacteria (5 x 105) were incubated in sera of human (A), mouse (B), or guinea pig (C) origin for 4 h. Values represent the percentages of CFU upon cultivation in 50% serum relative to bacterial counts in the inocula. The graph depicts average values ± standard deviations calculated from triplicates of two similar tests.

An ideal live vaccine strain combines efficient immunogenicity with minimal adverse effects of the carrier. In this study, we have shown that an isogenic rfaH mutant of S. enterica serovar Typhimurium prototype strain SL1344 fulfills both of these criteria: the mutant becomes attenuated and elicits protective immunity against a subsequent challenge with the wild-type strain. Interestingly, in vitro humoral immunoreactivity is not confined to the homologous strain; rather, it is directed towards multiple serovariants of S. enterica and even S. bongori. Further experiments are needed, however, to determine whether cross-reactivity of sera is concomitant with protection of mice from a challenge with a heterologous serovariant. The nature of Salmonella antigens that are targeted by cross-reactive antibodies raised during vaccination with the rfaH mutant of strain SL1344 needs also to be elucidated.

 
This study was supported by grants OTKA T037833 and ETT 086/2001 to L.E.

We are grateful to Zoltán Péterfi (Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary) for providing S. enterica strains. We thank R. Lajkó, M. Pápa, and B. Plaschke for excellent technical assistance.

 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Immunology, University of Pécs, Szigeti út 12, 7624 Pécs, Hungary. Phone: 36 72 536252. Fax: 36 72 536253. E-mail: levente.emody{at}aok.pte.hu.

Editor: A. D. O'Brien

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Infection and Immunity, July 2004, p. 4297-4301, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4297-4301.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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