Role of RpoS in Fine-Tuning the Synthesis of Vi Capsular Polysaccharide in Salmonella enterica Serotype Typhi

Regulation of the synthesis of Vi polysaccharide, a major virulencedeterminant in Salmonella enterica serotype Typhi, is underthe control of two regulatory systems, ompR-envZ and rscB-rscC,which respond to changes in osmolarity. Some serotype Typhistrains exhibit overexpression of Vi polysaccharide, which masksclinical detection of lipopolysaccharide O antigen. This variationin Vi polysaccharide and O antigen display (VW variation) hasbeen observed since the initial studies of serotype Typhi. Inthis study, we report that rpoS plays a role in this increasedexpression in Vi polysaccharide. We constructed a variety ofisogenic serotype Typhi mutants that differed in their expressionlevels of RpoS and examined the role of the rpoS product insynthesis of Vi polysaccharide under different osmolarity conditions.Vi polysaccharide synthesis was also examined in serotype Typhimutants in which the native promoter of the rpoS was replacedby an araCP_BAD cassette, so that the expression of rpoS wasarabinose dependent. The RpoS^– strains showed increasedsyntheses of Vi polysaccharide, which at low and medium osmolaritiesmasked O antigen detection. In contrast, RpoS⁺ strains showedlower syntheses of Vi polysaccharide, and an increased detectionof O antigen was observed. During exponential growth, when rpoSis unstable or present at low levels, serotype Typhi RpoS⁺ strainsoverexpress the Vi polysaccharide at levels comparable to thosefor RpoS^– strains. Our results show that RpoS is anotherregulator of Vi polysaccharide synthesis and contributes toVW variation in serotype Typhi, which has implications for thedevelopment of recombinant attenuated Salmonella vaccines inhumans.

INTRODUCTION

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Introduction

Salmonella enterica serotype Typhi is a facultative intracellularpathogen that causes typhoid enteric fever exclusively in humansand is among the organisms causing the most costly human infectionsin terms of both morbidity and mortality (51). The mechanismresponsible for the virulence of serotype Typhi is differentfrom those of other serovars of Salmonella, and in this regard,serotype Typhi produces the virulence capsular (Vi) polysaccharide,which is an important virulence determinant during infection(27). Vi polysaccharide is a polymer of ${alpha}$ -1 $->$ 4-galacturonic acidwith an N-acetyl group at position C-2 and a variable O acetylationat C-3 (69). Virtually all strains isolated from blood or bonemarrow samples from patients with acute typhoid fever and frombile samples or feces from those who carry serotype Typhi inthe gallbladder are found to express Vi polysaccharide antigenwhen tested in clinical microbiology laboratories (29, 39, 55).Vi-positive (Vi⁺) strains were shown to be more virulent thanVi^– mutant strains in experiments conducted with humanvolunteers (27), and the Vi⁺ strains were resistant to complement-mediatedkilling and phagocytosis (61) and survived in human serum (20).In addition, the Vi⁺ strain but not the Vi^– mutant straincan multiply in the human macrophage cell line THP-1 and themouse macrophage-like cell line J774.1 (22).

The genes required for the biosynthesis of the capsular antigenVi are located in a 133.5-kb chromosomal region called Salmonellapathogenicity island 7 (SPI-7). Locus viaB, which encodes Vipolysaccharide in serotype Typhi, consists of 10 genes: tviBCDEfor Vi polysaccharide biosynthesis; vexABCDE for the exportof the Vi antigen; tviA, which is activated by unlinked regulatorsrcsB-rcsC (1, 28); and ompR-envZ, which are themselves controlledby osmolarity levels (53).

The Vi polysaccharide is expressed or overexpressed in mediawith low or medium osmolarities, respectively, and often coversand masks the lipopolysaccharide (LPS) O antigen (1, 71). Strainsof serotype Typhi Ty2 grown in media with medium osmolarities(446 mosmol, $~$ 170 mM NaCl [71]) exhibit high-level productionof Vi antigen. When the Vi antigen is expressed, the bacteriaare less adherent to and invasive into epithelial cells (71)but are more resistant to killing by macrophages (22). In high-osmolaritymedia (676 mosmol, $~$ 300 mM of NaCl [71] or more, as in the intestinallumen), the Vi antigen is not expressed (1, 53, 80), exposingthe somatic antigen and liberating secretion proteins, whichare stockpiled inside the bacterial cell when Vi polysaccharideis expressed at the bacterial cell surface (1). In this stage,serotype Typhi is more invasive into epithelial cells but isless resistant to killing by macrophages (22). Therefore, theVi antigen of serotype Typhi is a negative factor for invasionbut a positive factor for survival and multiplication insidemacrophages (22). Since the Vi polysaccharide can block theaccess of antibodies to the underlying O antigen, sometimesagglutination with Salmonella somatic D₁ antiserum cannot bedemonstrated until the bacterial cells are boiled to removethe Vi polysaccharide (11).

From initial studies on serotype Typhi, variations in Vi andO antigen detection have been observed. Observations recordedby Kauffman (33) and confirmed by Felix and Pitt (14) demonstratedthe concept of VW variation in Vi and O antigen relationships(74). Serotype Typhi strains inagglutinable with O antiseraand agglutinable only with Vi antisera are called V-form strains,while serotype Typhi strains that lack the Vi antigen and agglutinateonly with O antisera are called W-form strains (74). The VWform, which is referenced as the most common form observed inclinical laboratories, is identified when both Vi and O antigenare detected by agglutination with the respective antisera (74).Coincident with the early work of Felix and Pitt (14), and sinceverified by others (16, 17, 22, 75), most virulent strains ofserotype Typhi were the VW form.

In Salmonella, the rpoS gene encodes an alternative sigma factor( ${sigma}$ ^s/RpoS) that is the master regulator in the general stressresponse and is required for survival under extreme conditions(including osmotic and oxidative stress, acid shock, and transitionto stationary phase) and for virulence of S. enterica serotypeTyphimurium (6, 13, 36, 47, 48). RpoS controls the expressionof the serotype Typhimurium virulence plasmid genes, spvRABCD(13, 47). In addition, RpoS regulates chromosomal genes requiredfor colonization of Peyer's patches and for persistence in mice(6, 47). Serotype Typhi does not contain a virulence plasmid,and the role of rpoS in the virulence of this serotype is unknown.However, rpoS might also contribute to the virulence of thisserotype because RpoS^– strains of serotype Typhi are lesscytotoxic than RpoS⁺ strains, but RpoS^– strains survivebetter inside resting THP-1 macrophages without apoptosis inductionand have higher capacities for resistance in the macrophage(34).

We deduced from our observations that there is a correlationbetween Vi polysaccharide overexpression and the allele stateof the rpoS gene. We report here that rpoS is involved in thesubtle overexpression of Vi polysaccharide at osmolarities lowerthan 667 mosmol resulting from the addition of 300 mM NaCl toculture media. We presume that the rpoS gene influences theVW form described in earlier studies. Furthermore, we discussthe implication of the effect of rpoS on the synthesis of Viantigen in serotype Typhi Ty2 and serotype Typhi ISP1820, whichare the most useful parental strains in designing live serotypeTyphi vaccines.

MATERIALS AND METHODS

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Materials and Methods

Salmonella strains and conditions of culture. Salmonella strains used in this study are listed in Table 1.The strains were routinely cultured at 37°C in LB medium(Bacto tryptone, 10 g/liter; Bacto yeast extract, 5 g/liter;NaCl, 10 g/liter) (44). Media were solidified with 1.5% (wt/vol)agar. When required, media were supplemented with chloramphenicol(Cm; 25 µg/ml), ampicillin (Ap; 100 µg/ml), 2,6-diaminopimelicacid (DAP; 50 µg/ml), L-arabinose (Ara; 0.2% [wt/vol]),or sucrose (5% [wt/vol]). The osmolarities of the LB agar plateswere changed by adding NaCl (0, 10, 85, 150, 300, 400, and 500mM) (71) or sucrose. The final pHs of the media were adjustedto 7.0 with NaOH before the autoclaving.

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TABLE 1. Salmonella strains and relevant characteristics

Characterization of serotype Typhi strains. The serotype Typhi strains were characterized for type I fimbriaein static broth cultures (35, 49) and for motility in motilitymedium (bioMérieux, Marcy I'Etoile, France). The presenceof LPS was evaluated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis when visualized by silver staining (23,72). Plasmid profiles were verified by alkaline lysis and agarosegel (0.5%) electrophoresis (31, 63). Fermentation patterns ofvarious carbohydrates and production of H₂S were determinedby using the API 20E system (bioMérieux, Marcy I'Etoile,France). The serotype Typhi RpoS^– strains gave API 20Ecode number 640454057 (a good identification as serotype Typhi[97.4%] and Salmonella spp. [1.8%]), whereas the RpoS⁺ strainsgave API 20E code number 440454057 (an excellent identificationas serotype Typhi [99.9%] and S. enterica serovar Choleraesuis[0.1%]). The expression levels of the Vi, O₉ (serotype Typhi-specific),D₁ (O₁, O₉, and O₁₂), and H_d antigens of serotype Typhi strainswere evaluated by the growth of strains on LB agar plates withdifferent osmolarities after 18 to 24 h.

Construction of serotype Typhi Ty2 RpoS⁺. The recombinant suicide vector pYA3467 (9.6 kb), carrying therpoS gene from serotype Typhi ISP1820 (993 bp) between flankingregions (338 bp 5' and 83 bp 3') of the rpoS ( ${sigma}$ ^s2/RpoS^–)allele in serotype Typhi Ty2 ( ${chi}$ 3769), was constructed as follows(Fig. 1A). The rpoS allele from serotype Typhi Ty2 representsa frameshift mutation caused by a guanosine insertion at position1313 that modified the amino acid sequence of the C-terminalpart of ${sigma}$ ^s/RpoS, resulting in a protein of 384 amino acids ratherthan 330 (58). The SmaI/PmeI fragment, harboring 338 bp of the5'-end upper region, 993 bp of the rpoS gene, an extra 83 bpof the 3'-end region, and $~$ 93 bp of multiple cloning sites (atotal of $~$ 1,450 bp), was inserted into the SmaI/PmeI sites ofpMEG-375 to generate pYA3467 (Fig. 1A). To construct serotypeTyphi Ty2 RpoS⁺, the suicide plasmid pYA3467 was conjugationallytransferred from Escherichia coli ${chi}$ 7213 (32) to serotype TyphiTy2 strain ${chi}$ 3769 in LB broth supplemented with DAP (67). Strainscontaining single-crossover plasmid insertions (serotype TyphiTy2 rpoS::pYA3467) (Fig. 1B) were isolated on LB agar platescontaining Cm without DAP. Loss of the suicide vector afterthe second recombination between homologous regions (i.e., allelicexchange) was selected by using the sacB-based sucrose sensitivitycounterselection system (18). The colonies were screened forCm^s and for positive catalase activity evidence by bubble productionafter addition of H₂O₂ (Fig. 1C). In particular, rpoS mutantsare impaired in the ability to resist H₂O₂ during stationaryphase. Indeed, two of the catalases produced by Salmonella (KatEand KatN) are expressed in stationary phase under the controlof rpoS (3, 60). To observe the presence of rpoS mutations,we added H₂O₂ to the bacterial colony mass removed from LB agar.The extent of bubbling indicated absence or reduction of catalaseproduction in strains with differing rpoS genotypes (57). Thepresence of the RpoS⁺ insertion in ${chi}$ 8438 serotype Typhi Ty2 RpoS⁺was confirmed by a catalase activity assay (Fig. 1C).

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FIG. 1. Construction of suicide vector pYA3467 and generation of RpoS⁺ strain in serotype Typhi Ty2. (A) Construction of suicide vector pYA3467 (9,618 bp) used to generate the serotype Typhi Ty2 RpoS⁺ strain. (B) Crossover between suicide vector pYA3467 and chromosome of serotype Typhi Ty2. (C) Catalase test for determination of RpoS activity.

Construction of serotype Typhi rpoS mutants. Strains ${chi}$ 9060 and ${chi}$ 9061 were constructed by P22HTint-mediatedtransduction of ${chi}$ 8438 and ${chi}$ 3744, respectively, to introduce theinactive rpoS allele (rpoS ${Omega}$ Ap; truncated RpoS) from ${chi}$ 4991 (47).The bacteriophage P22HTint was propagated in the serotype Typhimurium ${chi}$ 4991 donor strain by standard methods (65). Plaque assays wereperformed to determine phage titers. The concentrated phagelysate was prepared, using ${chi}$ 4991 as the host, as described bySantander and Robeson (64). Serotype Typhi was infected witha multiplicity of infection of 10 (phage/recipient). Transductantswere selected on LB agar containing Ap. Green indicator plates(54) and Evans blue uridine indicator plates (68) were usedto confirm that transductants were phage-free. The serotypeTyphi rpoS ${Omega}$ Ap strains were screened for Ap^r and absence of bubblingafter addition of H₂O₂ and expression of a truncated RpoS proteinby Western blot analysis (see Fig. 3).

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FIG. 3. Evaluation of RpoS expression at different osmolarities by Western blot analysis. (A) ${chi}$ 3744 serotype Typhi ISP1820 RpoS⁺. (B) ${chi}$ 3769 serotype Typhi Ty2 RpoS^–. (C) ${chi}$ 8438 serotype Typhi Ty2 RpoS⁺. (D) ${chi}$ 9060 serotype Typhi Ty2 rpoS ${Omega}$ Ap RpoS^–. (E) ${chi}$ 9061 serotype Typhi ISP1820 rpoS ${Omega}$ Ap RpoS^–. (F to H) Assays conducted in the absence and presence of arabinose (0.2%). (F) ${chi}$ 9066 serotype Typhi ISP1820 RpoS⁺ ${Delta}$ P_rpoS183::TTaraCP_BADrpoS. (G) ${chi}$ 9067 serotype Typhi Ty2 RpoS^– ${Delta}$ P_rpoS183::TTaraCP_BADrpoS. (H) ${chi}$ 9068 serotype Typhi Ty2 RpoS⁺ ${Delta}$ P_rpoS183::TTaraCP_BADrpoS.

Construction of serotype Typhi mutants with regulatable expression of rpoS. The recombinant suicide vector pYA3735 (7.3 kb), carrying aTTaraCP_BAD (1,329-bp) insertion between flanking regions (410bp 5' and 365 bp 3') deleting a 35-bp promoter sequence (rpoS–13 to –48), was constructed as follows (Fig. 2A).PCR primers 5'-TGCGAGCTCCTCGACTGCACGGATAAGCCCC-3' (primer 1)and 5'-CCGCTCGAGAGGAGCCACCTTATGCAGATTA-3' (primer 2) were designedto amplify the rpoS flanking region (410 bp). A SacI site wasincluded in primer 1 (underlined), and an XhoI site was includedin primer 2 (underlined). The nlpD flanking region (365 bp)was amplified, mediated by primers 5'-CATCTGCAGCTGGTTCCGCCGCTTTATCGCTG-3' (primer 3) and 5'-ACATGCATGCGGATGGCAAAGTGATCGAAA-3'(primer 4). A PstI site was included in primer 3 (underlined),and an SphI site was included in primer 4 (underlined). Theflanking regions were amplified from serotype Typhi Ty2 ${chi}$ 3769and were cloned into pYA3700 between the TTaraCP_BAD insertion(Fig. 2A). The resultant plasmid (designated p ${Delta}$ P_rpoSrpoS, 4,801bp), carrying the TTaraCP_BAD cassette between the flanking regions,was digested with SacI and SphI to yield a 2,068-bp fragmentthat was cloned into pRE112 digested with SacI and SphI (12).The resulting suicide vector was designated pYA3735. To constructserotype Typhi ${Delta}$ P_rpoS183::TTaraCP_BADrpoS mutants, the suicideplasmid pYA3735 was conjugationally transferred from Escherichiacoli ${chi}$ 7213 (32) to serotype Typhi strains in LB supplementedwith DAP (67). The strains containing single-crossover plasmidinsertions were isolated on LB agar plates containing Cm withoutDAP (Fig. 2B). Loss of the suicide vector after the second recombinationbetween homologous regions (i.e., allelic exchange) was selectedby using the sacB-based sucrose sensitivity counterselectionsystem (18). The colonies were screened for Cm^s and for positivecatalase activity with H₂O₂ (bubble production) only when grownin the presence of 0.2% Ara (Fig. 2D). The presence of the ${Delta}$ P_rpoS183::TTaraCP_BADrpoSinsertion in serotype Typhi strains was confirmed by PCR amplificationof DNA fragments bigger than those amplified from the parentalstrains. The P_rpoS primer set (primers 1 and 4) amplified 770-and 2,100-bp DNA fragments from the chromosomal DNA templatesof serotype Typhi (parental) and the serotype Typhi ( ${Delta}$ P_rpoS183::TTaraCP_BADrpoS)mutant, respectively (Fig. 2C). The regulation of the rpoS geneby araCP_BAD was confirmed using a catalase activity assay forcells grown with and without 0.2% Ara (Fig. 2D).

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FIG. 2. Construction of suicide vector pYA3735 and generation of serotype Typhi with regulated rpoS expression ( ${Delta}$ P_rpoS183::TTaraCP_BADrpoS). (A) Construction of suicide vector pYA3735 (7,281 bp) used to generate serotype Typhi ${Delta}$ P_rpoS183::TTaraCP_BADrpoS. (B) Crossover between suicide vector pYA3735 and chromosome of serotype Typhi. (C) Agarose gel (0.8%), a PCR product from chromosomal DNA from wild-type (770-bp) and ${Delta}$ P_rpoS183::TTaraCP_BADrpoS (2,100-bp) mutants. Lanes: 1, ${chi}$ 3744 serotype Typhi ISP1820 RpoS⁺; 2, ${chi}$ 3769 serotype Typhi Ty2 RpoS^–; 3, ${chi}$ 8438 serotype Typhi Ty2 RpoS⁺; 4, ${chi}$ 9066 serotype Typhi ISP1820 RpoS⁺ ${Delta}$ P_rpoS183::TTaraCP_BADrpoS; 5, ${chi}$ 9067 serotype Typhi Ty2 RpoS^– ${Delta}$ P_rpoS183:: TTaraCP_BADrpoS; 6, ${chi}$ 9068 serotype Typhi Ty2 RpoS⁺ ${Delta}$ P_rpoS183::TTaraCP_BADrpoS. (D) Catalase test for detection of ${Delta}$ P_rpoS183::TTaraCP_BADrpoS without and with growth in the presence of 0.2% Ara.

Construction of serotype Typhi Vi^– strains. Additionally, serotype Typhi Vi^– strains ( ${Delta}$ tviABCDE10)were constructed as controls. Deletion of the tviA, tviB, tviC,tviD, and tviE genes ( ${Delta}$ tviABCDE10) in the viaB loci of the serotypeTyphi strains was performed by allelic exchange. The recombinantsuicide vector pYA4009 (5.9 kb), carrying the flanking regions(361 bp 5' and 422 bp 3'), was constructed as follows. PCR primers5'-ACATGCATGCGAACGGTATTACTGTCAGTCACAAG-3' (primer 1) and 5'-TGCGACCTCATGAAAAAAATCATCATATTACTA-3'(primer 2) were designed to amplify the upstream tviA flankingregion (361 bp). An SphI site was included in primer 1 (underlined),and a SacI site was included in primer 2 (underlined). The vexAflanking region (422 bp) was amplified by primers 5'-TGCGAGCTCGAAGTCTCCTTATGCTGAAATAAC-3'(primer 3) and 5'-TCCCCCGGGCAGATTATTTCAAATACGATTAGG-3' (primer4). A SacI site was included in primer 3 (underlined), and aSmaI site was included in primer 4. The flanking regions wereamplified from serotype Typhi Ty2 ${chi}$ 3769 and were cloned intopRE112 digested with SphI and SmaI (12). The resulting suicidevector was designated pYA4009. The tviABCDE10 mutant strainswere constructed by allelic exchange and the sucrose sensitivitycounterselection system (18), as described above. The deletionwas confirmed by PCR and corroborated by the sensitivity ofthe mutant to the Vi-II phage and the lack of agglutinationwith Vi antisera.

Agglutination assays. Agglutination tests were performed on glass microscope slidesby mixing 50 µl of antisera against Vi, O₉, D₁ (O₁, O₉,and O₁₂), and H_d (Difco Laboratories, Detroit, MI) with suspensionsof single colonies. The reactions were visualized by phase contrastmicroscopy at 10x magnification. When the assays for O₉ andD₁ antigens were negative, the cells were boiled for 20 minand cooled prior to addition of O antisera for detection ofO antigens (39, 53).

Western blot analysis. The strains were grown in 3 ml of LB medium with different NaClconcentrations at 37°C with aeration. The samples were collectedwhen the absorbance values reached 1.5 (optical density at 600nm, 1.5; $~$ 1 x 10⁹ CFU/ml), which corresponds to stationary growthphase, known to stimulate rpoS expression. One milliliter ofculture was collected and centrifuged at 10,500 x g for 10 min.The supernatant was discarded, and the total bacterial proteinswere suspended in 150 µl of loading buffer and boiledfor 10 min. The total proteins were normalized by a nanodropspectrophotometer (ND-1000; NanoDrop) at 25 mg/µl, separatedby 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and transferred onto nitrocellulose membranes (63). Fat-freemilk powder solution (5% [wt/vol]) was used for blocking. Themembrane was incubated with a primary anti-mouse RpoS monoclonalantibody (1:1,000) (Neoclone), anti-rabbit H_d polyclonal (1:5,000)(Difco), or anti-rabbit GroEL polyclonal (1:10,000) (Sigma)for 1 h at room temperature, washed three times with phosphate-bufferedsaline supplemented with Tween (0.05%), and then incubated witha 1:10,000 dilution of alkaline phosphatase-conjugated anti-mouseimmunoglobulin G (Sigma) or anti-rabbit immunoglobulin G (Sigma).Color was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.

Rocket immune electrophoresis. The measurement of Vi polysaccharide expression was visualizedby rocket immune electrophoresis (38, 73) adapted for Vi antigen.The strains were grown under the same conditions described abovefor the Western blot assays. One milliliter of culture was centrifugedat 10,500 x g for 10 min. The supernatant was discarded, andthe total bacterial pellet was washed one time and resuspendedin 150 µl of barbital buffer (60 mM, pH 8.6) (Sigma).The cells were disintegrated by sonication on ice until thesuspension became clarified. Total protein concentration wasused to normalize the samples (25 mg/µl). Agarose (1%[wt/vol]) was prepared in barbital buffer, melted, and adjustedto 55°C. The Vi antiserum (Sigma) was mixed with the warmagarose to make 1% Vi antiserum (vol/vol) gels. The gel wasprepared in mini plates (Bio-Rad) 1.5 mm thick. After beingcooled, the gel was placed in a Gel-bond membrane (3 M). Holeswith 3-mm diameters were made 10 mm from the bottom of the gel,with 3 mm of distance between them. An aliquot of 20 µlof disrupted bacteria was placed in the holes. The electrophoresiswas run from the negative to the positive pole at 10 V/cm for3 h in barbital buffer. Paper filters were used as conductingbridges. Following the run, the gels were removed from the membraneand placed in NaCl (0.2 M) overnight. The gel was washed withdistilled water for 1 h and placed back in the Gel-bond membrane.The gels were dried at 30°C and stained with a solutionof Coomassie blue (0.5%) in acetic acid-methanol-water (1:5:5[vol/vol/vol]) at 37°C for 10 min. The excess stain wasremoved by washing the gels with the same solution without Coomassieblue. Purified serotype Typhi Vi polysaccharide vaccine (AventisPasteur, Lyon, France) was used as a standard for determinationof the Vi antigen synthesis of the serotype Typhi strains grownat different osmolarities.

Evaluation of Vi antigen expression in serotype Typhi during growth. The strains were grown in LB media with 150 mM of NaCl withaeration at 37°C. The growth was monitored by measurementof absorbance at 600 nm. The samples, taken at different times,were normalized to an optical density at 600 nm of 0.8. Then,1 ml of culture in duplicate (proteins and Vi antigen samples)was centrifuged at 10,500 x g for 10 min. The supernatant wasdiscarded, and the sample was frozen at –70°C overnight.Western blot assays were performed for RpoS and GroEL as explainedabove. Evaluation of Vi antigen was determined by rocket immuneelectrophoresis as described above.

RESULTS

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Results

Strain construction. Since in preliminary studies we observed that production ofVi antigen in serotype Typhi strains differed depending on theRpoS phenotype, we constructed a variety of strains that differedin their RpoS expression levels to further understand the relationshipbetween this sigma factor, Vi capsule production, and osmolarity.To be sure that the results observed with serotype Typhi Ty2strain ${chi}$ 3769, which has an rpoS mutation (57), were due to therpoS mutation, we first restored this strain to the RpoS⁺ phenotypeto yield ${chi}$ 8438. We then generated RpoS^– derivatives ofthe RpoS⁺ Ty2 ( ${chi}$ 8438) and ISP1820 ( ${chi}$ 3744) strains by insertionof rpoS ${Omega}$ Ap and selection for the Ap cassette to yield ${chi}$ 9060 and ${chi}$ 9061, respectively. The strains were Ap^r and catalase negativeand presented an expression of a truncated RpoS due to the Apinsertion cassette (Fig. 3). rpoS mutants with regulatable expressionof rpoS were also constructed by deleting P_rpoS and replacingit with an araCP_BAD cassette. The presence of RpoS⁺, rpoS ${Omega}$ Ap,and ${Delta}$ P_rpoS183::TTaraCP_BADrpoS mutations was verified by testingfor resistance to Ap, sensitivity to Cm, and formation of bubblesfrom H₂O₂ by PCR and by Western blot analysis as described inMaterials and Methods. All mutants conserved the phenotypesof the original strains, including smooth LPS, fimbrial typeI production, antigenic composition, and auxotrophic markers.

RpoS expression. Western blot assays were used to determine the RpoS expressionlevels during stationary phase growth under different osmolarityconditions. The expression levels of RpoS were relatively thesame under all osmolarity conditions (Fig. 3). RpoS^– strains ${chi}$ 3769 and ${chi}$ 9067 did not show expression of RpoS. The rpoS ${Omega}$ Ap strains,which express a truncated RpoS, showed lower-molecular-weightproteins than wild-type RpoS, as expected (Fig. 3D to E). Thestrains with regulatable rpoS showed expression of RpoS onlywhen grown in the presence of 0.2% arabinose (Fig. 3F to H;see also Table 3).

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TABLE 3. Synthesis of Vi polysaccharide (µg of Vi/500 mg of total serotype Typhi protein) of serotype Typhi strains grown on LB agar (pH 7) supplemented with different amounts of NaCl at 37°C^a

Effect of rpoS on Vi synthesis. Since the osmolarity of the growth media influences the synthesisof Vi antigen, we examined strains of different rpoS genotypesin media with different osmolarities for levels of Vi antigensynthesis. The RpoS⁺ strains grown at osmolarities less than676 mosmol ( $~$ 300 mM NaCl [71]) showed overexpression of Vi antigensufficient to cover the somatic O antigen. The phenotypicallyO antigen-negative strains were boiled, and the O antigen wassubsequently detected in all cases. When RpoS^– strainswere grown in media with 300 mM NaCl (676 mosmol) or more, theexpression of Vi polysaccharide was less or null and the O antigenwas exposed. Under this condition (>300 mM NaCl), RpoS⁺ strainshave the same synthesis of the Vi antigen and the O antigensare exposed (Table 2). The RpoS⁺ strains and regulatable rpoSmutants grown with Ara (0.2%) did not overexpress the Vi polysaccharide,and both Vi and O antigens were detected at osmolarities lowerthan 676 mosmol. However, at 676 mosmol or higher osmolarities,Vi synthesis was almost null in these strains (Table 2).

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TABLE 2. O₉ and Vi slide agglutination reactions of serotype Typhi strains grown on LB agar (pH 7) supplemented with different amounts of NaCl at 37°C overnight (18 to 24 h)^a

To make sure that the effects of osmolarity and rpoS genotype/expressionon Vi antigen synthesis and detection of O antigen were notdue to a consequence of using NaCl, identical evaluations weremade using sucrose to alter osmolarity. These results are essentiallyidentical to those presented in Table 2, with the exceptionthat osmolarities higher than 676 mosmol did not result in substantialVi antigen synthesis for masking O antigen detection (Table2), whereas sucrose did cause Vi antigen synthesis that maskedO antigen detection (data not shown). This effect of sucrosewas more pronounced in strains that were RpoS^– than instrains that were RpoS⁺.

Pickard et al. (53) showed that in serotype Typhi vaccine strains,Vi antigen synthesis is regulated by the ompB locus and is dependenton osmolarity. These authors also observed that serotype TyphiTy2 and serotype Typhi ISP1820 have different levels of Vi antigensynthesis when these strains were grown at different osmolarities.We observed the same results in these different strains of serotypeTyphi (Table 2). However, Pickard et al. (53) explained thedifferences among the Vi antigen syntheses in these strainsby the different times of isolation. On the other hand, an rpoSframeshift mutation in serotype Typhi Ty2 has been previouslyreported (58). These references and the results reported inthis study suggest that the rpoS allelic state, one of the majordifferences between serotype Typhi Ty2 and serotype Typhi ISP1820,is responsible for the subtle down-regulation of Vi polysaccharidesynthesis at osmolarities lower than 676 mosmol (300 mM of NaCl).

The rocket immune electrophoresis assays indicated that RpoS⁺strains down-regulated Vi antigen expression. The major productionof Vi polysaccharide in RpoS⁺ strains was at 150 mM of NaCl,even in the presence of RpoS, but these strains never exhibitedexpression levels higher than those of RpoS^– strains (Fig.4 and Table 3). The RpoS^– strains demonstrated overexpressionof Vi polysaccharide at all osmolarities tested. However, RpoS^–strains showed regulation of Vi polysaccharide in response toosmolarity changes (Fig. 4 and Tables 2 and 3), but with a substantialincrease in Vi synthesis (Fig. 4 and Table 3). These resultsindicated that RpoS down-regulated the Vi polysaccharide expressionin serotype Typhi.

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FIG. 4. Evaluation of the effect of RpoS on the synthesis of Vi polysaccharide in serotype Typhi. (A) Standards of Vi antigen. (B) Negative control, ${chi}$ 9197 serotype Typhi Ty2 RpoS^– ${Delta}$ tviAEBCD10. (C) Negative control, ${chi}$ 9198 serotype Typhi ISP1820 RpoS⁺ ${Delta}$ tviAEBCD10. (D) ${chi}$ 3744 serotype Typhi ISP1820 RpoS⁺. (E) ${chi}$ 3769 serotype Typhi Ty2 RpoS^–. (F) ${chi}$ 8438 serotype Typhi Ty2 RpoS⁺. (G) ${chi}$ 9060 serotype Typhi Ty2 rpoS ${Omega}$ Ap. (H) ${chi}$ 9061 serotype Typhi ISP1820 rpoS ${Omega}$ Ap. (I) Noninduced ${chi}$ 9066 serotype Typhi ${Delta}$ P_rpoS183::TTaraCP_BADrpoS RpoS. (J) ${chi}$ 9067 serotype Typhi Ty2 RpoS^– ${Delta}$ P_rpoS183::TTaraCP_BADrpoS RpoS induced by 0.2% arabinose. The strains were grown in LB media with 0, 0.15, 0.3, and 0.4 mM of NaCl. (K) Relation between Vi antigen concentration (µg) and length of rocket (cm).

Both RpoS⁺ and RpoS^– strains showed regulation of Vi polysaccharidein response to changes in osmolarity, suggesting that the repressionof Vi polysaccharide synthesis in response to osmolarity isnot under the control of RpoS (1, 53). However, RpoS may helpin the repression of Vi polysaccharide at high and low osmolarities,conditions under which rpoS is induced (6, 43).

Effects of rpoS on Vi antigen synthesis and H_d detection. The fliC gene is present only in serotype Typhi strains andencodes one type of flagellin, designated H_d (1). Serotype TyphiGIFU10007 appeared to require intrinsic, intact motility forinvading cultured epithelial cells, as nonmotile mutants werenot invasive (42). The production of the H_d flagellin in serotypeTyphi, as well as that of Vi antigen, is modulated by the RcsB-RcsCregulatory system in response to changes in the osmolarity ofthe growth medium (1). We examined H_d synthesis in strains ofdifferent rpoS genotypes in media with different osmolarities.Synthesis of the H_d flagellar antigen in RpoS⁺ strains was detectedat all osmolarities tested. In contrast, the H_d flagellar antigenwas not detected in RpoS^– strains at osmolarities lowerthan 10 mM of NaCl (Table 4). At osmolarities higher than 10mM NaCl, the H_d antigen was detected with a strong reactionin RpoS^– strains, the same as in RpoS⁺ strains. However,when the RpoS^– strains were grown at 0 and 10 mM NaCland then boiled, the H_d antigen was detected. These resultsshow that the Vi antigen also covers and masks the H_d antigenat low osmolarities in RpoS^– strains. These results collectivelysuggest that the rpoS gene causes a subtle down-regulation ofVi polysaccharide synthesis in serotype Typhi.

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TABLE 4. H_d flagellar antigen slide agglutination reactions of serotype Typhi strains grown on LB agar (pH 7) supplemented with different amounts of NaCl at 37°C overnight (18 to 24 h)^a

Although we observed similar results for masking detection ofthe H_d flagellar antigen with growth in media by adding sucroseto increase osmolarity, this masking in RpoS^– strainswas observed at much higher concentrations when sucrose wasused (data not shown) than when NaCl was used to alter osmolarity(Table 4).

Flagellar synthesis is dependent on the rcsB-rcsC loci, whichdecrease the syntheses of flagella at low osmolarities (1).Western blot analysis showed that H_d flagellin synthesis wasdecreased under low-osmolarity conditions, as expected (Fig.5). Similar results were obtained for rpoS arabinose-induciblemutants in the presence or absence of arabinose (Fig. 5). Expressionlevels of flagella were considerably lower when the strainswere grown at 0 mM of NaCl, but flagella were still detectedby agglutination in RpoS⁺ strains. However, in RpoS^– strains,flagella were not detected, due to overexpression of the Vipolysaccharide (Table 4).

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FIG. 5. Evaluation of H_d (serotype Typhi flagella factor) expression at different osmolarities by Western blot analysis. (A) ${chi}$ 3744 serotype Typhi ISP1820 RpoS⁺. (B) ${chi}$ 3769 serotype Typhi Ty2 RpoS^–. (C) ${chi}$ 8438 serotype Typhi Ty2 RpoS⁺. (D) ${chi}$ 9060 serotype Typhi Ty2 rpoS ${Omega}$ Ap RpoS^–. (E) ${chi}$ 9061 serotype Typhi ISP1820 rpoS ${Omega}$ Ap RpoS^–. (F to H) Assays conducted in the absence and presence of arabinose (0.2%). (F) ${chi}$ 9066 serotype Typhi ISP1820 RpoS⁺ ${Delta}$ P_rpoS183::TTaraCP_BADrpoS. (G) ${chi}$ 9067 serotype Typhi Ty2 RpoS^– ${Delta}$ P_rpoS183::TTaraCP_BADrpoS. (H) ${chi}$ 9068 serotype Typhi Ty2 RpoS⁺ ${Delta}$ P_rpoS183::TTaraCP_BADrpoS.

Evaluation of Vi polysaccharide synthesis in serotype Typhi RpoS⁺ during growth. RpoS is a key factor in the stress response during the transitionfrom the exponential growth phase to the stationary growth phase(43). RpoS is expressed at low levels or unstable at exponentialgrowth phase in E. coli (30, 46). Thus, Vi antigen could havedifferent expression levels in exponential phase, when RpoSis not present or is present at low levels, and in stationaryphase in serotype Typhi, when RpoS is maximally expressed. Wetested the effect of growth phase on the synthesis of Vi antigenin serotype Typhi RpoS⁺. RpoS was not detected in early exponentialphase (Fig. 6) but was detected at different levels from mid-exponential-growth-phasecultures and evidently expressed at stationary phase (Fig. 6),since bubble production in the catalase test was positive onlyin stationary phase. In concordance with RpoS expression levels,Vi polysaccharide synthesis was decreased (Fig. 6). These resultsindicated that serotype Typhi RpoS⁺ overexpressed the Vi antigenduring the early growth phase, when RpoS is not expressed orexpressed at low levels. This indicates that RpoS is the causeof the VW and V variation in serotype Typhi. RpoS^– strainsoverexpress the Vi polysaccharide without RpoS regulation, whichcauses a permanent V form. RpoS⁺ strains exhibit both forms,the V form during the early exponential growth, when RpoS isnot expressed or expressed at low levels, and the VW form whenRpoS is expressed. On the other hand, SPI-7, where the viaBlocus is located, is an unstable genetic element in serotypeTyphi (4) and strains that have lost this pathogenic islandexhibit the W form (Vi^–). Thus, this genetic instabilitycould be the cause of the W form.

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FIG. 6. Evaluation of Vi polysaccharide synthesis in serotype Typhi RpoS⁺ during growth. (A) RpoS expression during the growth curve. GroEL was used as a control. The strains were grown in LB medium with 150 mM of NaCl. (B) Vi polysaccharide expression during the growth. (C) Growth curve. *, the samples collected at each point were normalized as described in the text.

DISCUSSION

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Discussion

The expression of capsular polysaccharide by some Enterobacteriaceaeis highly regulated in response to different environments, beingactivated by certain signals and suppressed by others (77).Vi capsular expression in serotype Typhi is subject to regulationby at least two separate two-component systems, rcsB-rcsC (inthe viaA locus) (1) and ompR-envZ (in the ompB locus) (53).TviA (VipR), a positive regulator that activates its own promoterin the viaB locus, interacts with RcsB, independently of RcsAand Lon protease, to promote optimal transcription of genesinvolved in Vi antigen synthesis (1, 53, 76, 77). Vi is expressedpreferentially at low and medium osmolarities. Such environmentsmight include aqueous environments, which are thought to containnot more than 60 mM NaCl, and certain extracellular environments,such as blood and the cell cytoplasm, where the osmolaritiesare equivalent to 150 mM of NaCl (310 mosmol) (45, 37, 71).The latter environment is especially relevant to that encounteredby serotype Typhi in the infected host. It is possible thatthis preferential expression of Vi polysaccharide at low andmedium osmolarities serves to protect bacterial cells from thecomplement-mediated actions of the O antigen-specific antibody(61). Recent studies with serotype Typhi Ty2 grown under optimalconditions for Vi polysaccharide expression showed that Vi polysaccharidereduced TLR-dependent interleukin-8 production in human colonictissue explants, suggesting that the scarcity of neutrophilsin intestinal infiltrations of typhoid fever patients is dueto the Vi polysaccharide (56). In contrast, RcsB under low-osmolarityconditions, acting in association with the viaB locus-encodedTviA protein, negatively controls the transcription of iagA,invF, and sipB (encoding proteins involved in cell invasion)(1).

At high osmolarities, such as those in the intestinal lumen(with values believed to be equivalent to 300 mM of NaCl andgreater [71]), the transcriptions of iagA, invF, and sipB aremarkedly increased, whereas the transcriptions of genes involvedin Vi biosynthesis are markedly decreased (1). We observed thatthe synthesis of Vi antigen decreased as the osmolarity increased(Table 2 and Fig. 4), and when the osmolarity reached 676 mosmol(300 mM of NaCl), the Vi antigen no longer covered the O antigen(Table 2). Zhao et al. (80) showed that at 300 mM or more ofNaCl, serotype Typhi GIFU1007 had null expression of the Viantigen and exhibited a high invasion index in epithelial cells,together with a high secretion of SipC protein (80). Part ofthe stress response in the bacterial cell is the induction ofthe master stress response regulator protein RpoS (6, 43). Highosmolarity is one of the environmental signals that induce rpoS(6, 43). In accordance with these findings, our results showedthat RpoS down-regulates Vi polysaccharide synthesis, whichcould have importance for the outcome of infection. On the otherhand, serotype Typhi Ty2, which was isolated in 1918 (15) andis RpoS^– (58), exhibited maximal adherence to and invasivenessinto tissue culture cells when grown in media with 300 mM NaCl(676 mosmol) (71). We observed that the Vi polysaccharide didnot cover the O antigen when either RpoS^– or RpoS⁺ strainswere grown in media with $≥$ 300 mM of NaCl. However, at osmolaritieslower than those produced by 300 mM NaCl (676 mosmol), the Viantigen was overexpressed and the O antigens were covered andmasked in RpoS^– strains but not RpoS⁺ strains, which mayaffect the invasiveness. In addition, at osmolarities lowerthan 676 mosmol, adherence to and invasiveness into tissue culturecells have been found to be minimal in serotype Typhi Ty2 (71).On other hand, studies comparing serotype Typhi, serotype Typhimurium,and S. enterica serotype Dublin strains showed that serotypeTyphi, in this case ISP1820, is much more adherent to and invasiveinto Int-407 and murine MODE-K cell lines when grown in mediawith differing osmolarities, in contrast to the RpoS^–serotype Typhi Ty2 strain (79).

Additionally, our results imply that the rpoS gene is responsiblefor the VW variation in the display of Vi and O antigens inserotype Typhi as described in early studies (15, 74). The VWform, with a dual display of Vi and O antigens, is most commonlydisplayed by serotype Typhi strains from infected individuals,and the majority of such clinical isolates are also RpoS⁺ (58).

Serotype Typhi ISP1820, which is RpoS⁺, is more virulent inhumans than serotype Typhi Ty2, which is RpoS^– (70). Inconcordance, serotype Typhi CVD906, a derivate of the serotypeTyphi field isolate ISP1820 with deletion mutations in aroCand aroD, was highly immunogenic but caused fever and otheradverse reactions in humans (26), in contrast to the serotypeTyphi CVD908 Ty2 ${Delta}$ aroC ${Delta}$ aroD strain, which is RpoS^– (25).The live typhoid vaccine Ty21a, which is an RpoS^–, ${Delta}$ galE,and Vi^– (59) derivate of serotype Typhi Ty2 (58), hasbeen evaluated in several clinical trials and found to be welltolerated, although only modestly immunogenic, requiring threeor four doses to confer protection (40, 41, 66, 78). Furthermore,the live typhoid vaccine Ty21a has yielded poor results whenused as a live recombinant serotype Typhi vaccine (RAStyVs)expressing protective antigens against a diversity of pathogens(2, 9, 10, 21, 24, 50). The rpoS mutation could affect the immunogenicitiesof recombinant vaccines (1). It is thus possible that this poorimmunogenicity may be the result of the rpoS mutation in Ty2and its derivatives rather than Vi antigen deletion. On theother hand, the overexpression of Vi antigen in Ty2 could decreaseadherence to and invasion into intestinal tissues necessaryto colonize more internal lymphoid effector tissues (2). Inserotype Typhimurium, it has been demonstrated that chromosomalRpoS-regulated genes are necessary for invasion into and colonizationof the gut-associated lymphoid tissue (47). In accord with this,rpoS mutants of serotype Typhimurium, with or without the virulenceplasmid that possesses RpoS-regulated genes that play no rolein the colonization of the gut-associated lymphoid tissue (19,52), exhibit diminished immunogenicities (5, 7). These resultscollectively imply that serotype Typhi with RpoS⁺ genotype,with or without the ability to produce the Vi capsular antigen,might be superior to RpoS^– strains as a vector in thedevelopment of recombinant attenuated Salmonella vaccines forhumans (7, 8). This hypothesis requires further testing.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutesof Health (R01 AI24533, R01 AI057885, and R01 AI056289) andthe Bill and Melinda Gates Foundation (grant 37863).

We thank James Wilson (Biodesign Institute at Arizona StateUniversity), Vjollca Konjufca (Biodesign Institute at ArizonaState University), and Wei Xin (Biodesign Institute at ArizonaState University) for helpful advice and Erika Arch (BiodesignInstitute at Arizona State University) for her assistance withthe manuscript.

FOOTNOTES

* Corresponding author. Mailing address: The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, PO Box 875401, 1001 S. McAllister Avenue, Tempe, AZ 85287-5401. Phone: (480) 727-0445. Fax: (480) 727-0466. E-mail: rcurtiss{at}asu.edu.

Published ahead of print on 18 December 2006.

Editor: F. C. Fang

REFERENCES

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