Role of RpoS in fine-tuning the synthesis of Vi capsular polysaccharide in Salmonella enterica serotype Typhi

Abstract

Regulation of the synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi, is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some serotype Typhi strains exhibit overexpression of Vi polysaccharide, which masks clinical detection of lipopolysaccharide O antigen. This variation in Vi polysaccharide and O antigen display (VW variation) has been observed since the initial studies of serotype Typhi. In this study, we report that rpoS plays a role in this increased expression in Vi polysaccharide. We constructed a variety of isogenic serotype Typhi mutants that differed in their expression levels of RpoS and examined the role of the rpoS product in synthesis of Vi polysaccharide under different osmolarity conditions. Vi polysaccharide synthesis was also examined in serotype Typhi mutants in which the native promoter of the rpoS was replaced by an araCP(BAD) cassette, so that the expression of rpoS was arabinose dependent. The RpoS(-) strains showed increased syntheses of Vi polysaccharide, which at low and medium osmolarities masked O antigen detection. In contrast, RpoS(+) strains showed lower syntheses of Vi polysaccharide, and an increased detection of O antigen was observed. During exponential growth, when rpoS is unstable or present at low levels, serotype Typhi RpoS(+) strains overexpress the Vi polysaccharide at levels comparable to those for RpoS(-) strains. Our results show that RpoS is another regulator of Vi polysaccharide synthesis and contributes to VW variation in serotype Typhi, which has implications for the development of recombinant attenuated Salmonella vaccines in humans.

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.

FIG. 2.

Construction of suicide vector pYA3735 and generation of serotype Typhi with regulated rpoS expression (ΔP_rpoS183::TTaraCP_BADrpoS). (A) Construction of suicide vector pYA3735 (7,281 bp) used to generate serotype Typhi ΔP_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 ΔP_rpoS183::TTaraCP_BADrpoS (2,100-bp) mutants. Lanes: 1, χ3744 serotype Typhi ISP1820 RpoS⁺; 2, χ3769 serotype Typhi Ty2 RpoS⁻; 3, χ8438 serotype Typhi Ty2 RpoS⁺; 4, χ9066 serotype Typhi ISP1820 RpoS⁺ ΔP_rpoS183::TTaraCP_BADrpoS; 5, χ9067 serotype Typhi Ty2 RpoS⁻ ΔP_rpoS183:: TTaraCP_BADrpoS; 6, χ9068 serotype Typhi Ty2 RpoS⁺ ΔP_rpoS183::TTaraCP_BADrpoS. (D) Catalase test for detection of ΔP_rpoS183::TTaraCP_BADrpoS without and with growth in the presence of 0.2% Ara.

FIG. 3.

Evaluation of RpoS expression at different osmolarities by Western blot analysis. (A) χ3744 serotype Typhi ISP1820 RpoS⁺. (B) χ3769 serotype Typhi Ty2 RpoS⁻. (C) χ8438 serotype Typhi Ty2 RpoS⁺. (D) χ9060 serotype Typhi Ty2 rpoSΩAp RpoS⁻. (E) χ9061 serotype Typhi ISP1820 rpoSΩAp RpoS⁻. (F to H) Assays conducted in the absence and presence of arabinose (0.2%). (F) χ9066 serotype Typhi ISP1820 RpoS⁺ ΔP_rpoS183::TTaraCP_BADrpoS. (G) χ9067 serotype Typhi Ty2 RpoS⁻ ΔP_rpoS183::TTaraCP_BADrpoS. (H) χ9068 serotype Typhi Ty2 RpoS⁺ ΔP_rpoS183::TTaraCP_BADrpoS.

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, χ9197 serotype Typhi Ty2 RpoS⁻ ΔtviAEBCD10. (C) Negative control, χ9198 serotype Typhi ISP1820 RpoS⁺ ΔtviAEBCD10. (D) χ3744 serotype Typhi ISP1820 RpoS⁺. (E) χ3769 serotype Typhi Ty2 RpoS⁻. (F) χ8438 serotype Typhi Ty2 RpoS⁺. (G) χ9060 serotype Typhi Ty2 rpoSΩAp. (H) χ9061 serotype Typhi ISP1820 rpoSΩAp. (I) Noninduced χ9066 serotype Typhi ΔP_rpoS183::TTaraCP_BADrpoS RpoS. (J) χ9067 serotype Typhi Ty2 RpoS⁻ ΔP_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).

FIG. 5.

Evaluation of H_d (serotype Typhi flagella factor) expression at different osmolarities by Western blot analysis. (A) χ3744 serotype Typhi ISP1820 RpoS⁺. (B) χ3769 serotype Typhi Ty2 RpoS⁻. (C) χ8438 serotype Typhi Ty2 RpoS⁺. (D) χ9060 serotype Typhi Ty2 rpoSΩAp RpoS⁻. (E) χ9061 serotype Typhi ISP1820 rpoSΩAp RpoS⁻. (F to H) Assays conducted in the absence and presence of arabinose (0.2%). (F) χ9066 serotype Typhi ISP1820 RpoS⁺ ΔP_rpoS183::TTaraCP_BADrpoS. (G) χ9067 serotype Typhi Ty2 RpoS⁻ ΔP_rpoS183::TTaraCP_BADrpoS. (H) χ9068 serotype Typhi Ty2 RpoS⁺ ΔP_rpoS183::TTaraCP_BADrpoS.

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.