This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Mar;75(3):1382-92.
doi: 10.1128/IAI.00888-06. Epub 2006 Dec 18.

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

Affiliations

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

Javier Santander et al. Infect Immun. 2007 Mar.

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.

PubMed Disclaimer

Figures

FIG. 1.
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.
FIG. 2.
Construction of suicide vector pYA3735 and generation of serotype Typhi with regulated rpoS expression (ΔPrpoS183::TTaraCPBADrpoS). (A) Construction of suicide vector pYA3735 (7,281 bp) used to generate serotype Typhi ΔPrpoS183::TTaraCPBADrpoS. (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 ΔPrpoS183::TTaraCPBADrpoS (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+ ΔPrpoS183::TTaraCPBADrpoS; 5, χ9067 serotype Typhi Ty2 RpoS− ΔPrpoS183:: TTaraCPBADrpoS; 6, χ9068 serotype Typhi Ty2 RpoS+ ΔPrpoS183::TTaraCPBADrpoS. (D) Catalase test for detection of ΔPrpoS183::TTaraCPBADrpoS without and with growth in the presence of 0.2% Ara.
FIG. 3.
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+ ΔPrpoS183::TTaraCPBADrpoS. (G) χ9067 serotype Typhi Ty2 RpoS− ΔPrpoS183::TTaraCPBADrpoS. (H) χ9068 serotype Typhi Ty2 RpoS+ ΔPrpoS183::TTaraCPBADrpoS.
FIG. 4.
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 ΔPrpoS183::TTaraCPBADrpoS RpoS. (J) χ9067 serotype Typhi Ty2 RpoS− ΔPrpoS183::TTaraCPBADrpoS 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.
FIG. 5.
Evaluation of Hd (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+ ΔPrpoS183::TTaraCPBADrpoS. (G) χ9067 serotype Typhi Ty2 RpoS− ΔPrpoS183::TTaraCPBADrpoS. (H) χ9068 serotype Typhi Ty2 RpoS+ ΔPrpoS183::TTaraCPBADrpoS.
FIG. 6.
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.

References

    1. Arricau, N., D. Hermant, H. Waxin, C. Ecobichon, P. S. Duffey, and M. Y. Popoff. 1998. The RcsB-RcsC regulatory system of Salmonella typhi differentially modulates the expression of invasion proteins, flagellin and Vi antigen in response to osmolarity. Mol. Microbiol. 29:835-850. - PubMed
    1. Black, R. E., M. M. Levine, M. L. Clements, G. Losonsky, D. Herrington, S. Berman, and S. B. Formal. 1987. Prevention of shigellosis by a Salmonella typhi-Shigella sonnei bivalent vaccine. J. Infect. Dis. 155:1260-1265. - PubMed
    1. Buchmeier, N. A., S. J. Libby, Y. Xu, P. C. Loewen, J. Switala, D. G. Guiney, and F. C. Fang. 1995. DNA repair is more important than catalase for Salmonella virulence in mice. J. Clin. Investig. 95:1047-1053. - PMC - PubMed
    1. Bueno, S. M., C. A. Santiviago, A. A. Murillo, J. A. Fuentes, A. N. Trombert, P. I. Rodas, P. Youderian, and G. C. Mora. 2004. Precise excision of the large pathogenicity island, SPI7, in Salmonella enterica serovar Typhi. J. Bacteriol. 186:3202-3213. - PMC - PubMed
    1. Coynault, C., and F. Norel. 1999. Comparison of the abilities of Salmonella typhimurium rpoS, aroA and rpoS aroA strains to elicit humoral immune responses in BALB/c mice and to cause lethal infection in athymic BALB/c mice. Microb. Pathog. 26:299-305. - PubMed

Publication types

MeSH terms

Full text links
Atypon full text link Atypon Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /