Figure 1.
Overview of the viaB operon and Vi capsular polysaccharide biosynthesis pathway.
(A) Schematic diagram of the S. Typhi viaB operon. (B) The Vi antigen is a linear polymer of α-1,4-linked N-acetylgalatosaminuronate, nonstoichiometrically esterified with acetyl groups at the C-3. A TviB-catalyzed oxidation of UDP-N-acetylglucosamine (UDP-GlcNAc) followed by the TviC-catalyzed epimerization at the C-4 of UDP-N-acetylglucosaminuronate (UDP-GlcNAcA) results in the formation of UDP-N-acetylgalactosaminuronate (UDP-GalNAcA), the building block for Vi polymer formation. The Vi polymer is synthesized in the cytoplasm and assembly is dependent on TviD and TviE. The enzyme catalyzing the O-acetylation of the capsular polysaccharide has not yet been identified. Subsequent translocation of the polysaccharide to the cell surface follows an ATP-binding cassette (ABC) transporter-dependent process. The transporter consists of VexA, VexB, VexC and VexD. The precise function of VexE is equivocal although it might be involved in anchoring the Vi to the cell surface.
Figure 2.
Localization and Quantification of the Vi polysaccharide in different viaB transposon insertion mutants.
(A) Washed cell pellets (wc) and the corresponding supernatants (sn) of DH5α overnight cultures harboring the indicated plasmids were spotted onto nitrocellulose and blotted with anti-Vi antiserum. (B) Vi content was measured in cell culture supernatants (extracellular Vi), in a suspension of washed and fixed cells (cell associated Vi), and in cell lysates using a quantitative sandwich ELISA. Intracellular Vi was defined using the difference in Vi measured in cell lysates and fixed cells. The amount of Vi was normalized to the protein content measured in the cell pellets.
Table 1.
Overview of transposon insertion mutants analyzed in this study.
Figure 3.
Transmission electron micrographs of ultrathin sections from E. coli DH5α expressing the Vi polysaccharide.
The Vi has been labeled with the immunogold technique by using an anti-Vi antibody. (A) E. coli DH5α cells expressing the wild-type viaB operon (pGVXN158) are shown. The Vi is mainly localized on the cell surface (CS) but is also stained in the periplasmic space (PP) and inside inclusion-like bodies within the cytoplasm (IB). (B) E. coli DH5α cells containing the viaB operon with a transposon insertion in vexA (pGVXN158vexA::Tn5), the outer membrane polysaccharide export (OPX) protein, are shown. Vi was mainly stained within large inclusion-like bodies (IB) and sometimes clustered at the inside of the inner membrane (CM). Very occasionally, filamentous Vi erupting from the cell surface was labeled. (C) E. coli DH5α cells containing the viaB operon with a transposon insertion in vexC (pGVXN158vexC::Tn5), the ATP-binding protein of the ABC transporter, are shown. The Vi polysaccharide was labeled in large inclusion bodies in the cytoplasm. (D) E. coli DH5α cells containing the viaB operon with a transposon insertion in vexE (pGVXN158vexE::Tn5) are shown. The Vi polysaccharide was labeled in large inclusion bodies in the cytoplasm. (E) As a control E. coli DH5α cells containing the empty plasmid pGVXN157 are shown.
Figure 4.
Detection of the cytosolic protein GroEL in cell culture supernatants of individual vex mutants.
Either whole cells (wc) or supernatants (sn) of DH5α cell cultures carrying the indicated plasmids were separated by SDS-PAGE and after transfer to a nitrocellulose membrane, GroEL was detected by western blot using an anti-GroEL antibody. Supernatant samples were normalized by TCA precipitation to the optical density of the cell culture. In DH5α cells expressing the wild-type viaB operon (pGVXN158), GroEL is mainly detected in the cell pellet. In contrast, the supernatant of a vexA transposon insertion mutant (pGVXN158vexA::Tn5) contains substantial amounts of the cytosolic marker protein, indicating cell instability.
Figure 5.
Size exclusion chromatography of exported Vi produced by wild type and vexE transposon insertion mutant.
DH5α(pGVXN158) and DH5α(pGVXN158vexE::Tn5) cells were metabolically labeled with [3H]GlcNAc and the Vi polysaccharide was purified from the cell culture supernatants. Purified [3H]Vi polysaccharide was analyzed by size exclusion chromatography using a Sephacryl S-1000 column. The elution profiles of the [3H]Vi preparations are shown. The void volume (vo) and the inclusion volume (vi) of the column are indicated.
Table 2.
Incorporation of [3H] into purified samples (counts/total mg protein).
Table 3.
O-acetyl content of Vi.
Table 4.
Strains and plasmids used in this study.