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The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus
S Kolyva1, H Waxin, M Y Popoff
1Institut Pasteur, Service des Entérobactéries, Unité INSERM, Paris, France.
Abstract:
Strains of Salmonella typhi isolated from the blood of patients with typhoid fever invariably express a capsular polysaccharide, termed the Vi antigen. Vi antigen expression is controlled by two separate chromosomal loci, viaA and viaB. The viaA locus is commonly found in enteric bacteria. In contrast, the viaB locus appears to be specific to Vi-expressing strains of Salmonella and Citrobacter. Here the cloning, expression and analysis of viaB determinants from S. typhi Ty2 is described. Whole-cell DNA from strain Ty2 was size-fractionated and cloned into the pLA2917 cosmid vector. A recombinant cosmid, pVT1, conferring a Vi-positive phenotype upon Escherichia coli and upon the Vi-non-expressing strain Ty21a of S. typhi, was characterized and used for further studies. Transposon Tn5 insertion mutagenesis demonstrated that the Vi-antigen-encoding region on pVT1 consisted of a 15 kb fragment. A subclone, designated pVT3, which contained an 18 kb insert, was sufficient to confer Vi antigen expression upon E. coli and S. typhi Ty21a. Results of recombination experiments indicated that this DNA sequence was the viaB locus of S. typhi Ty2. In E. coli SE5000 maxicells, the viaB determinants encoded at least eight polypeptides, with molecular masses of 80, 65, 59, 48, 44, 39, 35 and 28 kDa. Functional characterization of viaB mutations in S. typhi Ty2 suggested that the 80 and 65 kDa proteins were required for cell-surface localization of the Vi antigen.
Insights
Researchers cloned and analyzed the viaB locus from Salmonella typhi, identifying specific genes responsible for Vi antigen expression and cell-surface localization in typhoid fever.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Salmonella typhi causes typhoid fever and expresses a capsular Vi antigen.
- Vi antigen expression is regulated by two chromosomal loci: viaA and viaB.
- The viaB locus is specific to Vi-expressing Salmonella and Citrobacter strains.
Purpose of the Study:
- To clone, express, and analyze the viaB determinants from S. typhi Ty2.
- To identify the genetic region responsible for Vi antigen production.
- To understand the role of viaB-encoded proteins in Vi antigen localization.
Main Methods:
- Size-fractionation and cosmid cloning of S. typhi Ty2 DNA into pLA2917.
- Functional analysis of recombinant cosmids in E. coli and S. typhi Ty21a.
- Transposon Tn5 mutagenesis and subclone analysis of the viaB locus.
- Maxicell analysis to identify viaB-encoded polypeptides.
- Recombination experiments to confirm the viaB locus.
Main Results:
- A recombinant cosmid, pVT1, conferred Vi-positive phenotype on E. coli and S. typhi Ty21a.
- The Vi-antigen-encoding region was mapped to a 15 kb fragment, with an 18 kb subclone (pVT3) sufficient for expression.
- Recombination experiments confirmed pVT3 contained the viaB locus.
- The viaB locus encoded at least eight polypeptides (80, 65, 59, 48, 44, 39, 35, and 28 kDa).
- Mutational analysis indicated 80 and 65 kDa proteins are essential for cell-surface localization of Vi antigen.
Conclusions:
- The viaB locus of S. typhi Ty2 has been cloned and characterized.
- Specific genes within the viaB locus are responsible for Vi antigen synthesis and localization.
- The 80 and 65 kDa proteins are crucial for the functional expression of Vi antigen on the bacterial surface.