Intragenic breakpoints localized by array CGH in a t(2;6) familial translocation.

A 244K genome-wide array based comparative genomic hybridization study was carried out in a familial translocation t(2;6)(p25;p21) balanced in the mother and unbalanced in her daughter. In the past, this translocation has allowed us to localize the HLA multigene cluster to chromosome 6. With microarray technology, confirmation of the chromosome localization of the HLA system was easily obtained, showing that such approach may be applied to the breakpoint localizations of other familial structural changes when they are unbalanced. The disruption of genes at the translocation breakpoints that did not have any phenotypic consequences in the parent will allow the generation of a map of 'haplotolerant genes'. In addition, many genomic variants were detected with this technology, enlarging the possibility of analyzing their possible contribution to phenotypic diversity.

The RcsB-RcsC regulatory system of Salmonella typhi differentially modulates the expression of invasion proteins, flagellin and Vi antigen in response to osmolarity.

Entry into intestinal epithelial cells is an essential feature in the pathogenicity of Salmonella typhi, which causes typhoid fever in humans. This process requires intact motility and secretion of the invasion-promoting Sip proteins, which are targets of the type III secretion machinery encoded by the inv, spa and prg loci. During our investigations into the entry of S. typhi into cultured epithelial cells, we observed that the secretion of Sip proteins and flagellin was impaired in Vi-expressing strains. We report here that the production of Sip proteins, flagellin and Vi antigen is differentially modulated by the RcsB-RcsC regulatory system and osmolarity. This regulation occurs at both transcriptional and post-translational levels. Under low-osmolarity conditions, the transcription of iagA, invF and sipB genes is negatively controlled by the RcsB regulator, which probably acts in association with the viaB locus-encoded TviA protein. The cell surface-associated Vi polysaccharide, which was maximally produced under these growth conditions, prevented the secretion of Sip proteins and flagellin. As the NaCl concentration in the growth medium was increased, transcription of iagA, invF and sipB was found to be markedly increased, whereas transcription of genes involved in Vi antigen biosynthesis was greatly reduced. The expression of iagA, whose product is involved in invF and sipB transcription, occurred selectively during the exponential growth phase and was maximal in the presence of 300mM NaCl. At this osmolarity, large amounts of Sips and flagellin were secreted in culture supernatants. As expected from these results, and given the essential role of Sip proteins and motility in entry, RcsB and osmolarity modulated the invasive capacity of S. typhi. Together, these findings might reflect the adaptive response of S. typhi to the environments encountered during the different stages of pathogenesis.

Molecular characterization of the Salmonella typhi StpA protein that is related to both Yersinia YopE cytotoxin and YopH tyrosine phosphatase.

Analysis of the nucleotide sequence of a 4-kb DNA fragment located between the sip and iag loci on Salmonella typhi chromosome revealed three open reading frames, termed sipF, ctpA and stpA. The 82-amino-acid (aa) sipF product showed extensive similarity to the lacP protein from S. typhimurium. The StpA protein (535 aa) exhibited significant similarity to both Yersinia enterocolitica YopE cytotoxin and YopH tyrosine phosphatase. The CtpA polypeptide (130 aa) might be the molecular chaperone of the StpA protein.

Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis.

Synthesis of Vi antigen, a capsular polysaccharide expressed by Salmonella typhi, is controlled by the viaA and viaB chromosomal loci. It was previously shown that Vi antigen expression was regulated by a system similar to the rcs regulatory system involved in colanic acid synthesis in Escherichia coli. We have cloned the rcsA, rcsB, and rcsC genes from S. typhi. The predicted amino sequences of the RcsA and RcsB proteins showed a high degree of similarity to their E. coli homologs. The nucleotide sequence of the rcsC gene was partially determined and was shown to be homologous to that of its E. coli counterpart. Complementation experiments indicated that rcsB and rcsC were encompassed within the viaA locus. The RcsA protein was not involved in Vi antigen synthesis. In contrast, the RcsB protein acted as a positive regulator of Vi polysaccharide expression. By mRNA and gene fusion analyses, we studied the role of RcsB and TviA, a via-B-encoded regulatory protein characterized previously, in regulating Vi antigen synthesis. The transcriptional start point of tviA mRNA was not influenced by RcsB or TviA. In the absence of RcsB or TviA protein, transcription of tviA gave rise to only a monocistronic tviA-specific mRNA. The presence of RcsB and TriA not only increased the amount of monocistronic tviA-specific mRNA but also resulted in countranscription of tviA and tviB, which is located immediately downstream of tviA on the viaB locus. In addition, TviA protein did not appear to be subject to degradation by the Lon protease. These results strongly suggest that TviA might act in concert with RcsB at the tviA promoter to activate transcription of the genes involved in Vi polymer synthesis in S. typhi in a Lon-independent manner.

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection.

Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.

Role of the viaB locus in synthesis, transport and expression of Salmonella typhi Vi antigen.

The Vi antigen is a capsular polysaccharide expressed by Salmonella typhi, the agent of human typhoid fever. Expression of this antigen is controlled by the viaA and viaB chromosomal loci. The viaB locus is composed of 11 genes designated tviA-tviE (typhi Vi), vexA-vexE (Vi antigen export) and ORF11. We constructed S. typhi Ty2 strains carrying non-polar mutations in ten genes located at the viaB locus and examined the individual contribution of each gene to Vi phenotype. Phenotypes of the mutants and complementation experiments suggested that synthesis of Vi antigen monomer was catalysed by the TviB and TviC polypeptides. Subsequent polymerization of the polysaccharide might be catalysed by the TviE protein, but required functional TviD product. Proteins encoded by vexA, vexB and vexC directed transport of the polymer to the bacterial cell surface. Anchoring of the Vi antigen at the bacterial cell surface was dependent of the VexE protein. The TviA protein was not essential for Vi polymer synthesis. However, disruption of the tviA gene on S. typhi Ty2 chromosome strongly decreased expression of Vi antigen. This defect was fully complemented by providing tviA in trans on a recombinant plasmid. By using lacZ transcriptional fusions, it was shown that the TviA product positively regulated co-transcription of the tviA and tviB genes from a promoter located upstream of tviA. Moreover, we showed that a tviAB-lacZ fusion was not expressed in a viaA (rcsB) mutant of S. typhi.(ABSTRACT TRUNCATED AT 250 WORDS)

The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus.

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.