The PmlI-PmlR quorum-sensing system in Burkholderia pseudomallei plays a key role in virulence and modulates production of the MprA protease.

Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infection of humans and animals. The virulence of this pathogen is thought to depend on a number of secreted proteins, including the MprA metalloprotease. We observed that MprA is produced upon entry into the stationary phase, when the cell density is high, and this prompted us to study cell density-dependent regulation in B. pseudomallei. A search of the B. pseudomallei genome led to identification of a quorum-sensing system involving the LuxI-LuxR homologs PmlI-PmlR. PmlI directed the synthesis of an N-acylhomoserine lactone identified as N-decanoylhomoserine lactone. A B. pseudomallei pmlI mutant was significantly less virulent than the parental strain in a murine model of infection by the intraperitoneal, subcutaneous, and intranasal routes. Inactivation of pmlI resulted in overproduction of MprA at the onset of the stationary phase. A wild-type phenotype was restored following complementation with pmlI or addition of cell-free culture supernatant. In contrast, there was no significant difference between the virulence of a B. pseudomallei mprA mutant and the virulence of the wild-type strain. These results suggest that the PmlI-PmlR quorum-sensing system of B. pseudomallei is essential for full virulence in a mouse model and downregulates the production of MprA at a high cell density.

Supplement 2000 (no. 44) to the Kauffmann-White scheme.

This supplement reports the characterization of 12 new Salmonella serovars recognized in 2000 by the WHO Collaborating Centre for Reference and Research on Salmonella: nine were assigned to S. enterica subsp. enterica, two to subspecies salamae, and one to subspecies diarizonae.

Supplement 1998 (no. 42) to the Kauffmann-White scheme.

This supplement reports the characterization of 14 new Salmonella serovars recognized in 1998 by the WHO Collaborating Centre for Reference and Research on Salmonella: 11 were assigned to S. enterica subsp. enterica, one to subspecies salamae, one to subspecies diarizonae, and one to subsp. indica. In addition, the antigenic factor H:z88 is described.

Supplement 1999 (no. 43) to the Kauffmann-White scheme.

This supplement reports the characterization of 26 new Salmonella serovars recognized in 1999 by the WHO Collaborating Centre for Reference and Research on Salmonella: 15 were assigned to S. enterica subsp. enterica, seven to subspecies salamae, two to subspecies diarizonae, and one to subsp. houtenae; and one to S. bongori. In addition, the antigenic factor H:z89 is described.

Supplement 1997 (no. 41) to the Kauffmann-White scheme.

This supplement reports the characterization of 15 new Salmonella serovars recognized in 1997 by the WHO Collaborating Centre for Reference and Research on Salmonella: 8 were assigned to S. enterica subsp. enterica, 4 to subspecies salamae, 2 to subspecies diarizonae, and 1 to subsp. houtenae. In addition, the antigenic factors H:z85 and H:z87 are described and one modification to the Kauffmann-White scheme is reported.

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.

Supplement 1996 (no. 40) to the Kauffmann-White scheme.

This supplement reports the characterization of 13 new Salmonella serovars recognized in 1996 by the WHO Collaborating Centre for Reference and Research on Salmonella: 8 were assigned to S. enterica subsp. enterica, 3 to subspecies salamae and 2 to subspecies diarizonae.

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)

Supplement 1994 (no. 38) to the Kauffmann-White scheme.

This supplement reports the characterization of 24 new Salmonella serovars recognized in 1994 by the WHO Collaborating Centre for Reference and Research on Salmonella: 11 were assigned to S. enterica subsp. enterica, 6 to subspecies salamae, 6 to subspecies diarizonae and 1 to subspecies houtenae. In addition, the antigenic factor H:z83 is described.

Salmonella typhimurium TnphoA mutants with increased sensitivity to biological and chemical detergents.

Salmonella typhimurium is a ubiquitous pathogenic bacterium able to sustain the environmental conditions of the gastrointestinal tract, including biliary salts. To understand the mechanisms involved in bile salt resistance and, more generally, detergent resistance, we investigated S. typhimurium mutants produced with the random mutagenic TnphoA transposon. A total of 3,000 transpositional mutants were isolated. Three strains among the 1,432 first mutants lost the ability to grow in the presence of biological and chemical detergents. They were prototrophic and exhibited normal lipopolysaccharide and outer membrane protein profiles after SDS-PAGE. They did not show sensitivity to dyes but showed very different sensitivities to antibiotics. For each mutant strain, Southern blotting analysis revealed a unique TnphoA insertion at different chromosomal locations. These observations were confirmed by transduction experiments.

Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells.

A Salmonella typhi chromosomal locus composed of five adjacent genes, designated sipEBCDA, was identified by transposon mutagenesis as being essential for cell invasion. Products of the sip genes exhibit extensive sequence similarities to the effectors of Shigella entry into epithelial cells encoded by the virulence plasmid-borne ipa operon. Expression of sipE and sipB in a Shigella non-invasive ipaB mutant restored the ability to invade epithelial cells. The structural and functional conservation of the Sip and Ipa proteins suggests that Salmonella and Shigella entry processes are promoted by similar effectors.

Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate.

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella. One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto animated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale.