Michel Popoff

Reproducibility and correlation study of three deoxyribonucleic acid hybridization procedures

Reproducibility of relative binding ratios (RBR) was studied with 26 labeled DNA-unlabeled DNA hybridization reactions carried out at two temperatures (60 and 75°C) and following three procedures: the S1-nuclease method with precipitation of S1-resistant DNA by trichloroacetic acid (S1-TCA), the S1-nuclease method with adsorption of S1-resistant DNA onto DE81 filters (S1-DE81), and the hydroxyapatite (HA) method. Analyses of variance indicated that different hybridization methods give different RBR, but similar percent divirgence (ΔTm) results. A curvilinear relationship was found between RBR data obtained with S1-TCA and HA procedures at 60 or 75°C, and between 60°C RBR data obtained with S1-DE81 and HA procedures. At 75°C, RBR obtained with S1-DE81 and HA methods are comparable without any transformation of the data.

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 300 mM 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.

Clostridium Perfringens Epsilon-Toxin Shows Structural Similarity to the Pore-Forming Toxin Aerolysin

ε-Toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of ε-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.

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.

Polynucleotide sequence relatedness among motileAeromonas species

Relatedness among 55 motileAeromonas strains was assessed by determining the extent of reassociation in heterologous DNA preparations. S1 nuclease and diethylaminoethyl-cellulose filters were used to separate reassociated nucleotide sequences from nonreassociated sequences and to determine the thermal stability of related nucleotide sequences. The motile aeromonads would presently consist of three species:A. hydrophila (type strain ATCC 7966),A. caviae (type strain ATCC 15468), andA. sobria (type strain CIP 7433). These three species are clearly separated on the basis of both biochemical characteristics and similarities in DNA. Each of these three species contains more than one hybridization group: three groups inA. hydrophila; two groups inA. caviae; and at least two groups inA. sobria. DNA hybridization groups are biochemically similar within each species. When additional data is available, these separate DNA hybridization groups may merit designation. Any decision to delineate species in terms of DNA hybridization groups must await a phenotypic basis for their separation.

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 2001 (no. 45) to the Kauffmann–White scheme

This supplement reports the characterization of 22 new Salmonella serovars recognized in 2001 by the WHO Collaborating Centre for Reference and Research on Salmonella: 14 were assigned to S. enterica subspecies enterica, 2 to subspecies salamae, 1 to subspecies arizonae, 4 to subspecies diarizonae and 1 to subspecies indica.

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion.

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds β-catenin, which then recruits α-catenin. α-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of α-catenin. Using a two-hybrid screen, we identified a new partner of α-catenin: ARHGAP10. This protein colocalized with α-catenin at cell–cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and α-catenin recruitment at cell–cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced α-catenin and cortical actin levels at cell–cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell–cell junctions that controls α-catenin recruitment and has a key role during L. monocytogenes uptake.

Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

ABSTRACT Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.

Molecular relationships between virulence plasmids of Salmonella serotypes typhimurium and Dublin and large plasmids of other Salmonella serotypes

All studied isolates of Salmonella serotypes abortusovis (16 strains), enteritidis (30 strains), paratyphi C (29 strains), and 2 out of 10 isolates of serotype newport harboured large 54-76-Kb plasmids. No such plasmids were found in the following serotypes: agona, bovismorbificans, heidelberg, infantis, panama, paratyphi A, paratyphi B, saintpaul, senftenberg and typhi. These plasmids and the virulence-associated plasmids of Salmonella serotypes typhimurium and dublin were compared at the molecular level. Plasmids from the same serotype usually showed similar HindIII endonuclease patterns. Plasmids from different serotypes displayed markedly different cleavage patterns. Using the 3H-labelled plasmid from serotype typhimurium strain C5 as a probe, nitrocellulose filter hybridization showed that all these plasmids shared homologous sequences distributed throughout the plasmid molecule. With the S1-nuclease method, all plasmids were 61 to 88% related to the virulence plasmid of serotype typhimurium strain C5. The large plasmids in Salmonella serotypes abortusovis, enteritidis, paratyphi C, newport and the virulence-associated plasmids in serotypes typhimurium and dublin thus constitute a single group of homology and represent a family of related plasmids. We suggest that this plasmid group may contribute to the pathogenic potential of host serotypes.