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Gilles Prévost

Publications and source records attributed to Gilles Prévost.

15 recordsLinked to original sources
PubMed2006

Engineered covalent leucotoxin heterodimers form functional pores: insights into S-F interactions.

The staphylococcal alpha-toxin and bipartite leucotoxins belong to a single family of pore-forming toxins that are rich in beta-strands, although the stoichiometry and electrophysiological characteristics of their pores are different. The different known structures show a common beta-sandwich domain that plays a key role in subunit-subunit interactions, which could be targeted to inhibit oligomerization of these toxins. We used several cysteine mutants of both HlgA (gamma-haemolysin A) and HlgB (gamma-haemolysin B) to challenge 20 heterodimers linked by disulphide bridges. A new strategy was developed in order to obtain a good yield for S-S bond formation and dimer stabilization. Functions of the pores formed by 14 purified dimers were investigated on model membranes, i.e. planar lipid bilayers and large unilamellar vesicles, and on target cells, i.e. rabbit and human red blood cells and polymorphonuclear neutrophils. We observed that dimers HlgA T28C-HlgB N156C and HlgA T21C-HlgB T157C form pores with similar characteristics as the wild-type toxin, thus suggesting that the mutated residues are facing one another, allowing pore formation. Our results also confirm the octameric stoichiometry of the leucotoxin pores, as well as the parity of the two monomers in the pore. Correctly assembled heterodimers thus constitute the minimal functional unit of leucotoxins. We propose amino acids involved in interactions at one of the two interfaces for an assembled leucotoxin.

Amino Acid Sequence↗
PubMed2006

LukM/LukF'-PV is the most active Staphylococcus aureus leukotoxin on bovine neutrophils.

Staphylococcus aureus is a ubiquitous pathogen causing infections in humans and domestic animals. It is often associated with bovine mastitis. Among secreted virulence factors, the leukotoxins constitute a family of toxins composed of two distinct subunits (class S and F proteins) which induce first Ca2+ influx and subsequent pore formation that allows ethidium entry. As mastitis-causing isolates harbor the genes of at least two, and often three leukotoxins, we compared the biological activities of the purified leukotoxins whose genes are found in mastitis-causing isolates on bovine polymorphonuclear neutrophils (PMN): spreading on a solid support, calcium influx and ethidium entry. In the spreading assay, the homologous pair LukM/LukF'-PV was the most active leukotoxin. Within each class, either S or F, subunits were interchangeable and generated leukotoxins with different specific activity. LukM was also very active when associated with heterologous F subunits. A similar ranking of homologous pairs was also found in the ethidium entry assay: LukM/LukF'-PV > HlgA/HlgB > HlgC/HlgB > LukE/LukD = LukEv/LukDv. In the Ca2+ flux assay, LukM/F'-PV was the most active pair, but gamma-hemolysin (Hlg) was also very efficient. LukEv/Dv was more active (twofold) than LukE/D in the spreading assay, but the two variants showed similar activities in the other two assays. Supposing that spreading and ethidium entry (pore formation) reflect toxic activities on bovine PMN, and Ca2+ influx cell activation, LukM/F'-PV was by far the most cytotoxic leukotoxin, but it was closely followed by gamma-hemolysin for PMN activation. These results suggest that LukM/F'-PV may constitute a particular virulence attribute of mastitis-causing S. aureus strains.

Animals↗
PubMed2006

Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore.

Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor-acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA-HlgB or HlgB-HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA-HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB-HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.

Amino Acid Substitution↗
PubMed2006

Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidine-rich peptides at acidic pH.

The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state (2)H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.

Amino Acid Sequence↗
PubMed2005

Healthcare-associated Staphylococcus aureus bacteremia and the risk for methicillin resistance: is the Centers for Disease Control and Prevention definition for community-acquired bacteremia still appropriate?

OBJECTIVE: To evaluate a new classification for bloodstream infections that differentiates hospital acquired, healthcare associated, and community acquired in patients with blood cultures positive for Staphylococcus aureus. DESIGN: Prospective, observational study. SETTING: Three tertiary-care, university-affiliated hospitals in Dublin, Ireland, and Strasbourg, France. PATIENTS: Two hundred thirty consecutive patients older than 18 years with blood cultures positive for S. aureus. METHODS: S. aureus bacteremia (SAB) was defined as hospital acquired if the first positive blood culture was performed more than 48 hours after admission. Other SABs were classified as healthcare associated or community acquired according to the definition proposed by Friedman et al. When available, strains of methicillin-resistant Staphylococcus aureus (MRSA) were analyzed by pulsed-field gel electrophoresis (PFGE). RESULTS: Eighty-two patients were considered as having community-acquired bacteremia according to the Centers for Disease Control and Prevention (CDC) classification. Of these 82 patients, 56% (46) had healthcare-associated SAB. MRSA prevalence was similar in patients with hospital-acquired and healthcare-associated SAB (41% vs 33%; P > .05), but significantly lower in the group with community-acquired SAB (11%; P < .03). PFGE of MRSA strains showed that most community-acquired and healthcare-associated MRSA strains were similar to hospital-acquired MRSA strains. On multivariate analysis, Friedman's classification was more effective than the CDC classification for predicting MRSA. CONCLUSION: These results support the call for a new classification for community-acquired bacteremia that would account for healthcare received outside the hospital by patients with SAB.

Aged↗
PubMed2005

Sensitive and specific detection of staphylococcal epidermolysins A and B in broth cultures by flow cytometry-assisted multiplex immunoassay.

Two of the most common bacterial skin infections of young infants and children are bullous impetigo due to Staphylococcus aureus and its more acute form, staphylococcal scalded skin syndrome. Epidermolysin A (ETA), ETB and, possibly, ETD are responsible for these diseases, which may appear as epidemics in pediatric patients. We tested the reliability of a flow cytometry-assisted multiplex immunoassay (Bio-Plex system) for the detection of ETA and ETB. The Bio-Plex system was found to be highly specific and highly sensitive for toxin concentrations of between 2 and 80,000 pg/ml. The results of this assay were 100% identical to the results of a PCR-based method. We demonstrated that this test did not generate any cross-reactions with ETD-producing isolates. The level of detection of ETB by this test differed according to culture conditions and from isolate to isolate; these results must be taken into account for diagnostic purposes.

Exfoliatins↗
PubMed2005

Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils.

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.

Apoptosis↗
PubMed2004

Crystal structure of leucotoxin S component: new insight into the Staphylococcal beta-barrel pore-forming toxins.

Staphylococcal leucocidins and gamma-hemolysins (leucotoxins) are bi-component toxins that form lytic transmembrane pores. Their cytotoxic activities require the synergistic association of a class S component and a class F component, produced as water-soluble monomers that form hetero-oligomeric membrane-associated complexes. Strains that produce the Panton-Valentine leucocidin are clinically associated with cutaneous lesions and community-acquired pneumonia. In a previous study, we determined the crystal structure of the F monomer from the Panton-Valentine leucocidin. To derive information on the second component of the leucotoxins, the x-ray structure of the S protein from the Panton-Valentine leucocidin was solved to 2.0 angstrom resolution using a tetragonal crystal form that contains eight molecules in the asymmetric unit. The structure demonstrates the different conformation of the domain involved in membrane contacts and illustrates sequence and tertiary structure variabilities of the pore-forming leucotoxins. Mutagenesis studies at a key surface residue (Thr-28) further support the important role played by these microheterogeneities for the assembly of the bipartite leucotoxins.

Amino Acid Sequence↗
PubMed2004

Crystallization and preliminary crystallographic data of a leucotoxin S component from Staphylococcus aureus.

Class S proteins of staphylococcal bicomponent pore-forming leucotoxins play an important role in membrane targetting and cell specificity. Wild-type and recombinant S components of the Panton-Valentine leucocidin (LukS-PV) were expressed in Staphylococcus aureus and Escherichia coli, respectively, and purified. Both proteins were crystallized in two crystal forms with Jeffamine M-600 as the precipitant at 285 K using the hanging-drop vapour-diffusion method and seeding techniques. Crystals belong to space group P2 (or P2(1)) and P4(1) (or P4(3)), with unit-cell parameters a = 72.3, b = 95.1, c = 108.1 A, beta = 106.4 degrees and a = b = 94.8, c = 306.2 A, respectively. A full set of X-ray diffraction data was collected to 2.1 A from a single tetragonal crystal of the wild-type protein at 100 K.

Crystallography, X-Ray↗
PubMed2004

Regulation of virulence determinants in Staphylococcus aureus: complexity and applications.

The virulence of Staphylococcus aureus is essentially determined by cell wall associated proteins and secreted toxins that are regulated and expressed according to growth phases and/or growth conditions. Gene expression is regulated by specific and sensitive mechanisms, most of which act at the transcriptional level. Regulatory factors constitute numerous complex networks, driving specific interactions with target gene promoters. These factors are largely regulated by two-component regulatory systems, such as the agr, saeRS, srrAB, arlSR and lytRS systems. These systems are sensitive to environmental signals and consist of a sensor histidine kinase and a response regulator protein. DNA-binding proteins, such as SarA and the recently identified SarA homologues (SarR, Rot, SarS, SarT, SarU), also regulate virulence factor expression. These homologues might be intermediates in the regulatory networks. The multiple pathways generated by these factors allow the bacterium to adapt to environmental conditions rapidly and specifically, and to develop infection. Precise knowledge of these regulatory mechanisms and how they control virulence factor expression would open up new perspectives for antimicrobial chemotherapy using key inhibitors of these systems.

Amino Acid Sequence↗
PubMed2004

Aeromonas simiae sp. nov., isolated from monkey faeces.

Two Aeromonas strains, IBS S6874(T) and IBS S6652, were isolated from the faeces of two healthy monkeys (Macaca fascicularis) from Mauritius that were kept in quarantine in the Centre for Primatology, Strasbourg, France. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates formed an unknown genetic lineage within the genus Aeromonas. The two isolates had nearly identical sequences (0.1 % nucleotide substitution) that were related closely to those of recognized Aeromonas species (1.7-3.5 % nucleotide substitution). DNA-DNA hybridization showed that strains IBS S6874(T) and IBS S6652 had high DNA-DNA similarity (89 %) to each other and a low level of DNA-DNA similarity to closely related taxa (18 % relatedness to Aeromonas trota and 16 % relatedness to Aeromonas schubertii). Phenotypically, the two monkey isolates differed from most previously described mesophilic Aeromonas species by their lack of haemolysis on sheep-blood agar and inability to produce indole, gas from glucose or acid from mannitol. They differed from the most closely related species, A. schubertii, by their ability to produce acid from D-cellobiose and D-sucrose and by their pyrazinamidase activity. The name Aeromonas simiae sp. nov. is proposed for these isolates; strain IBS S6874(T) (=CIP 107798(T)=CCUG 47378(T)) is the type strain.

Aeromonas↗
PubMed2003

Staphylococcal epidermolysins.

PURPOSE OF REVIEW: Staphylococcal epidermolysins are the major causative toxins of bullous impetigo and staphylococcal scalded skin syndrome. This disease is characterized by the splitting of the epidermis between two cell layers resulting in exfoliation. It predominantly affects newborn babies and exposes them to secondary infections. This leads to the risk of epidemics, especially in nurseries. With only an experimental model which consists of skin injections in newborn mice and the recent determination of three-dimensional structures, the essential function of these toxins remained controversial, split between that of specific proteases and that of superantigens. RECENT FINDINGS: Staphylococcal epidermolysins now constitute a family of toxins, with the recent characterizations of two new serotypes: ETC and ETD. They may be secreted by sensitive or methicillin-resistant strains. Four molecules were also identified in Staphylococcus hyicus responsible for exudative epidermitis in swine. While different observations suggested a proteolytic action to these toxins, the histological parallel made with pemphigus foliaceus greatly helped in the characterization of the targets for epidermolysins ETA, ETB, ETD: desmoglein-1, a desmosome-constitutive protein, and incidentally melanocyte-stimulating hormones, which accounts for the blisters observed clinically. SUMMARY: The growing complexity in staphylococcal toxins has to be taken into account both for their association with diseases and for diagnosis purposes. Even though cases of staphylococcal scalded skin syndrome in adults are rare, they raise further questions about the pathogenic features of the disease such as individual sensitivity and distribution of the toxins into the body.

Adult↗
PubMed2002

Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal gamma-haemolysins in lipid membranes.

Staphylococcal gamma-haemolysins are bicomponent toxins in a family including other leucocidins and alpha-toxin. Two active toxins are formed combining HlgA or HlgC with HlgB. Both open pores in lipid membranes with conductance, current voltage characteristics and stability similar to alpha-toxin, but different selectivity (cation instead of anion). Structural analogies between gamma-haemolysins and alpha-toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in alpha-toxin, but either positive or negative in gamma-haemolysins. Four mutants were produced (HlgA D44K, HlgB D47K, HlgB D49K and HlgB D47K/D49K) converting those negative charges to positive in HlgA and HlgB. When all charges were positive, the pores had the same selectivity and conductance as alpha-toxin, suggesting that the cluster may form an entrance electrostatic filter. As mutated HlgC-HlgB pores were less affected, additional charges in the lumen of the pore were changed (HlgB E107Q, HlgB D121N, HlgB T136D and HlgA K108T). Removing a negative charge from the lumen made the selectivity of both HlgA-HlgB D121N and HlgC-HlgB D121N more anionic. Residue D121 of HlgB is compensated by a positive residue (HlgA K108) in the HlgA-HlgB pore, but isolated in the more cation-selective HlgC-HlgB pore. Interestingly, the pore formed by HlgA K108T-HlgB, in which the positive charge of HlgA was removed, was as cation selective as HlgC-HlgB. Meanwhile, the pore formed by HlgA K108T-HlgB D121N, in which the two charge changes compensated, retrieved the properties of wild-type HlgA-HlgB. We conclude that the conductance and selectivity of the gamma-haemolysin pores depend substantially on the presence and location of charged residues in the channel.

Amino Acid Sequence↗
PubMedDate not supplied

Staphylococcus aureus bicomponent gamma-hemolysins, HlgA, HlgB, and HlgC, can form mixed pores containing all components.

Staphylococcal gamma-hemolysins are bicomponent toxins forming a protein family with leucocidins and alpha-toxin. Two active toxins (AB and CB) can be formed combining one of the class-S components, HlgA or HlgC, with the class-F component HlgB. These two gamma-hemolysins form pores with marked similarities to alpha-toxin in terms of conductance, nonlinearity of the current-voltage curve, and channel stability in the open state. AB and CB pores, however, are cation-selective, whereas alpha-toxin is anion-selective. gamma-Hemolysins' pores are hetero-oligomers formed by three or four copies of each component (indicated as 3A3B and 3C3B or 4A4B and 4C4B). Point mutants located on a beta-strand of the class-S component that forms part of the protomer-protomer contact region can prevent oligomer assembly. Interestingly, these mutants inhibit growth of pores formed not only by their natural components but also by nonstandard components. This lead to the hypothesis that mixed ABC pores could also be formed. By studying the conductance of pores, assembled in the presence of all three components (in different ratios), it was observed that the magnitudes expected for mixed pores were, indeed, present. We conclude that the gamma-hemolysin/leucocidin bicomponent toxin family may form a larger than expected number of active toxins by cross-combining various S and F components.

Erythrocytes↗