Gilles Prévost

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.

Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry identifies 90% of bacteria directly from blood culture vials

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now widely used for marker/multi-biomarker detection in medical diagnosis. We tested a new protocol for bacterial identification from blood culture broths in hospital routine by using collection tubes with separator gels on 503 included samples examined over 3 months, where 1.5 mL was injected by a syringe into BD Vacutainer tubes from BACTEC-positive bottles, before processing for bacterial protein extraction. Samples were loaded in duplicate onto the MALDI MS target, allowing a series of 12 samples to be processed in duplicate within 80 min by using Biflex III and BioTyper 2.0 software (Bruker). Including polymicrobial samples, 193 of 213 of Gram-negative bacteria (91.08%) and 284 of 319 of Gram-positive bacteria (89.02%) were correctly identified at the species level. Enterobacteriaceae constituted 35.15% of all species found, Staphylococaceae 37.96%, Streptococaceae and Enterococaceae 20.85%, Pseudomonadaceae 1.69%, and anaerobes 2.44%. In most of the polymicrobial samples, one of the species present was identified (80.9%). Seven isolates remained misidentified as Streptococcus pneumoniae, all belonging to Streptococcus mitis. Staphylococcus aureus was identified better when grown on anaero-aerobic medium, and MALDI BioTyper identification scores as low as 1.4 were pertinent, provided that four successive proposals of the same species were given. This new protocol correlates with conventional microbiology procedures by up to 90%, and by >95% for only monomicrobial samples, and provides a decreased turn-around time for identification of bacteria isolated from blood cultures, making this technology suitable also for blood cultures, with less delay and cost decreases in bacterial diagnostics, and favouring better care of patients.

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins

BACKGROUND Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Ion channels and bacterial infection: the case of β-barrel pore-forming protein toxins of Staphylococcus aureus

Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore‐forming protein family formed by the single‐component α‐hemolysin and the bicomponent leukocidins and γ‐hemolysins. The last comprise two protein elements, S and F, that co‐operatively form the active toxin. α‐Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more specifically involved in a few diseases. X‐ray crystallography of the α‐hemolysin pore has shown it is a mushroom‐shaped, hollow heptamer, almost entirely consisting of β‐structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre‐pore can trigger Ca2+‐mediated activation of some white cells, initiating an inflammatory response. Within the bicomponent toxins, γ‐hemolysins define three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like α‐hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. γ‐Hemolysin pores seem to be organized as α‐hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.

Mode of action of β-barrel pore-forming toxins of the staphylococcal α-hemolysin family

Staphylococcal alpha-hemolysin is the prototype of a family of bacterial exotoxins with membrane-damaging function, which share sequence and structure homology. These toxins are secreted in a soluble form which finally converts into a transmembrane pore by assembling an oligomeric beta-barrel, with hydrophobic residues facing the lipids and hydrophilic residues facing the lumen of the channel. Besides alpha-hemolysin the family includes other single chain toxins forming homo-oligomers, e.g. beta-toxin of Clostridium perfringens, hemolysin II and cytotoxin K of Bacillus cereus, but also the staphylococcal bi-component toxins, like gamma-hemolysins and leucocidins, which are only active as the combination of two similar proteins which form hetero-oligomers. The molecular basis of membrane insertion has become clearer after the determination of the crystal structure of both the oligomeric pore and the soluble monomer. Studies on this family of beta-barrel pore-forming toxins are important for many aspects: (i) they are involved in serious pathologies of humans and farmed animals, (ii) they are a good model system to investigate protein-membrane interaction and (iii) they are the basic elements for the construction of nanopores with biotechnological applications in various fields.

The interaction of Staphylococcus aureus bi-component γ-hemolysins and leucocidins with cells and lipid membranes

Staphylococcus aureus gamma-hemolysins (HlgA, HlgB and HlgC) and Panton-Valentine leucocidins (LukS-PV and LukF-PV) are bi-component toxins forming a protein family with some relationship to alpha-toxin. Active toxins are couples formed by taking one protein from each of the two subfamilies of the S-components (LukS-PV, HlgA and HlgC) and the F-components (LukF-PV and HlgB). We compared the mode of action of the six possible couples on leukocytes, red blood cells and model lipid membranes. All couples were leucotoxic on human monocytes, whereas only four couples (HlgA+HlgB, HlgC+HlgB, LukS-PV+HlgB and HlgA+LukF-PV) were hemolytic. Toxins HlgA+HlgB and HlgC+HlgB were also able to induce permeabilisation of model membranes by forming pores via oligomerisation. The presence of membrane-bound aggregates, the smallest and most abundant of which had molecular weight and properties similar to that formed by alpha-toxin, was detected by SDS-PAGE. By infrared spectroscopy in the attenuated total reflection configuration (FTIR-ATR), the secondary structure of both components and of the aggregate were determined to be predominantly beta-sheet and turn with small variations among different toxins. Polarisation experiments indicated that the structure of the membrane complex was compatible with the formation of a beta-barrel oriented perpendicularly to the plane of the membrane, similar to that of porins. The couple LukS-PV+LukF-PV was leucotoxic, but not hemolytic. When challenged against model membranes it was able to bind to the lipid vesicles and to form the aggregate with the beta-barrel structure, but not to increase calcein permeability. Thus, the pore-forming effect correlated with the hemolytic, but not with the complete leucotoxic activity of these toxins, suggesting that other mechanisms, like the interaction with endogenous cell proteins, might also play a role in their pathogenic action.

Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding

ABSTRACT The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (Kd = 0.07 ± 0.02 nM, n = 5) and monocytes (Kd = 0.020 ± 0.003 nM,n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.

The structure of Staphylococcus aureus epidermolytic toxin A, an atypic serine protease, at 1.7 A resolution.

BACKGROUND Staphylococcal epidermolytic toxins A and B (ETA and ETB) are responsible for the staphylococcal scalded skin syndrome of newborn and young infants; this condition can appear just a few hours after birth. These toxins cause the disorganization and disruption of the region between the stratum spinosum and the stratum granulosum--two of the three cellular layers constituting the epidermis. The physiological substrate of ETA is not known and, consequently, its mode of action in vivo remains an unanswered question. Determination of the structure of ETA and its comparison with other serine proteases may reveal insights into ETAs catalytic mechanism. RESULTS The crystal structure of staphylococcal ETA has been determined by multiple isomorphous replacement and refined at 1.7 A resolution with a crystallographic R factor of 0.184. The structure of ETA reveals it to be a new and unique member of the trypsin-like serine protease family. In contrast to other serine protease folds, ETA can be characterized by ETA-specific surface loops, a lack of cysteine bridges, an oxyanion hole which is not preformed, an S1 specific pocket designed for a negatively charged amino acid and an ETA-specific specific N-terminal helix which is shown to be crucial for substrate hydrolysis. CONCLUSIONS Despite very low sequence homology between ETA and other trypsin-like serine proteases, the ETA crystal structure, together with biochemical data and site-directed mutagenesis studies, strongly confirms the classification of ETA in the Glu-endopeptidase family. Direct links can be made between the protease architecture of ETA and its biological activity.

Structural Determinants of Antimicrobial and Antiplasmodial Activity and Selectivity in Histidine-rich Amphipathic Cationic Peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using d-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2

Background Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.