Didier A. Colin

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.

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.

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.

Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding

ABSTRACT In this study, the binding of F components of the staphylococcal bicomponent leukotoxins Panton-Valentine leucocidin (LukF-PV) and gamma-hemolysin (HlgB) on polymorphonuclear neutrophils (PMNs), monocytes, and lymphocytes was determined using labeled mutants and flow cytometry. Leukotoxin activity was evaluated by measuring Ca2+ entry or pore formation using spectrofluorometry or flow cytometry. Although HlgB had no affinity for cells in the absence of an S component, LukF-PV had high affinity for PMNs (dissociation constant [Kd], 6.2 ± 1.9 nM; n = 8), monocytes (Kd, 2.8 ± 0.8 nM; n = 7), and lymphocytes (Kd, 1.2 ± 0.2 nM; n = 7). Specific binding of HlgB was observed only after addition of LukS-PV on PMNs (Kd, 1.1 ± 0.2 nM; n = 4) and monocytes (Kd, 0.84 ± 0.31 nM; n = 4) or after addition of HlgC on PMNs, monocytes, and lymphocytes. Addition of LukS-PV or HlgC induced a second specific binding of LukF-PV on PMNs. HlgB and LukD competed only with LukF-PV molecules bound after addition of LukS-PV. LukF-PV and LukD competed with HlgB in the presence of LukS-PV on PMNs and monocytes. Use of antibodies and comparisons between binding and activity time courses showed that the LukF-PV molecules that bound to target cells before addition of LukS-PV were the only LukF-PV molecules responsible for Ca2+ entry and pore formation. In contrast, the active HlgB molecules were the HlgB molecules bound after addition of LukS-PV. In conclusion, LukF-PV must be linked to LukS-PV and to a binding site of the membrane to have toxin activity.

Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

Background Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. Methodology The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. μ opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. Principal Findings We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca2+. LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca2+. LPS treatment increased μ opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. Conclusions Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.

Discoupling the Ca2+‐activation from the pore‐forming function of the bi‐component Panton‐Valentine leucocidin in human PMNs

The consecutive cell activation, including Ca2+‐channel opening, and pore formation leading to human neutrophil lysis were the two functions of the staphylococcal Panton‐Valentine leucocidin attempted to be discoupled by site‐directed mutagenesis. In a first approach consisting in deletions of the cytoplasmic extremity of the transmembranous domain, we produced a LukF‐PV ΔSer125‐Leu128 with a slightly reduced Ca2+ induction but with a significantly lowered lytic activity when combined with its synergistic protein LukS‐PV. The second approach consisted in the modification of charges and/or introduction of a steric hindrance inside the pore, which also led to interesting mutated proteins: LukF‐PV G131D, G131W and G130D. The latter had an intact Ca2+ induction ability while the lytic one was 20‐fold diminished. Binding properties and intrinsic pore diameters of these discoupled toxins remained comparable to the wild‐type protein. The mutated proteins promoted interleukin‐8 secretion, but they were rather inactive in an experimental model. New insights are brought concerning the role of the two functions in the virulence of this bi‐component leucotoxin.

Control of the Oxidative Burst of Human Neutrophils by Staphylococcal Leukotoxins

ABSTRACT The ability of staphylococcal two-component leukotoxins to induce an oxidative burst and/or to prime human polymorphonuclear cells (PMNs) was studied by using spectrofluorometry or flow cytometry. At sublytic concentrations, the HlgA-HlgB, HlgA-LukF-PV, LukS-PV-LukF-PV, and HlgC-LukF-PV combinations of leukotoxins, but not the LukS-PV-HlgB and HlgC-HlgB combinations, were able to induce H2O2 production similar to the H2O2 production induced by 1 μM N-formyl-Met-Leu-Phe (fMLP). In addition, when added at sublytic concentrations, all of the leukotoxin combinations primed PMNs for H2O2 production induced by fMLP. Leukotoxin activation was dependent on the presence of Ca2+ and was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but not by N-methyl-l-arginine, an inhibitor of NO generation, which eliminates the possibility that NO plays a role in the action of leukotoxins. At higher concentrations, all leukotoxins inhibited H2O2 production by PMNs activated by fMLP, phorbol 12-myristate 13-acetate (PMA), or the leukotoxins themselves. This inhibition was not related to the pore formation induced by leukotoxins. Intracellular release of H2O2 induced by fMLP and PMA was not primed by leukotoxins but was inhibited. It seems that leukotoxin inhibition of H2O2 release is independent of pore formation but secondary to an intracellular event, as yet unknown, triggered by leukotoxins.

Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal γ‐haemolysins in lipid membranes

Staphylococcal γ‐haemolysins are bicomponent toxins in a family including other leucocidins and α‐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 α‐toxin, but different selectivity (cation instead of anion). Structural analogies between γ‐haemolysins and α‐toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in α‐toxin, but either positive or negative in γ‐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 α‐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 γ‐haemolysin pores depend substantially on the presence and location of charged residues in the channel.

Retrieving biological activity from LukF-PV mutants combined with different S components implies compatibility between the stem domains of these staphylococcal bicomponent leucotoxins.

ABSTRACT Bicomponent leucotoxins, such as Panton-Valentine leucocidin, are composed of two classes of proteins, a class S protein such as LukS-PV, which bears the cell membrane binding function, and a class F protein such as LukF-PV, which interacts to form a bipartite hexameric pore. These leucotoxins induce cell activation, linked to a Ca2+ influx, and pore formation as two consecutive and independently inhibitable events. Knowledge of the LukF-PV monomer structure has indicated that the stem domain is folded into three antiparallel β-strands in the water-soluble form and has to refold into a transmembrane β-hairpin during pore formation. To investigate the requirements for the cooperative assembly of the stems of the S and F components to produce biological activity, we introduced multiple deletions or single point mutations into the stem domains of LukF-PV and HlgB. While the binding of the mutated proteins was weakly dependent on these changes, Ca2+ influx and pore formation were affected differently, confirming that they are independent events. Ca2+ entry into human polymorphonuclear cells requires oligomerization and may follow the formation of a prepore. The activity of some of the LukF-PV mutants, carrying the shorter deletions, was actually improved. This demonstrated that a crucial event in the action of these toxins is the transition of the prefolded stem into the extended β-hairpins and that this step may be facilitated by small deletions that remove some of the interactions stabilizing the folded structure.