Ivo Konopásek

Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin MEMBRANE TRANSLOCATION OF AC DOMAIN POLYPEPTIDE PROMOTES CALCIUM INFLUX INTO CD11b+ MONOCYTES INDEPENDENTLY OF THE CATALYTIC AND HEMOLYTIC ACTIVITIES

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the αMβ2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC– toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC– toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

Time-resolved polarized fluorescence studies of the temperature adaptation in Bacillus subtilis using DPH and TMA-DPH fluorescent probes

The validity of the concept of homeoviscous adaptation was tested for bacteria Bacillus subtilis. The Bacillus subtilis grown at 20 degrees C (referred to as Bs20) exhibit a considerable increase of branched anteiso-C15, the major fatty acid component of membrane lipids, relative to membranes grown at 40 degrees C (Bs40). The time-resolved fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) showed that these changes in the lipid composition are accompanied by changes in a mean lipid order. In particular, the DPH order parameters and measured in Bs20 membranes at 18 degrees C and in Bs40 membranes at 45 degrees C, respectively, tend to be equal. This effect was less pronounced for TMA-DPH. Our observations suggest that a physical parallel to the changes of lipid composition is the maintenance of an optimal lipid order in the hydrophobic core of the cytoplasmic membranes. It can be interpreted as a tendency of Bacillus subtilis to keep the lateral pressure in its membranes at an optimal value, independent of the temperature of cultivation.

Fluorescence decay and depolarization in membranes

In optically discontinuous and/or anisotropic environments such as lipid membranes, the radiative decay rate is dependent on the orientation of the emission dipole. A theory of fluorescence decay and depolarization in membranes has been developed that takes the orientational dependence into account. The theory predicts nonexponential total emission intensity decay and emission anisotropy decay different than the one in optically isotropic systems. 1,6‐diphenyl‐1,3,5‐hexatriene fluorescence in L‐α‐dipalmitoyl‐phosphatidylcholine large unilamellar vesicles has been analyzed in terms of this theory. It has been shown that the orientational dependence of the radiative rate is sufficient to account for the complexity of the intensity decay in membranes. The analysis also allowed the determination of the refractive index, orientational order, and rotational dynamics in the membrane.

Cold shock in Bacillus subtilis: different effects of benzyl alcohol and ethanol on the membrane organisation and cell adaptation.

A temperature shift-down of Bacillus subtilis from 40 to 20 degrees C induces an 80 min growth lag. Benzyl alcohol reduced this period to 51 min, whereas ethanol prolonged it up to 102 min. The effect of the two alcohols on the membrane state was investigated by measuring the steady-state fluorescence anisotropy and analysing the lifetime distribution of diphenylhexatriene (DPH) and its polar derivative, TMA-DPH. As followed from the fluorescence anisotropy, the two alcohols exerted similar (fluidizing) effects on the cytoplasmic membranes of B. subtilis. However, benzyl alcohol significantly shortened the main DPH lifetime component and widened its distribution, while ethanol had no effect. The benzyl alcohol activity was interpreted in terms of an increased membrane hydration due to disordering of the membrane structure. Such an effect imitates the cold shock induced synthesis of unsaturated fatty acids in B. subtilis. The fatty acid analysis revealed that ethanol hindered this adaptive synthesis of fatty acids. At the same time, its effect on the membrane state (membrane order) was very low and could not substitute the physiological response as was the case with benzyl alcohol. It can thus be concluded that the adaptation of the membrane physical state contributes significantly to the cold shock response of B. subtilis.

THE TRANSMEMBRANE GRADIENT OF THE DIELECTRIC CONSTANT INFLUENCES THE DPH LIFETIME DISTRIBUTION

The fluorescence lifetime distribution of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) and 1‐[4‐(trimethylamino)phenyll‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH) in egg‐phosphatidylcholine liposomes was measured in normal and heavy water. The lower dielectric constant (by ∼12%) of heavy water compared with normal water was employed to provide direct evidence that the drop of the dielectric constant along the membrane normal shifts the centers of the distribution of both DPH and TMA‐DPH to higher values and sharpens the widths of the distribution. The profile of the dielectric constant along the membrane normal was not found to be a linear gradient (in contrast to [1]) but a more complex function. Presence of cholesterol in liposomes further shifted the center of the distributions to higher value and sharpened them. In addition, it resulted in a more gradient‐like profile of the dielectric constant (i.e. linearization) along the normal of the membrane. The effect of the change of dielectric constant on the membrane proteins is discussed.

Differences in Cold Adaptation of Bacillus subtilis under Anaerobic and Aerobic Conditions

Bacillus subtilis, which grows under aerobic conditions, employs fatty acid desaturase (Des) to fluidize its membrane when subjected to temperature downshift. Des requires molecular oxygen for its activity, and its expression is regulated by DesK-DesR, a two-component system. Transcription of des is induced by the temperature downshift and is decreased when membrane fluidity is restored. B. subtilis is also capable of anaerobic growth by nitrate or nitrite respiration. We studied the mechanism of cold adaptation in B. subtilis under anaerobic conditions that were predicted to inhibit Des activity. We found that in anaerobiosis, in contrast to aerobic growth, the induction of des expression after temperature downshift (from 37 degrees C to 25 degrees C) was not downregulated. However, the transfer from anaerobic to aerobic conditions rapidly restored the downregulation. Under both aerobic and anaerobic conditions, the induction of des expression was substantially reduced by the addition of external fluidizing oleic acid and was fully dependent on the DesK-DesR two-component regulatory system. Fatty acid analysis proved that there was no desaturation after des induction under anaerobic conditions despite the presence of high levels of the des protein product, which was shown by immunoblot analysis. The cold adaptation of B. subtilis in anaerobiosis is therefore mediated exclusively by the increased anteiso/iso ratio of branched-chain fatty acids and not by the temporarily increased level of unsaturated fatty acids that is typical under aerobic conditions. The degrees of membrane fluidization, as measured by diphenylhexatriene fluorescence anisotropy, were found to be similar under both aerobic and anaerobic conditions.

The origin of the diphenylhexatriene short lifetime component in membranes and solvents

Abstract Fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) was found to be bicomponental in lipid bilayers and isotropic solvents. Mean lifetime values of both components correlated with the permittivity of the solvents. Decomposition of time-resolved spectra of DPH in phospholipid vesicles revealed a significant red shift of the spectrum of the short-lived component with respect to the spectrum of the long-lived component. This indicated that part of DPH was in a hydrophilic environment, which was supported by fluorescence energy transfer experiments. We showed that the increased fraction of the short-lived component in lipid bilayers originates from the population of DPH at the membrane–water interface.

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: alphahemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size - 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities alpha for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV(1000)) were found to be more suitable for distinguishing between high alpha values whereas smaller ones (LUV(100)) were more appropriate for discriminating an all-or-none leakage (alpha=0) from the graded leakage with low values of alpha. While disrupting LUV(1000), CyaA caused a highly cation-selective leakage (alpha~15) whereas its mutated form with decreased channel K(+)/Cl(-) selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K+E516K) exhibited much lower selectivity (alpha approximately 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.