R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Colicin U from Shigella boydii Forms Voltage-Dependent Pores.

Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.

Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane.

Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl2) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in B. subtilis cells using fluorescence methods [PI and DiSC3(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.

Simple Way to Detect Trp to Tb3+ Resonance Energy Transfer in Calcium-Binding Peptides Using Excitation Spectrum.

The sensitized phosphorescence of Tb3+ is often used for the assessment of the ion binding to various chelating agents or natural Ca2+-binding proteins. The detailed structure of the Tb3+ excitation spectrum gives a special advantage for analysis; any extra absorption peak can be easily detected which provides simple and direct evidence that resonance energy transfer occurs. By employing the Tb3+ phosphorescence, we characterized the Ca2+-binding sites of two related peptides - self-processing module of the FrpC protein produced by bacterium Neisseria meningitidis and the shorter peptide derived from FrpC. Here we show that while the increase of direct Tb3+ excitation at 243 nm generally corresponds to Tb3+ association with various binding sites, the excitation enhancement in the 250-300 nm band signifies Tb3+-binding in the close proximity of aromatic residues. We demonstrate that the presence of resonance energy transfer could be easily detected by inspecting Tb3+ excitation spectra. Additionally, we show that the high level of specificity of Tb3+ steady state detection on the spectral level could be reached at very low Tb3+ concentrations by taking advantage of its narrow phosphorescence emission maximum at 545 nm and subtracting the averaged autofluorescence intensities outside this peak, namely at 525 and 565 nm.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches.

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis.

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry. We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.

Probing the Ca(2+)-assisted π-π interaction during Ca(2+)-dependent protein folding.

Protein folding is governed by a balance of non-covalent interactions, of which cation-π and π-π play important roles. Theoretical calculations revealed a strong cooperativity between cation-π involving alkali and alkaline earth metal ions and π-π interactions, but however, no experimental evidence was provided in this regard. Here, we characterized a Ca(2+)-binding self-processing module (SPM), which mediates a highly-specific Ca(2+)-dependent autocatalytic processing of iron-regulated protein FrpC secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis. The SPM undergoes a Ca(2+)-induced transition from an intrinsically unstructured conformation to the compact protein fold that is ultimately stabilized by the π-π interaction between two unique tryptophan residues arranged in the T-shaped orientation. Moreover, the pair of tryptophans is located in a close vicinity of a calcium-binding site, suggesting the involvement of a Ca(2+)-assisted π-π interaction in the stabilization of the tertiary structure of the SPM. This makes the SPM an excellent model for the investigation of the Ca(2+)-assisted π-π interaction during Ca(2+)-induced protein folding.

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 Ca²âº into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²âº 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 Ca²âº influx promoted by molecules locked in a Ca²âº-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.

Outer membrane and phospholipid composition of the target membrane affect the antimicrobial potential of first- and second-generation lipophosphonoxins.

Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli. We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.

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.

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.

Bacillus subtilis cardiolipin protects its own membrane against surfactin-induced permeabilization.

Surfactin, a cyclic lipoheptapeptide produced by Bacillus subtilis, is a surface-active antimicrobial that targets the barrier function of lipid membranes. It inserts itself into the membrane, where it forms conductive pores. Depending on its concentration, it eventually disintegrates the membrane in a detergent-like manner. The molecular details of this activity are not yet sufficiently understood, nor are the mechanisms that the surfactin producer employs to resist its own toxic product. We have previously shown that B. subtilis modifies its membrane lipid composition upon the onset of surfactin production, mainly increasing the cardiolipin content. Here we show that the increased cardiolipin content leads to a decreased surfactin-induced leakage of liposomes reconstituted from lipids isolated from the surfactin producer. This stabilizing effect of cardiolipin is concentration-dependent. Using a propidium iodide-based cell permeabilization assay, we further confirmed that the cytoplasmic membrane of the mutant B. subtilis strain lacking cardiolipin was substantially more susceptible to the action of surfactin, even though the amount of bound surfactin was the same as in the wild-type strain. We propose that membrane remodelling; due to the increase in cardiolipin content, contributes to the surfactin tolerance of B. subtilis.

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation.

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 degrees C) and low (20 degrees C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T(m) (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 degrees C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T(m) was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.

Diamond nanoparticles suppress lateral growth of bacterial colonies.

Diamond nanoparticles (DNPs) of various types have been recently reported to possess antibacterial properties. Studies have shown a decrease of the colony forming ability on agar plates of the bacteria that had been previously co-incubated with DNPs in the suspension. Before plating, bacteria with DNPs were adequately diluted in order to obtain a suitable number of colony forming units. However, residual DNPs were still present on an agar plate, concentrated on the surface during the plating process; this introduces a potential artifact which might affect colony growth. The effect of DNPs remaining on the surface, alongside growing bacteria, has not been previously investigated. In this work, we present the experiments designed to investigate the effect of DNPs on bacterial survival and on the growth of the bacterial colony on a solid media. We employed Escherichia coli and Bacillus subtilis as models of Gram-negative and Gram-positive bacteria, respectively, and Proteus mirabilis as a model of bacterium exhibiting swarming motility on the surfaces. We analyzed the number, area, and weight of bacterial colonies grown on the agar surface covered with DNPs. We did not observe any bactericidal effect of such applied DNPs. However, in all bacterial species used in this work, we observed the appreciable reduction of colony area, which suggests that DNPs obstruct either bacterial growth or motility. The most obvious effect on colony growth was observed in the case of motile P. mirabilis. We show that DNPs act as the mechanical barrier blocking the lateral colony growth.

Different structural requirements for adenylate cyclase toxin interactions with erythrocyte and liposome membranes.

The bifunctional Bordetella adenylate cyclase toxin-hemolysin (ACT) penetrates target cell membranes, forms cation-selective channels and subverts cellular signaling by catalyzing uncontrolled conversion of ATP to cAMP. While primarily targeting phagocytes expressing the alphaMbeta2 integrin (CD11b/CD18), the toxin can also penetrate mammalian erythrocytes lacking the receptor and membrane endocytosis. We sought here to analyze the membrane interactions of ACT in a liposome model. Insertion of ACT into liposome membranes required calcium and caused leakage of entrapped fluorescent probes due to liposome disruption, as indicated by similar release kinetics for the approximately 398 Da FITC probe and its approximately 4400 Da dextran conjugate. However, the non-acylated proACT, which does not penetrate cellular membranes, exhibited higher capacity to bind and lyze liposomes than the mature toxin, showing that the fatty-acyl modification was not required for penetration of ACT into the lipid bilayer. Individual deletions within the channel-forming, acylation and repeat domains of ACT abolished its capacity to disrupt both liposomes and erythrocytes. In contrast to erythrocyte binding, however, the liposome binding was only lost upon a simultaneous deletion of both the channel-forming and acylation domains, suggesting that the acylation domain was also involved in liposome penetration of ACT. Moreover, substitutions of glutamates 509 and 516 by lysines, which strongly enhanced the channel-forming and hemolytic activity of ACT, did not affect its capacity to disrupt liposomes. This shows that the mechanism of ACT action in cellular membranes is not fully reproduced in liposome membranes.

Short-lived fluorescence component of DPH reports on lipid--water interface of biological membranes.

Fluorescence measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) in large unilamellar phospholipid vesicles were performed to characterize the influence of the membrane physical properties on the short-lived lifetime component of the fluorescence decay. We have found that the short-lived component of DPH significantly shortens when the membrane undergoes a temperature-induced phase transition as it is known for the long-lived component of DPH. We induced membrane phase transitions also by alcohols, which are reported to be distributed different way in the membrane--ethanol close to the membrane-water interface and benzyl alcohol in the membrane core. A different effect of the respective alcohol on the short and long decay component was observed. Both the time-resolved fluorescence spectra of DPH taken during lipid vesicle staining and the lifetime dependences caused by changes of temperature and/or induced by the alcohols show that the short-lived fluorescence originates from the population of dye molecules distributed at the membrane-water interface.

Sensitivity of bacteria to diamond nanoparticles of various size differs in gram-positive and gram-negative cells.

In this study, the influence of the size and surface termination of diamond nanoparticles (DNPs) on their antibacterial activity against Escherichia coli and Bacillus subtilis was assessed. The average size and distribution of DNPs were determined by dynamic light scattering and X-ray diffraction techniques. The chemical composition of the DNPs studied by X-ray photoelectron spectroscopy showed that DNPs > 5 nm and oxidized particles have a higher oxygen content. The antibacterial potential of DNPs was assessed by the viable count method. In general, E. coli exhibited a higher sensitivity to DNPs than B. subtilis. However, in the presence of all the DNPs tested, the B. subtilis colonies exhibited altered size and morphology. Antibacterial activity was influenced not only by DNP concentration but also by DNP size and form. Whereas untreated 5-nm DNPs were the most effective against E. coli, the antibacterial activity of 18-50-nm DNPs was higher against B. subtilis. Transmission electron microscopy showed that DNPs interact with the bacterial surface, probably affecting vital cell functions. We propose that DNPs interfere with the permeability of the bacterial cell wall and/or membrane and hinder B. subtilis colony spreading.

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 alpha(M)beta2 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.

Acylation of lysine 860 allows tight binding and cytotoxicity of Bordetella adenylate cyclase on CD11b-expressing cells.

The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.

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.