Author Correction: Enhanced tenacity of mycobacterial aerosols from necrotic neutrophils.

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Enhanced tenacity of mycobacterial aerosols from necrotic neutrophils.

The tuberculosis agent Mycobacterium tuberculosis is primarily transmitted through air, but little is known about the tenacity of mycobacterium-containing aerosols derived from either suspensions or infected neutrophils. Analysis of mycobacterial aerosol particles generated from bacterial suspensions revealed an average aerodynamic diameter and mass density that may allow distant airborne transmission. The volume and mass of mycobacterial aerosol particles increased with elevated relative humidity. To more closely mimic aerosol formation that occurs in active TB patients, aerosols from mycobacterium-infected neutrophils were analysed. Mycobacterium-infected intact neutrophils showed a smaller particle size distribution and lower viability than free mycobacteria. In contrast, mycobacterium-infected necrotic neutrophils, predominant in M. tuberculosis infection, revealed particle sizes and viability rates similar to those found for free mycobacteria, but in addition, larger aggregates of viable mycobacteria were observed. Therefore, mycobacteria are shielded from environmental stresses in multibacillary aggregates generated from necrotic neutrophils, which allows improved tenacity but emphasizes short distance transmission between close contacts.

Synthetic anti-endotoxin peptides interfere with Gram-positive and Gram-negative bacteria, their adhesion and biofilm formation on titanium.

AIMS: Implant-associated infections arise from the formation of bacterial biofilms, which are difficult to be treated with conventional antibiotics. Therefore, there is a need for new implant functionalizations, which inhibit biofilm formation. The aim of the present study was to characterize the effect of synthetic peptides to assess their applicability for this purpose. METHODS AND RESULTS: Two synthetic anti-endotoxin peptides, Pep19-2.5 and Pep19-4LF (Aspidasept I and II) were tested against both Gram-positive (Staphylococcus aureus and Streptococcus oralis) and Gram-negative (Pseudomonas aeruginosa and Aggregatibacter actinomycetemcomitans) bacteria associated with implant infections. Their activity was evaluated against different states of biofilm formation on the implant material titanium using CFU, live/dead fluorescence staining and confocal microscopy. Both peptides inhibited planktonic bacteria growth, impacted initial bacterial adhesion, reduced biofilm volume and increased the proportion of dead cells. Additionally, cytotoxicity analyses showed that neither peptide harmed human gingival fibroblasts nor osteoblasts at lower concentrations. CONCLUSION: A concentration-dependent antibacterial activity of both peptides against biofilms of four clinically relevant bacteria could be demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study serve as a promising basis for the improvement of these peptides in order to finally achieve a peptide-equipped antibacterial implant surface.

Effects of antimicrobial peptides on methanogenic archaea.

As members of the indigenous human microbiota found on several mucosal tissues, Methanobrevibacter smithii and Methanosphaera stadtmanae are exposed to the effects of antimicrobial peptides (AMPs) secreted by these epithelia. Although antimicrobial and molecular effects of AMPs on bacteria are well described, data for archaea are not available yet. Besides, it is not clear whether AMPs affect them as the archaeal cell envelope differs profoundly in terms of chemical composition and structure from that of bacteria. The effects of different synthetic AMPs on growth of M. smithii, M. stadtmanae, and Methanosarcina mazei were tested using a microtiter plate assay adapted to their anaerobic growth requirements. All three tested methanoarchaea were highly sensitive against derivatives of human cathelicidin, of porcine lysin, and a synthetic antilipopolysaccharide peptide (Lpep); however, sensitivities differed markedly among the methanoarchaeal strains. The potent AMP concentrations affecting growth were below 10 µM, whereas growth of Escherichia coli WBB01 was not affected at peptide concentrations up to 10 µM under the same anaerobic growth conditions. Atomic force microscopy and transmission electron microscopy revealed that the structural integrity of the methanoarchaeal cells is destroyed within 4 h after incubation with AMPs. The disruption of the cell envelope of M. smithii, M. stadtmanae, and M. mazei within a few minutes of exposure was verified by using LIVE/DEAD staining. Our results strongly suggest that the release of AMPs by eukaryotic epithelial cells is a potent defense mechanism targeting not only bacteria, but also methanoarchaea.

Endotoxins: relationship between structure, function, and activity.

Endotoxins as amphiphilic components of the outer layer of the outer membrane of Gram-negative bacteria exert their immunostimulatory activity after release from bacterial cells. Thus, the characterization of the physicochemical properties of this glycolipid in physiological fluids is of utmost importance for an understanding of cell activation processes. Here, the essential physicochemical parameters describing endotoxins such as critical micellar concentration, acyl chain fluidity, intramolecular conformation, supramolecular structures, and size as well as morphology of the aggregates are discussed and assessed with respect to their importance for an understanding of the interaction mechanisms with immunorelevant cells. The reviewed data clearly indicate that knowledge of these parameters is essential for understanding the bioactivity of not only endotoxins, but also endotoxin-like amphiphiles.

Structural prerequisites for endotoxic activity in the Limulus test as compared to cytokine production in mononuclear cells.

The structural prerequisites for lipopolysaccharide (LPS) and its partial structures for the activation of the Limulus clotting cascade (Limulus amebocyte lysate [LAL] test) are described and compared with the corresponding requirements for the activation of human immune cells such as mononuclear cells. A necessary, but not sufficient, structural motif for this is the presence of the 4(')-phosphate-diglucosamine backbone recognition structure ('epitope') in lipid A. High activity is only expressed by assemblies of endotoxins, but this is largely independent of the type of supramolecular aggregate structure. A particular conformation of the epitope within the lipid A assembly must be present, which is influenced by addition of further saccharide units to the lipid A moiety, but also reacts slightly to the acylation pattern. In contrast, the cytokine production of human immune cells induced by LPS sensitively depends on the type of its aggregate structure. In the case of a hexa-acylated bisphosphorylated lipid A structure, high activity is only observed with cubic inverted aggregates. Furthermore, addition of antimicrobial agents (such as polymyxin B) leads to a nearly complete inhibition of cytokine production, whereas the reduction in the Limulus assay is much lower. These data are important since a reliable determination of endotoxin concentrations, in particular with respect to its ability to elicit severe infections, is of high interest.

Phase diagrams of monoacylated amide-linked disaccharide glycolipids.

A series of monoacylated glycolipids with even-numbered acyl chain lengths ranging from saturated C11 to C15 and an unsaturated C17:1 fatty acid connected by an amide in linkage to the disaccharide head groups maltose, melibiose and lactose were synthesized. The structural polymorphism of the glycolipids was investigated using Fourier-transform infrared spectroscopy and differential scanning calorimetry for the detection of the gel to liquid-crystalline acyl chain melting behaviour and small-angle X-ray scattering for the elucidation of the physical structure of the lipid aggregates. Also, the phase morphology was studied by polarizing microscopy in contact preparations. The data clearly show the existence of uni- and multilamellar structures. Although only one acyl chain is present, there is no evidence for the existence of micelles - of spherical or of cylindrical (H(I)) type - or of interdigitated phases. The preference for lamellar phases seems to be correlated with the intrinsic high conformational order of the amide linkage of these compounds which inhibits the formation of highly curved structures.

The role of membrane-bound LBP, endotoxin aggregates, and the MaxiK channel in LPS-induced cell activation.

We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists (e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-alpha and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations.

Dual role of lipopolysaccharide (LPS)-binding protein in neutralization of LPS and enhancement of LPS-induced activation of mononuclear cells.

The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

Interaction between lipopolysaccharide (LPS), LPS-binding protein (LBP), and planar membranes.

The mechanism of interaction of the lipopolysaccharide (LPS)-binding protein, LBP, with differently composed symmetric and asymmetric planar lipid bilayers was investigated in electrical measurements (membrane current, potential, capacitance). From a change of the inner membrane potential difference, binding of LBP to membranes was deduced. After addition of LBP to one side of the membrane, binding of anti-LBP antibodies and LPS to LBP on both sides of the bilayer was observed. Effects resulting from an interaction of anti-LBP antiserum with membrane-bound LBP depend on the side of addition of the antiserum, indicating a directed intercalation of LBP into the membrane. Addition of LPS to the same side as LBP may induce a change of the conformation of LBP or its orientation in the membrane. Based on these observations, we propose that LBP intercalates in a directed orientation into negatively-charged membranes and assumes a transmembrane configuration. Moreover, pre-incubated complexes of LPS and LBP do not interact with membranes. These experiments show that reconstituted planar membranes are a suitable tool for investigations of the interaction of non pore-forming proteins that are involved in signal transduction.

Interaction of CAP18-derived peptides with membranes made from endotoxins or phospholipids.

Antimicrobial peptides with alpha-helical structures and positive net charges are in the focus of interest with regard to the development of new antibiotic agents, in particular against Gram-negative bacteria. Interaction between seven polycationic alpha-helical CAP18-derived peptides and different types of artificial membranes composed of phosphatidylcholine or lipopolysaccharide of the Gram-negative bacterium Escherichia coli were investigated using different biophysical techniques. Results obtained from fluorescence energy transfer spectroscopy with liposomes, monolayer measurements on a Langmuir trough, and electrophysiological measurements on planar reconstituted asymmetric bilayer membranes including the lipid matrix of the outer membrane of E. coli were correlated, and these data were, furthermore, correlated with structural parameters of the peptides (net charge, alpha-helical content, hydrophobic moment, and hydrophobicity). All peptides induced current fluctuations in planar membranes due to the formation of transient lesions above a peptide- and lipid-specific minimal clamp voltage. Antibacterial activity was exhibited only by those peptides that induced lesion formation in the reconstituted outer membrane at clamp voltages below the transmembrane potential of the natural membrane. Thus, we propose that the physicochemical properties of both the peptides as well as of the target membranes are important for antibacterial activity.

Mechanisms of action of rabbit CAP18 on monolayers and liposomes made from endotoxins or phospholipids.

We have investigated the mechanism of action of the cationic antimicrobial protein (18 kDa) CAP18 on liposomes and monolayers made from phospholipids and enterobacterial lipopolysaccharides (LPS). CAP18 intercalates into lipid matrices composed of LPS from sensitive strains, weaker into those made of LPS from a resistant strain (Proteus mirabilis strain R45) or negatively charged phospholipids, but not into those composed of neutral phosphatidylcholine. From the combination of data obtained with fluorescence resonance energy transfer and Fourier-transform infrared spectroscopy and film balance measurements, it can be concluded that structural differences in the LPS determine the depth of intercalation of CAP18 into the respective lipid matrices. Thus, we identified the L-Arap4N linked to the first Kdo of the LPS of P. mirabilis strain R45 to be responsible for the CAP18 resistance of this strain. These data provide insight into CAP18-mediated effects on the integrity of the outer membrane of Gram-negative bacteria and led to an improved model for rabbit CAP18 membrane interaction.

Molecular mechanisms of interaction of rabbit CAP18 with outer membranes of gram-negative bacteria.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.

Molecular mechanisms of polymyxin B-membrane interactions: direct correlation between surface charge density and self-promoted transport.

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoyl-phosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d = 2.4 nm +/- 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction.