Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments.

Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.     

Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Here we report the crystal structure of Skp from E. coli. The structure of the Skp trimer resembles a jellyfish with alpha-helical tentacles protruding from a beta barrel body defining a central cavity. The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC, a cytosolic chaperone present in eukaria and archea, that binds unfolded substrates in its central cavity. The ability of Skp to prevent the aggregation of model substrates in vitro is independent of ATP. Skp can interact directly with membrane lipids and lipopolysaccharide (LPS). These interactions are needed for efficient Skp-assisted folding of membrane proteins. We have identified a putative LPS binding site on the outer surface of Skp and propose a model for unfolded substrate binding.     

LPS targets host guanylate‐binding proteins to the bacterial outer membrane for non‐canonical inflammasome activation

Pathogenic and commensal Gram‐negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase‐11 non‐canonical inflammasome. Here, we show that OMV‐induced inflammasome activation requires TLR4‐TRIF signaling, the production of type I interferons, and the action of guanylate‐binding proteins (GBPs), both in macrophages and in vivo. Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram‐negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell‐autonomous immunity and innate immunity.     

Spectroscopic investigations of the binding mechanisms between antimicrobial peptides and membrane models of Pseudomonas aeruginosa and Klebsiella pneumoniae

CD spectroscopy was used to investigate the interactions of a series of synthetic AMPs with LPS isolated from Pseudomonas aeruginosa and Klebsiella pneumoniae, as well as with various phospholipids to better approximate the chemical composition of the membranes of these two strains of Gram-negative bacteria. This investigation was conducted in order to probe how the contributions of key physicochemical properties of an AMP vary in different regions of the membranes of these two bacteria. The conclusions from this study are as follows. (1) The binding interactions between the AMP and the membranes are defined by the complementarity of delocalization of positive charge density of the basic amino side chains (i.e., electrostatics), molecular flexibility of the peptide backbone, and overall hydrophobicity. (2) The binding interactions of these AMPs to LPS seem to be predominantly with the lipid A region of the LPS. (3) Incorporation of phospholipids into the LPS containing SUVs resulted in dramatic changes in the conformational equilibrium of the bound AMPs. (4) For the LPS-phospholipid models of Pseudomonas aeruginosa, delocalization of the side chain positive charge plays a major role in determining the number of conformers that contribute to the binding conformational equilibrium. This relationship was not observed for the models of the outer and inner membranes of Klebsiella pneumoniae.     

Thermodynamic analysis of the lipopolysaccharide-dependent resistance of gram-negative bacteria against polymyxin B

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay

Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydiacetylene (PDA) chromatic biomimetic membranes. Colorimetric and fluorescence analyses determined that an indolicidin analog with a proline and tryptophan residue substituted with lysines showed more pronounced bilayer surface interactions, while indolicidin and particularly an indolicidin analog in which all prolines were replaced with alanine residues exhibited deeper insertion into the lipid bilayer. The colorimetric data demonstrated that more pronounced blue-red transitions were observed when the chromatic vesicles incorporated lipopolysaccharide (LPS) within the lipid bilayer, indicating that LPS promoted preferred binding and incorporation of the peptides at the lipid/water interface. The fluorescence quenching experiments further confirmed this outcome. The results indicate that the antibacterial activity of indolicidin most likely requires initial binding to the LPS moieties within bacterial membranes, as well as disruption of the bilayer interface. The degree of hemolysis induced by the analogs, on the other hand, correlated to the extent of penetration into the hydrophobic core of the lipid assembly.     

Interaction of recombinant surfactant protein D with lipopolysaccharide: conformation and orientation of bound protein by IRRAS and simulations

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.     

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy

Lipopolysaccharide (LPS) on gram‐negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano‐resolution approaches in microbiology.     

Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Mechanism of membrane insertion of a multimeric beta-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational changes

Perfringolysin O, a bacterial cytolytic toxin, forms unusually large pores in cholesterol-containing membranes by the spontaneous insertion of two of its four domains into the bilayer. By monitoring the kinetics of domain-specific conformational changes and pore formation using fluorescence spectroscopy, the temporal sequence of domain-membrane interactions has been established. One membrane-exposed domain does not penetrate deeply into the bilayer and is not part of the actual pore, but is responsible for membrane recognition. This domain must bind to the membrane before insertion of the other domain into the bilayer is initiated. The two domains are conformationally coupled, even though they are spatially separated. Thus, cytolytic pore formation is accomplished by a novel mechanism of ordered conformational changes and interdomain communication.     

Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrin-based peptide

Treatment of Gram-negative bacterial infections with antimicrobial agents can cause release of the endotoxin lipopolysaccharide (LPS), the potent initiator of sepsis, which is the major cause of mortality in intensive care units worldwide. Structural information on peptides bound to LPS can lead to the development of more effective endotoxin neutralizers. Short linear antimicrobial and endotoxin-neutralizing peptide LF11, based on the human lactoferrin, binds to LPS, inducing a peptide fold with a "T-shaped" arrangement of a hydrophobic core and two clusters of basic residues that match the distance between the two phosphate groups of LPS. Side chain arrangement of LF11 bound to LPS extends the previously proposed LPS binding pattern, emphasizing the importance of both electrostatic and hydrophobic interactions in a defined geometric arrangement. In anionic micelles, the LF11 forms amphipathic conformation with a smaller hydrophobic core than in LPS, whereas in zwitterionic micelles, the structure is even less defined. Protection of tryptophan fluorescence quenching in the order SDS>LPS>DPC and hydrogen exchange protection indicates the decreasing extent of insertion of the N terminus and potential role of peptide plasticity in differentiation between bacterial and eukaryotic membranes.     

The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ～3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release.     

Insertion of the bacterial type III translocon: not your average needle stick

Bacterial type III secretion systems are thought to translocate virulence proteins directly from the bacterial cytoplasm into host cells through a continuous molecular channel. Little is known about how the apparatus itself interacts with membranes and whether insertion of this structure into the host membrane has consequences for the bacteria apart from its beneficial role in delivering virulence proteins. New evidence suggests that membrane insertion of the bacterial type III apparatus might turn on a calcium-dependent signaling pathway resulting in phagolysosomal fusion.     

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X = 0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Structuring and interactions of human beta-defensins 2 and 3 with model membranes.

beta-Defensins play an important role in both innate and adaptive immunity, displaying a direct anti-microbial activity against a wide variety of micro-organisms as well as interesting immuno-modulatory effects on host cells. Interaction with biological membranes appears to be a central theme in modulating these activities, leading to different consequences such as membrane lysis, translocation into the cytoplasm or transfer to a receptor. We have investigated the structuring of human beta-defensins (hBD2 and hBD3) and rationally designed variants, in relation to their interactions with real and model membranes. Biophysical methods, such as circular dichroism (CD), transmission or reflection IR and dye release were used to probe their structure/activity in the presence of model membranes, while fluorimetric and flow cytometric assays were used to investigate the effects on prokaryotic cells. Our results indicate that structural features, such as the helical N-terminal domains and oligomerisation at the membrane surface, may modulate the efficiency of membrane insertion and selectivity for microbial or host-cell membranes. We propose that both peptides interact with membranes as extended beta-sheet platforms that present amphipathic helices for insertion into the lipid bilayer.     

Single-Cell Force Spectroscopy of Interaction of Lipopolysaccharides from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis> with J774 Macrophage Membrane Using Optical Tweezers

In order to investigate quantitatively the role of lipopolysaccharides (LPS) from outer bacterial membrane at the initial state of bacterium adhesion to a host cell membrane, a model system for single cell force spectroscopy was developed and used. The system comprised of an LPS-coated microsphere placed into optical trap and a J774 macrophage being approached the microsphere to initiate their binding and then moved back to rupture the bond. An “object shadow” phenomenon was discovered, manifested as large-scale variations of the signal of photodetector registering the trapped microsphere displacement, such variations emerging long before the actual interaction between the macrophage and microsphere. The theory and the measurements technique were developed for registration of the force required for detachment of bounded microsphere from the object investigated by means of optical tweezers under the “object shadow” conditions. Characteristic spectra of binding force between J774 macrophage and microspheres functionalized with various LPS, as well as LPS plus complementary antibodies preparations were obtained at the rate of detachment force application of 3–6 pN/s. Force spectrum characteristic of Yersinia pseudotuberculosis LPS possessing O-antigen had a maximum at ~14 pN with half-width of ~23 pN. The treatment of O-antigen with complementary antibodies resulted in transformation of this spectrum into a spectrum with maximum at ~10 pN and half-width of ~14 pN, being almost identical to the spectrum of Y. pestis LPS devoid of O-antigen, with a maximum at ~9 pN and half-width of ~13 pN. A possible mechanism of force spectra formation has been proposed under assumptions of nonspecific binding of O-antigen and probable receptor-type binding of LPS core region to the macrophage surface. The elastic modulus of macrophage envelope, as estimated using analysis of displacement of the contacting microsphere as an indenter, was ≈0.17 pN/nm.     

Progressive structuring of a branched antimicrobial Peptide on the path to the inner membrane target

In recent years, interest has grown in the antimicrobial properties of certain natural and non-natural peptides. The strategy of inserting a covalent branch point in a peptide can improve its antimicrobial properties while retaining host biocompatibility. However, little is known regarding possible structural transitions as the peptide moves on the access path to the presumed target, the inner membrane. Establishing the nature of the interactions with the complex bacterial outer and inner membranes is important for effective peptide design. Structure-activity relationships of an amphiphilic, branched antimicrobial peptide (B2088) are examined using environment-sensitive fluorescent probes, electron microscopy, molecular dynamics simulations, and high resolution NMR in solution and in condensed states. The peptide is reconstituted in bacterial outer membrane lipopolysaccharide extract as well as in a variety of lipid media mimicking the inner membrane of Gram-negative pathogens. Progressive structure accretion is observed for the peptide in water, LPS, and lipid environments. Despite inducing rapid aggregation of bacteria-derived lipopolysaccharides, the peptide remains highly mobile in the aggregated lattice. At the inner membranes, the peptide undergoes further structural compaction mediated by interactions with negatively charged lipids, probably causing redistribution of membrane lipids, which in turn results in increased membrane permeability and bacterial lysis. These findings suggest that peptides possessing both enhanced mobility in the bacterial outer membrane and spatial structure facilitating its interactions with the membrane-water interface may provide excellent structural motifs to develop new antimicrobials that can overcome antibiotic-resistant Gram-negative pathogens.     

Contribution of histidine residues to oligomerization of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin

Theta-toxin (perfringolysin O) modified by diethyl pyrocarbonate, a histidine-specific reagent, lost its hemolytic activity. The modified toxin retains the activities of binding to and insertion into cholesterol-containing membranes but lacks the ability to form oligomers. These results suggest that histidine residues of theta-toxin contribute their share to cytolysis, especially the oligomerization process.     

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.