The core lipopolysaccharide of Escherichia coli is a ligand for the dendritic-cell-specific intercellular adhesion molecule nonintegrin CD209 receptor

The dendritic-cell-specific intercellular adhesion molecule nonintegrin (DC-SIGN) CD209 is a receptor for Escherichia coli K-12 that promotes bacterial adherence and phagocytosis. However, the ligand of E. coli for DC-SIGN has not yet been identified. In this study, we found that DC-SIGN did not mediate the phagocytosis of several pathogenic strains of E. coli, including enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, and uropathogenic E. coli, in dendritic cells or HeLa cells expressing human DC-SIGN antigen. However, we showed that an outer core lipopolysaccharide (LPS) (rough) mutant, unlike an inner core LPS (deep rough) mutant or O-antigen-expressing recombinant of E. coli K-12 was phagocytosed. These results demonstrate that the host cells expressing DC-SIGN can phagocytose E. coli in part by interacting with the complete core region of the LPS molecule. These results provide a mechanism for how O antigen acts as an antiphagocytic factor.     

The effect of composition of the core region of Escherichia coli K-12 lipopolysaccharides on the surface properties of cells

The pH dependences of electrokinetic potentials (EKP) of the cells of two Escherichia coli K-12 strains (D21 and JM 103) with known lipopolysaccharide (LPS) core composition have been determined by the method of microelectrophoresis. At pH 4.6-5.2, the negative surface charge of the cells with Re core LPS was reliably higher. It was shown that the interaction of bacteria with lysozyme results in a decrease of optical density of suspensions due to higher sensitivity of the cells with complete LPS core to hypotonic shock. LPS release from bacterial cell wall depended also on bacterial LPS core composition and increased with LPS core extension. Electrokinetic measurements and the study of the interaction of cells with lysozyme suggest that higher negative surface charge of E. coli JM 103 cells (Re type LPS) is associated with higher quantity and density of LPS packing in the cell wall as compared with the cells of E. coli D21 (Ra type LPS).     

Passive immunization to outer membrane proteins MLP and PAL does not protect mice from sepsis

Multiple older studies report that immunoglobulin directed to rough mutant bacteria, such as E. coli J5, provides broad protection against challenge with heterologous strains of Gram-negative bacteria. This protection was initially believed to occur through binding of immunoglobulin to bacterial lipopolysaccharide (LPS). However, hundreds of millions of dollars have been invested in attempting to develop clinically-effective anti-LPS monoclonal antibodies without success, and no study has shown that IgG from this antiserum binds LPS. Identification of the protective mechanism would facilitate development of broadly protective human monoclonal antibodies for treating sepsis. IgG from this antiserum binds 2 bacterial outer membrane proteins: murein lipoprotein (MLP) and peptidoglycan-associated lipoprotein (PAL). Both of these outer membrane proteins are highly conserved, have lipid domains that are anchored in the bacterial membrane, are shed from bacteria in blebs together with LPS, and activate cells through Toll-like receptor 2. Our goal in the current work was to determine if passive immunization directed to MLP and PAL protects mice from Gram-negative sepsis. Neither monoclonal nor polyclonal IgG directed to MLP or PAL conferred survival protection in 3 different models of sepsis: cecal ligation and puncture, an infected burn model, and an infected fibrin clot model mimicking peritonitis. Our results are not supportive of the hypothesis that either anti-MLP or anti-PAL IgG are the protective antibodies in the previously described anti-rough mutant bacterial antisera. These studies suggest that a different mechanism of protection is involved.     

Structural requirements for TLR4-mediated LPS signalling: a biological role for LPS modifications

Cells of the mucosal lining are the first to encounter invading bacteria during infection, and as such, they have developed numerous ways of detecting microbial intruders. Recently, we showed that epithelial cells recognize lipopolysaccharide (LPS) through the CD14-Toll-like receptor (TLR)-4 complex. Here, we identify the substructures of LPS that are recognized by the TLR4 receptor complex. In contrast to lipid A, the O-antigen does not mediate an inflammatory response; rather it interferes with the lipid A recognition. An Escherichia coli strain genetically modified to express penta-acylated lipid A not only showed reduced immunogenicity, but was also found to inhibit pro-inflammatory signalling induced by wild-type E. coli (hexa-acylated lipid A) as well as LPS from other bacteria of the Enterobacteriaceae family. Furthermore, penta-acylated LPS from Pseudomonas aeruginosa acted as an antagonist to hexa-acylated E. coli LPS, as did E. coli, as shown by its inhibitory effect on IL-8 production in stimulated cells. Hypo-acylated lipid A, such as that of P. aeruginosa, is found in several species within the gut microflora as well as in several bacteria causing chronic infections. Thus, our results suggest that the composition of the microflora may be important in modulating pro-inflammatory signalling in epithelial cells under normal as well as pathologic conditions.     

Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.     

Cell wall receptor for bacteriophage Mu G(+).

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.     

The effect of composition of the core region of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 lipopolysaccharides on the surface properties of cells

The pH dependences of electrokinetic potentials (EKP) of the cells of two Escherichia coli K-12 strains (D21 and JM 103) with known lipopolysaccharide (LPS) core composition have been determined by the method of microelectrophoresis. At pH 4.6–5.2, the negative surface charge of the cells with Re core LPS was reliably higher. It was shown that the interaction of bacteria with lysozyme results in a decrease of optical density of suspensions due to higher sensitivity of the cells with complete LPS core to hypotonic shock. LPS release from bacterial cell wall depended also on bacterial LPS core composition and increased with LPS core extension. Electrokinetic measurements and the study of the interaction of cells with lysozyme suggest that higher negative surface charge of E. coli JM 103 cells (Re type LPS) is associated with higher quantity and density of LPS packing in the cell wall as compared with the cells of E. coli D21 (Ra type LPS).     

The disaccharide L-alpha-D-heptose1-->7-L-alpha-D-heptose1-->of the inner core domain of Salmonella lipopolysaccharide is accessible to antibody and is the epitope of a broadly reactive monoclonal antibody.

We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS.     

Rhizobium sin-1 lipopolysaccharide (LPS) prevents enteric LPS-induced cytokine production

Endotoxin (lipopolysaccharide (LPS)), a component of Gram-negative bacteria, is among the most potent proinflammatory substances known. The lipid-A region of this molecule initiates the production of multiple host-derived inflammatory mediators, including cytokines (e.g. tumor necrosis factor-alpha (TNFalpha)). It has been a continuous effort to identify methods of interfering with the interaction between enteric LPS and inflammatory cells using natural and synthetic LPS analogs. Some of these LPS analogs (e.g. Rhodobacter spheroides LPS/lipid-A derivatives) are antagonists in human cells but act as potent agonists with cells of other species. Data reported here indicate that structurally novel LPS from symbiotic, nitrogen-fixing bacteria found in association with the root nodules of legumes do not stimulate human monocytes to produce TNFalpha. Furthermore, LPS from one of these symbiotic bacterial species, Rhizobium sp. Sin-1, significantly inhibits the synthesis of TNFalpha by human cells incubated with Escherichia coli LPS. Rhizobium Sin-1 LPS exerts these effects by competing with E. coli LPS for binding to LPS-binding protein and by directly competing with E. coli LPS for binding to human monocytes. Rhizobial lipid-A differs significantly from previously characterized lipid-A analogs in phosphate content, fatty acid acylation patterns, and carbohydrate backbone. These structural differences define the rhizobial lipid-A compounds as a potentially novel class of LPS antagonists that might well serve as therapeutic agents for the treatment of Gram-negative sepsis.     

Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice

Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice. In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice. Similar results of early LPS-induced PMN infiltration and enhanced clearance of E. coli were seen in Toll-like receptor (TLR) 4-deficient mice. Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice. In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.     

Surface topology of the Escherichia coli K-12 ferric enterobactin receptor.

Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D. A total of 11 distinct FepA epitopes were identified. The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA. These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein. Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands. Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400. One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E. coli K-12 LPS core sugars. Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin. In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified. Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.     

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.     

Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.     

Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus

The recruitment of polymorphonuclear leukocytes (PMNs) from the vascular space into the lung interstitium and airspace is an early step in the host innate immune response to bacterial invasion of these sites. To determine the ability of intact bacteria to directly elicit PMN migration across an endothelial monolayer, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli, as well as to purified E. coli LPS. Bacterial induction of PMN migration was dose dependent and elicited by > or =10(4) bacteria/ml of each of the species tested. Pretreatment of PMNs with blocking Abs to CD18 significantly inhibited migration of PMN in response to all stimuli tested, but had the most profound effect on migration to S. pneumoniae and S. aureus. Intact E. coli were 10 times more potent in inducing transmigration of PMNs than a corresponding amount of purified LPS. Bacterial induction of PMN migration did not correlate with up-regulation of surface endothelial ICAM-1 expression (purified LPS > intact E. coli > S. aureus and S. pneumoniae) nor up-regulation of VCAM-1 and E-selectin. Neutralizing Ab to ICAM-1 had no effect on PMN migration to any of the bacteria or to purified LPS. These findings demonstrate that diverse bacterial pathogens induce PMN migration across a pulmonary microvascular endothelial cell monolayer in a fashion that appears to be organism specific. In addition, intact bacteria elicit PMN-endothelial cell interactions distinct from those seen when purified bacterial products are used as agonists.     

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.     

The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes.

Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.     

Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram-negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O-polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O-antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K-12 have served as prototypes for studies on the LPS and lipo-oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.     

Binding of DC-SIGN to the Hemagglutinin of Influenza A Viruses Supports Virus Replication in DC-SIGN Expressing Cells

Dendritic cells express lectins receptors, like DC-SIGN, which allow these cells to sense glycans that are present on various bacterial and viral pathogens. Interaction of DC-SIGN with carbohydrate moieties induces maturation of dendritic cells and promotes endocytosis of pathogens which is an important property of these professional antigen presenting cells. Uptake of pathogens by dendritic cells may lead to cross-presentation of antigens or infection of these cells, which ultimately results in activation of virus-specific T cells in draining lymph nodes. Little is known about the interaction of DC-SIGN with influenza A viruses. Here we show that a virus with a non-functional receptor binding site in its hemagglutinin, can replicate in cells expressing DC-SIGN. Also in the absence of sialic acids, which is the receptor for influenza A viruses, these viruses replicate in DC-SIGN expressing cells including human dendritic cells. Furthermore, the efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin.     

The functional association of polymyxin B with bacterial lipopolysaccharide is stereospecific: studies on polymyxin B nonapeptide

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.