Characterization of anti-ECA antibodies in rabbit antiserum against rough <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> O:3

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA_PG) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA_LPS); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA_PG and ECA_LPS. To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain “monovalent” anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECAPG, and this suggested that in Y. enterocolitica O:3 ECA_LPS only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA_PG and ECA_LPS expression were found to be regulated by temperature and repressed at 37°C.     

A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria

A procedure is described to determine 2-keto-3-deoxyoctonate (KDO) present in lipopolysaccharide (LPS) of gram-negative bacteria. The method involves the treatment of LPS with 0.2 n H₂SO₄ at 100°C for 30 min to release KDO, followed by its reaction with periodic acid, sodium arsenite, and thiobarbituric acid. The red chromophore thus formed is kept in solution at room temperature by adding dimethylsulfoxide to the reaction mixture. The final color is stable for days at room temperature and facilitates accurate determination of KDO in microgram quantities. KDO contents of cell surface antigens and glycolipids from gram-negative bacteria are presented as illustrations of the accuracy and sensitivity of the assay.     

Protective effects of antimicrobial peptide S-thanatin against endotoxic shock in mice introduced by LPS

Sepsis continues to be a major unresolved medical challenge of the present. Severe sepsis and septic shock are the leading causes of multiple organ failure and mortality in noncoronary intensive care units (ICUs). The primary reason of septic shock is the activation of host effecter cells by endotoxin and lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the key point of treatment is removing LPS. S-thanatin (Ts), an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against gram-negative and gram-positive bacteria. We have reported its LPS-binding and -neutralizing activity in vitro. The aim of this study is to examine the LPS-neutralizing activities and the protective effects of S-thanatin in vivo. Every mice was injected intraperitoneally with LPS (from Escherichia coli O111:B4) 150 μg before injected intraperitoneally or vena caudalis with 3 mg/kg, 6 mg/kg and 12 mg/kg, and measured endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, as well as lethality. The results showed that S-thanatin can significantly reduce endotoxin and TNF-α level in plasma, at the same time resulting in the highest survival rates.     

Toll-like receptor activation of human cells by synthetic triacylated lipid A-like molecules

Recognition of microbial molecules by mammalian host receptors is essential to mount an immune response. Hexaacylated LPS is the prototypic example of a bacterial molecule recognized by the receptor complex TLR4/MD-2 with its lipid A moiety, whereas bacterial lipopeptides are recognized by TLR2. Here we show that a series of synthetic triacylated lipid A-like molecules are weak Toll-like receptor (TLR) agonists (mainly TLR2 agonists) but very potent TLR4/MD-2 antagonists (submicromolar range). Not only do they block human cell responses to LPS but also to whole gram-negative bacteria, and they inhibit the phagocytosis of gram-negative bacteria. These compounds may represent promising immunomodulatory agents.     

Targeting LPS biosynthesis and transport in gram-negative bacteria in the era of multi-drug resistance

Gram-negative bacteria pose a major threat to human health in an era fraught with multi-drug resistant bacterial infections. Despite extensive drug discovery campaigns over the past decades, no new antibiotic target class effective against gram-negative bacteria has become available to patients since the advent of the carbapenems in 1985. Antibiotic discovery efforts against gram-negative bacteria have been hampered by limited intracellular accumulation of xenobiotics, in large part due to the impermeable cell envelope comprising lipopolysaccharide (LPS) in the outer leaflet of the outer membrane, as well as a panoply of efflux pumps. The biosynthesis and transport of LPS are essential to the viability and virulence of most gram-negative bacteria. Thus, both LPS biosynthesis and transport are attractive pathways to target therapeutically. In this review, we summarize the LPS biosynthesis and transport pathways and discuss efforts to find small molecule inhibitors against targets within these pathways.     

Deacylated lipopolysaccharide inhibits plasminogen activator inhibitor-1, prostacyclin, and prostaglandin E2 induction by lipopolysaccharide but not by tumor necrosis factor-alpha

Bacterial LPS and TNF induce vascular endothelial cells to express a variety of response molecules. LPS that is partially deacylated (dLPS) by a human neutrophil enzyme blocks the ability of LPS, but not TNF, to augment one of these responses, the expression of endothelial cell surface molecules that promote neutrophil adherence (J. Exp. Med. 1987; 165:1393-1402). We show that dLPS can inhibit the ability of LPS, but not TNF, to elicit the expression of plasminogen activator inhibitor-1 (PAI-1), prostacyclin, and PGE2 by human umbilical vein endothelial cells. dLPS also prevented the accumulation of specific PAI-1 mRNA in response to LPS, but not to TNF. Neither the LPS- or TNF-induced expression of PAI-1 nor the dLPS inhibition of the LPS response was mediated by prostanoids. These results indicate that dLPS can specifically block a variety of endothelial cell responses to LPS and provide support for the hypotheses 1) that dLPS and LPS may interact with a common target molecule on or in endothelial cells, and 2) that dLPS, produced by enzymatic deacylation of LPS in vivo, could inhibit endothelial cell stimulation by LPS and thereby limit the host inflammatory response to invasive gram-negative bacteria.     

Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen.

It was demonstrated that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen with a molecular weight ranging from 42,000 to 48,000. Western blotting (immunoblotting) with an antibody specific to a 43-kDa membrane protein of Mycoplasma fermentans showed the existence of this protein antigen in all Mycoplasma spp. tested (14 species), Acholeplasma laidlawii (1 strain), and gram-negative bacteria (8 species) but only in Staphylococcus aureus of four gram-positive species tested. Neither Ureaplasma urealyticum nor mammalian cell cultures showed any cross-reactions with this antibody. These proteins were found in both cytoplasmic and membrane fractions of mycoplasma cells but were not exposed on the surface of mycoplasmal or bacterial cells.     

Investigation of a Providencia rettgeri strain that has enterobacterial common antigen in the immunogenic state

Antigenic material obtained by phenol-water extraction from Providencia rettgeri strains, Escherichia coli O:14 strains, and mutants of the E. coli O:14 strain were examined by the passive (indirect) hemagglutination technique, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by immune blotting (lipopolysaccharide (LPS) blotting). Providencia rettgeri 965, like E. coli O:14, was demonstrated to have an enterobacterial common antigen (ECA) in the immunogenic form but, unlike E. coli O:14, it possessed characteristics of a smooth strain. Two populations of molecules were observed to occur in P. rettgeri 965 phenol-water extracts: one consisting of LPS identifiable with specific O antisera and the other of ECA molecules identifiable with E. coli O:14 antiserum or with a monoclonal antibody against ECA.     

Properties of bacteria that trigger hemocytopenia in the Atlantic blue crab, Callinectes sapidus

In the blue crab Callinectes sapidus, injection with the bacterial pathogen Vibrio campbellii causes a decrease in oxygen consumption. Histological and physiological evidence suggests that the physical obstruction of hemolymph flow through the gill vasculature, caused by aggregations of bacteria and hemocytes, underlies the decrease in aerobic function associated with bacterial infection. We sought to elucidate the bacterial properties sufficient to induce a decrease in circulating hemocytes (hemocytopenia) as an indicator for the initiation of hemocyte aggregation and subsequent impairment of respiration. Lipopolysaccharide (LPS), the primary component of the gram-negative bacterial cell wall, is known to interact with crustacean hemocytes. Purified LPS was covalently bound to the surfaces of polystyrene beads resembling bacteria in size. Injection of these "LPS beads" caused a decrease in circulating hemocytes comparable to that seen with V. campbellii injection, while beads alone failed to do so. These data suggest that in general, gram-negative bacteria could stimulate hemocytopenia. To test this hypothesis, crabs were injected with different bacteria--seven gram-negative and one gram-positive species--and their effects on circulating hemocytes were assessed. With one exception, all gram-negative strains caused decreases in circulating hemocytes, suggesting an important role for LPS in the induction of this response. However, LPS is not necessary to provoke the immune response given that Bacillus coral, a gram-positive species that lacks LPS, caused a decrease in circulating hemocytes. These results suggest that a wide range of bacteria could impair metabolism in C. sapidus.     

Hepatic prolactin binding is rapidly altered by endotoxin in lactating mice

Endotoxin or lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, produces profound physiologic changes in most mammals. The effects of LPS on ovine prolactin (oPRL) binding by hepatic membranes of lactating mice is explored in this report. Specific 125I-oPRL binding by liver membranes from LPS-responder C3HfB/HeN mice increased two-fold within fifteen minutes of the injection of LPS, while no change was observed in the non-responder C3H/HeJ mice. Specific 125I-insulin binding did not change. Scatchard analysis of equilibrium binding of oPRL to C3HfB/HeN liver membranes indicated that within fifteen minutes of LPS injection, a receptor of differing binding affinity appears and then disappears by one hour post-injection. We propose that these rapid alterations in the specific binding of oPRL by liver membranes from LPS-injected, lactating C3HfB/HeN mice are due to the transient creation or unmasking of a novel class of PRL receptor.     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.     

Structures of β-hairpin antimicrobial protegrin peptides in lipopolysaccharide membranes: mechanism of gram selectivity obtained from solid-state nuclear magnetic resonance

The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both gram-positive and gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains gram-positive activity but is 30-fold less active against gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of gram-positive bacteria. (31)P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. (13)C chemical shifts and (13)C-(1)H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. Indeed, (1)H spin diffusion from lipid chains to the peptides shows that PG-1 fully inserts into the LPS-rich membrane whereas the mutant does not. The (13)C-(31)P distances between the most hydrophobically embedded Arg of PG-1 and the lipid (31)P are significantly longer in the LPS membrane than in the POPE/POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against gram-negative bacteria. The data provide a rational basis for controlling gram selectivity of AMPs by adjusting the charge densities.     

Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research.

The peptide antibiotic Polymyxin B (PMB) binds to bacterial endotoxin (lipopolysaccharide, LPS). We prepared covalent conjugates of PMB and horseradish peroxidase (HRP) by periodation of HRP-linked oligosaccharides followed by direct condensation with PMB. In addition we prepared monoclonal antibodies (Mabs) to PMB. The PMB-HRP conjugates and anti-PMB Mabs were used to study in ELISA the binding of PMB to LPS from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, PMB-HRP was used to quantify lipid A in ELISA, and to stain gram-negative bacteria histochemically. For the study of PMB-LPS interaction, PMB-HRP proved to be superior to the anti-PMB Mabs. PMB-HRP conjugates are useful general probes to detect or measure lipid A and LPS of various species using very simple methods and to stain bacteria, and they may obviate the need for many specific antisera. Thus, PMB-HRP conjugates are useful probes for endotoxin research.     

Innate immune defense of the sponge Suberites domuncula against bacteria involves a MyD88-dependent signaling pathway. Induction of a perforin-like molecule

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates gram-negative bacteria; it is inactive against gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.     

Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide.

In order to examine the immunochemistry of the core-lipid A region of Pseudomonas aeruginosa lipopolysaccharide (LPS), monoclonal antibodies (MAbs) specific for this region were produced in mice. Immunogen was prepared by coating a rough mutant of P. aeruginosa with column-purified core oligosaccharide fractions in order to enhance the immune response to the LPS core-lipid A region. Fourteen hybridoma clones were isolated, characterized, and further divided into three groups on the basis of their reactivities to rough LPS antigens in both enzyme-linked immunosorbent assays and Western immunoblots. In addition, another MAb, 18-19, designated group 1, was included in this study for defining core-lipid A epitopes. MAb 18-19 recognizes the LPS core-plus-one O-repeat unit of the serologically cross-reactive P. aeruginosa O2, O5, and O16. Group 2 MAbs are specific for the LPS outer core region and reacted with P. aeruginosa O2, O5, O7, O8, O10, O16, O18, O19, and O20, suggesting that these serotypes share a common outer core type. Group 3 MAbs recognize the inner core region and reacted with all 20 P. aeruginosa serotypes as well as with other Pseudomonas species, revealing the conserved nature of this region. Group 4 MAbs are specific for lipid A and reacted with all gram-negative organisms tested. Immunoassays using these MAbs and well-defined rough mutants, in addition to the recently determined P. aeruginosa core structures, have allowed us to precisely define immunodominant epitopes within the LPS core region.     

Homologous and heterologous antibody responses to lipopolysaccharide after enterohemorrhagic Escherichia coli infection

To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli(EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme-linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.     

Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis

It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.     

Serological diversity and chemical structures of Campylobacter jejuni low-molecular-weight lipopolysaccharides.

Low-Mr lipopolysaccharides (LPS) of Campylobacter jejuni reference strains for serotypes O:1, O:4, O:23, and O:36 were examined through the liberation of core oligosaccharides by mild acid cleavage of the ketosidic linkage of 3-deoxy-D-manno-2-octulosonic acid residues to the lipid A moiety. The liberated oligosaccharides were examined for chemical structure by compositional analysis and methylated linkage analysis in conjunction with fast atom bombardment-mass spectrometry of permethylated oligosaccharide derivatives. The results showed (i) that the LPS contained short oligosaccharide chains of branched nonrepetitive structure, to many of which N-acetylneuraminic acid residues remained attached by 2----3 linkages to 4-linked D-galactose residues in the core structure; (ii) that serotypical differences, which are not readily defined through qualitatively similar compositions, are clearly reflected in variations in linkage types and sequences of sugar residues in the outer core attached to an inner region of invariable structure; but (iii) that the presence or absence of NeuAc residues does not appear to be a basis for serotypical differences. The results also showed that oligosaccharide chains from LPS of serotypes O:1 and O:4 are distinctly different and are distinct again from those of the cross-reacting serotypes O:23 and O:36, between whose core oligosaccharide chains no differences were found. It is concluded that the structurally variable low-Mr LPS from C. jejuni show greater similarities to the lipooligosaccharides from Neisseria spp. than to the highly conserved core regions of Salmonella species. Those strains (serotypes O:23 and O:36) which also furnish high-Mr LPS are unique among gram-negative bacteria in possessing both low-Mr molecules of the Neisseria lipooligosaccharide type and high-Mr LPS of the Salmonella smooth type.     

Analysis of the immune response to lipopolysaccharide. Existence of an interspecies cross-reactive idiotype associated with anti-lipid A antibodies

LPS is the major surface glycolipid on gram-negative bacteria. In this work, we have idiotypically characterized the antibody response against LPS in different species. To do this, we have produced mAb against LPS. Binding of many of these antibodies to LPS could be inhibited by LPS and lipid A, indicating that the monoclonals are specific for lipid A, the toxic moiety of the LPS molecule. One anti-lipid A antibody, IC9, proved protective against gram-negative bacteremia and endotoxic shock in murine protection models. We generated anti-idiotypic antibodies against IC9. The binding of several of these anti-Id to IC9 was specifically inhibited by lipid A. We used these anti-Id to characterize the anti-LPS response, and the results revealed that the IC9 Id is conserved in different species. The importance of an interspecies cross-reactive Id in the response to endotoxin and its relevance in vaccine development for septic shock are discussed.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.