Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification.

Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

A TLR4 Agonist Induces Osteosarcoma Regression by Inducing an Antitumor Immune Response and Reprogramming M2 Macrophages to M1 Macrophages.

Osteosarcoma (OsA) has limited treatment options and stagnant 5-year survival rates. Its immune microenvironment is characterized by a predominance of tumor-associated macrophages (TAMs), whose role in OsA progression remain unclear. Nevertheless, immunotherapies aiming to modulate macrophages activation and polarization could be of interest for OsA treatment. In this study, the antitumor effect of a liposome-encapsulated chemically detoxified lipopolysaccharide (Lipo-MP-LPS) was evaluated as a therapeutic approach for OsA. Lipo-MP-LPS is a toll-like receptor 4 (TLR4) agonist sufficiently safe and soluble to be IV administered at effective doses. Lipo-MP-LPS exhibited a significant antitumor response, with tumor regression in 50% of treated animals and delayed tumor progression in the remaining 50%. The agent inhibited tumor growth by 75%, surpassing the efficacy of other immunotherapies tested in OsA. Lipo-MP-LPS modulated OsA's immune microenvironment by favoring the transition of M2 macrophages to M1 phenotype, creating a proinflammatory milieu and facilitating T-cell recruitment and antitumor immune response. Overall, the study demonstrates the potent antitumor effect of Lipo-MP-LPS as monotherapy in an OsA immunocompetent model. Reprogramming macrophages and altering the immune microenvironment likely contribute to the observed tumor control. These findings support the concept of immunomodulatory approaches for the treatment of highly resistant tumors like OsA.

A systemically administered detoxified TLR4 agonist displays potent antitumor activity and an acceptable tolerance profile in preclinical models.

Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.

Correction: Leptospiral LPS escapes mouse TLR4 internalization and TRIF-associated antimicrobial responses through O antigen and associated lipoproteins.

[This corrects the article DOI: 10.1371/journal.ppat.1008639.].

Leptospiral LPS escapes mouse TLR4 internalization and TRIF­associated antimicrobial responses through O antigen and associated lipoproteins.

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.

Regulation of waaH by PhoB during Pi Starvation Promotes Biofilm Formation by Escherichia coli O157:H7.

In open environments such as water, enterohemorrhagic Escherichia coli O157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. This activates the phosphate-specific transport (Pst) system that contains a high-affinity Pi transporter. In the Δpst mutant, PhoB is constitutively activated and regulates the expression of genes in the Pho regulon. Here, we show that Pi starvation and deletion of the pst system enhance E. coli O157:H7 biofilm formation. Among differentially expressed genes of EDL933 grown under Pi starvation conditions and in the Δpst mutant, we have found that a member of the PhoB regulon, waaH, predicted to encode a glycosyltransferase, was highly expressed. Interestingly, WaaH contributed to biofilm formation of E. coli O157:H7 during both Pi starvation and in the Δpst mutant. In the Δpst mutant, the presence of waaH was associated with lipopolysaccharide (LPS) R3 core type modifications, whereas in E. coli O157:H7, waaH overexpression had no effect on LPS structure during Pi starvation. Therefore, waaH participates in E. coli O157:H7 biofilm formation during Pi starvation, but its biochemical role remains to be clarified. This study highlights the importance of the Pi starvation stress response to biofilm formation, which may contribute to the persistence of E. coli O157:H7 in the environment.IMPORTANCE Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen that causes bloody diarrhea that can result in renal failure. Outside of mammalian hosts, E. coli O157:H7 survives for extended periods of time in nutrient-poor environments, likely as part of biofilms. In E. coli K-12, the levels of free extracellular Pi affect biofilm formation; however, it was unknown whether Pi influences biofilm formation by E. coli O157:H7. Our results show that upon Pi starvation, PhoB activates waaH expression, which favors biofilm formation by E. coli O157:H7. These findings suggest that WaaH is a target for controlling biofilm formation. Altogether, our work demonstrates how adaptation to Pi starvation allows E. coli O157:H7 to occupy different ecological niches.

Structure function relationships in three lipids A from the Ralstonia genus rising in obese patients.

The identification of a functional molecular moiety relating the lipopolysaccharides (LPSs) to their capacity to induce inflammation-mediated metabolic diseases needed to be performed. We previously described a proportional increase in the relative abundance of the 16 SrDNA bacterial gene from the genus Ralstonia, within the microbiota from the adipose tissue stroma vascular fraction of obese patients, suggesting a causal role of the bacteria. Therefore, we first characterized the structures of the lipids A, the inflammatory inducing moieties of LPSs, of three Ralstonia species: Ralstonia eutropha, R. mannitolilytica and R. pickettii, and then compared each, in terms of in vitro inflammatory capacities. R. pickettii lipid A displaying only 5 Fatty Acids (FA) was a weaker inducer of inflammation, compared to the two other species harboring hexa-acylated lipids A, despite the presence of 2 AraN substituents on the phosphate groups. With regard to in vitro pro-inflammatory activities, TNF-α and IL-6 inducing capacities were compared on THP-1 cells treated with LPSs isolated from the three Ralstonia. R. pickettii, with low inflammatory capacities, and recently involved in nosocomial outcomes, could explain the low inflammatory level reported in previous studies on diabetic patients and animals. In addition, transmission electron microscopy was performed on the three Ralstonia species. It showed that the R. pickettii under-acylated LPSs, with a higher level of phosphate substitution had the capacity of producing more outer membrane vesicles (OMVs). The latter could facilitate transfer of LPSs to the blood and explain the increased low-grade inflammation observed in obese/diabetic patients.

A comparative study of the complete lipopolysaccharide structures and biosynthesis loci of Bordetella avium, B. hinzii, and B. trematum.

A dozen species of human and animal pathogens have been described to date in the Bordetella genus, with the majority being respiratory tract pathogens. Bordetella avium lipopolysaccharides have been shown to be important virulence factors for this bird pathogen. B. hinzii is closely related to the B. avium species, but has also been isolated from humans. B. trematum is associated to ear and blood infections in humans. Its lipid A structure, the biological active moiety of LPS, was found to be closely related to those of B. avium and B. hinzii. It is important to unveil the subtle structural modifications orchestrated during the LPS biosynthetic pathway to better understand host adaptation. The present data are also important in the context of deciphering the virulence pathways of this important genus containing the major pathogens B. pertussis and B. parapertussis, responsible for whooping cough. We recently reported the isolated lipid A structures of the three presented species, following the previously identified O-chain structures. In the present study, we provide details on the free and O-chain-linked core oligosaccharides which were required to characterize the complete LPS structures. Data are presented here in relation to relevant biosynthesis genes. The present characterization of the three species is well illustrated by Matrix Assisted Laser Desorption Mass Spectrometry experiments, and data were obtained mainly on native LPS molecules for the first time.

Fine structure analysis of lipopolysaccharides in bacteriophage-resistant Pseudomonas aeruginosa PAO1 mutants.

Pseudomonas aeruginosa lipopolysaccharides (LPS) serve as primary receptors for many bacteriophages and, consequently, their biosynthesis is frequently affected in phage-resistant mutants. We previously isolated phage-resistant PAO1 mutants using three different phages, and showed that they were affected in the synthesis of LPS. Here we have investigated in detail the effect of mutations in seven genes involved in different steps of the production of core and oligosaccharide chains. The band profile of purified LPS was analysed by PAGE, and we further characterized the O-chains and core structures by MALDI mass spectrometry (MS). Mild LPS extraction conditions and native LPS MS analyses helped unveil lipid A molecular species with three phosphate residues in the close vicinity of the already highly charged inner-core region. No other MS direct analysis has allowed this peculiarity to be demonstrated for native lipid A high-molecular-weight molecular species, in normal growth conditions and without involving separation techniques. The present results shed light on the possible interactions between the phages and the LPS structures in the early phase of infection.

Micromethods for Isolation and Structural Characterization of Lipid A, and Polysaccharide Regions of Bacterial Lipopolysaccharides.

Lipopolysaccharides (LPS) are major components of the external membrane of most Gram-negative bacteria, providing them with an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region (PS) linked to a hydrophobic one, termed lipid A. The LPS polysaccharide moiety is divided into the core oligosaccharide (OS) and O-chain repetitive elements. Depending on their individual variable fine structures, LPS may be potent immunomodulators. The lipid A structure is a key determinant for LPS activity. However, the presence of the core region, or at least of the highly charged 3-deoxy-d-manno-oct-2-ulosonic acid molecules, is also important for preserving the native lipid A conformation within individual LPS molecules. We describe herein four rapid and practical micromethods for LPS, lipid A, and core OS structural analyses. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids enabling the characterization of their substitution position in the lipid A backbone, to be determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The third one is a microscale procedure for the mass spectra screening of LPS, lipid A, and PS using triethylamine and citric acid. The fourth method is a chromatography procedure for Rough-type LPS on thin-layer-chromatography. These methods were developed to be coupled to mass-spectrometry (e.g., MALDI-MS) but can also be used with other analytical techniques (e.g., chromatography). Examples are given with reference to two major human pathogens: Bordetella pertussis and Pseudomonas aeruginosa; to one porcine pathogen: Actinobacillus pleuropneumoniae; and to commercial samples of Salmonella Minnesota Re595 LPS.

Bordetella holmesii: Lipid A Structures and Corresponding Genomic Sequences Comparison in Three Clinical Isolates and the Reference Strain ATCC 51541.

Bordetella holmesii can cause invasive infections but can also be isolated from the respiratory tract of patients with whooping-cough like symptoms. For the first time, we describe the lipid A structure of B. holmesii reference strain ATCC 51541 (alias NCTC12912 or CIP104394) and those of three French B. holmesii clinical isolates originating from blood (Bho1) or from respiratory samples (FR4020 and FR4101). They were investigated using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The analyses revealed a common bisphosphorylated ß-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Similar to B. avium, B. hinzii and B. trematum lipids A, the hydroxytetradecanoic acid at the C-2' position are carrying in secondary linkage a 2-hydroxytetradecanoic acid residue resulting of post-traductional biosynthesis modifications. The three clinical isolates displayed characteristic structural traits compared to the ATCC 51541 reference strain: the lipid A phosphate groups are more or less modified with glucosamine in the isolates and reference strain, but the presence of 10:0(3-OH) is only observed in the isolates. This trait was only described in B. pertussis and B. parapertussis strains, as well as in B. petrii isolates by the past. The genetic bases for most of the key structural elements of lipid A were analyzed and supported the structural data.

Structural and biological characteristics of different forms of V. filiformis lipid A: use of MS to highlight structural discrepancies.

Vitreoscilla filiformis is a Gram-negative bacterium isolated from spa waters and described for its beneficial effects on the skin. We characterized the detailed structure of its lipopolysaccharide (LPS) lipid A moiety, an active component of the bacterium that contributes to the observed skin activation properties. Two different batches differing in postculture cell recovery were tested. Chemical analyses and mass spectra, obtained before and after mild-alkali treatments, revealed that these lipids A share the common bisphosphorylated ß-(1→6)-linked d-glucosamine disaccharide with hydroxydecanoic acid in an amide linkage. Short-chain FAs, hydroxydecanoic and dodecanoic acid, were found in a 2:1 ratio. The two lipid A structures differed by the relative amount of the hexa-acyl molecular species and phosphoethanolamine substitution of the phosphate groups. The two V. filiformis LPS batches induced variable interleukin-6 and TNF-α secretion by stimulated myelomonocytic THP-1 cells, without any difference in reactive oxygen species production or activation of caspase 3/7. Other different well-known highly purified LPS samples were characterized structurally and used as standards. The structural data obtained in this work explain the low inflammatory response observed for V. filiformis LPS and the previously demonstrated beneficial effects on the skin.

Antimicrobial Peptide Resistance Genes in the Plant Pathogen Dickeya dadantii.

Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves the products of the dltXABCD genes. These genes are widespread in Gram-positive bacteria, and they are also found in a few Gram-negative bacteria. Notably, these genes are present in all soft-rot enterobacteria (Pectobacterium and Dickeya) whose dltDXBAC operons have been sequenced. We studied the function and regulation of these genes in Dickeya dadantii dltB expression was induced in the presence of the AMP polymyxin. It was not regulated by PhoP, which controls the expression of some genes involved in AMP resistance, but was regulated by ArcA, which has been identified as an activator of genes involved in AMP resistance. However, arcA was not the regulator responsible for polymyxin induction of these genes in this bacterium, which underlines the complexity of the mechanisms controlling AMP resistance in D. dadantii Two other genes involved in resistance to AMPs have also been characterized, phoS and phoH dltB, phoS, phoH, and arcA but not dltD mutants were more sensitive to polymyxin than the wild-type strain. Decreased fitness of the dltB, phoS, and phoH mutants in chicory leaves indicates that their products are important for resistance to plant AMPs. IMPORTANCE: Gram-negative bacteria can modify their lipopolysaccharides (LPSs) to resist antimicrobial peptides (AMPs). Soft-rot enterobacteria (Dickeya and Pectobacterium spp.) possess homologues of the dlt genes in their genomes which, in Gram-positive bacteria, are involved in resistance to AMPs. In this study, we show that these genes confer resistance to AMPs, probably by modifying LPSs, and that they are required for the fitness of the bacteria during plant infection. Two other new genes involved in resistance were also analyzed. These results show that bacterial resistance to AMPs can occur in bacteria through many different mechanisms that need to be characterized.

Structure activity characterization of Bordetella petrii lipid A, from environment to human isolates.

Bordetella petrii, a facultative anaerobic species, is the only known member of the Bordetella genus with environmental origin. However it was also recently isolated from humans. The structures of the B. petrii lipid A moieties of the endotoxins were characterized here for the first time for an environmental strain and compared to that of human isolates. Characterization was achieved using chemical analyses, gas chromatography-mass spectrometry, and Matrix Assisted Laser Desorption Ionisation mass spectrometry. The analyses revealed that the different lipid A structures contain a common bisphosphorylated ß-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide as well at the C-3' in ester linkages. Similar to Bordetella pertussis and Bordetella bronchiseptica lipids A, the hydroxytetradecanoic acid at the C-2' position was substituted by tetradecanoic acid. Unlike B. pertussis, the hydroxytetradecanoic acid at the C-2 position was substituted with either 12:0 or 14:0 and/or their 2-OH forms. Depending on the environmental or human origin the structures differed in the length and degree of fatty acid acylation and impacted the IL-6 and TNF-α inflammatory responses tested. In one isolate we showed the presence at the C-3 position of the short-chain 10:0(3-OH), which according to our previous analyses is more characteristic of the human pathogens in the genus like B. pertussis and Bordetella parapertussis.

Desulfovibrio desulfuricans isolates from the gut of a single individual: structural and biological lipid A characterization.

The levels of sulfate-reducing bacteria (SRB), including Desulfovibrionaceae, in the gut increase following a fat-enriched diet. Endotoxins from gut microbiota contribute to the inflammation process, leading to metabolic diseases. Thus, we sought to characterize the lipid A structures of Desulfovibrionaceae lipopolysaccharides (LPS) that are associated with the microbiota inflammatory properties. LPS variants were obtained from two SRB isolates from the gut of a single individual. These LPS variants shared similar lipid A moieties with Enterobacterial LPS, but differed from one another with regard to fatty-acid numbers and endotoxic activity. This first complete structural characterization of Desulfovibrio lipid A gives new insights into previously published data on Desulfovibrio lipid A biosynthesis. LPS microdiversity within SRBs illustrates how adaptation can influence pro-inflammatory potential.

Biofilms formed by gram-negative bacteria undergo increased lipid a palmitoylation, enhancing in vivo survival.

UNLABELLED: Bacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increases in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria. IMPORTANCE: Bacterial communities called biofilms display characteristic properties compared to isolated (planktonic) bacteria, suggesting that some molecules could be more particularly produced under biofilm conditions. We investigated biofilm-associated modifications occurring in the lipopolysaccharide (LPS), a major component of all Gram-negative bacterial outer membrane. We showed that all tested commensal and pathogenic biofilm bacteria display high incorporation of a palmitate acyl chain into the lipid A part of LPS. This lipid A palmitoylation is mediated by the PagP enzyme, whose expression in biofilm is controlled by the regulatory proteins H-NS and SlyA. We also showed that lipid A palmitoylation in biofilm bacteria reduces host inflammatory response and enhances their survival in an animal model of biofilm infections. While these results provide new insights into the biofilm lifestyle, they also suggest that the level of lipid A palmitoylation could be used as an indicator to monitor the development of biofilm infections on medical surfaces.

Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era.

Endotoxins are lipopolysaccharides (LPS), and major constituents of the outer membrane of Gram-negative bacteria. Bordetella pertussis LPS were the only major antigens, of this agent of whooping-cough, that were not yet analyzed on isolates from the pre- and post-vaccination era. We compared here the LPS structures of four clinical isolates with that of the vaccine strain BP 1414. All physico-chemical analyses, including SDS-PAGE, TLC, and different MALDI mass spectrometry approaches were convergent. They helped demonstrating that, on the contrary to some other B. pertussis major antigens, no modification occurred in the dodecasaccharide core structure, as well as in the whole LPS molecules. These results are rendering these major antigens good potential vaccine components. Molecular modeling of this conserved LPS structure also confirmed the conclusions of previous experiments leading to the production of anti-LPS monoclonal antibodies and defining the main epitopes of these major antigens.

Minor modifications to the phosphate groups and the C3' acyl chain length of lipid A in two Bordetella pertussis strains, BP338 and 18-323, independently affect Toll-like receptor 4 protein activation.

Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

A new rapid and micro-scale hydrolysis, using triethylamine citrate, for lipopolysaccharide characterization by mass spectrometry.

Endotoxin (lipopolysaccharide, LPS) is, in general, composed of two moieties: a hydrophilic polysaccharide linked to a hydrophobic lipid A terminal unit and forms a major surface component of gram-negative bacteria. The structural features of LPS moieties play a role in pathogenesis and also involve immunogenicity and diagnostic serology. The major toxic factor of LPS resides in the lipid A moiety, anchored in the outer layer of the bacterium, and its relative biological activity is critically related to fine structural features within the molecule. In establishing relationships between structural features and biological activities of LPS it is of the utmost importance to develop new analytical methods that can be applied to the complete unambiguous characterization of a specific LPS molecule. Herein is presented a practical rapid and sensitive analytical procedure for the mass spectral screening of LPS using triethylamine citrate as an agent for both disaggregation and mild hydrolysis of LPS. It provides improved matrix-assisted laser desorption/ionization (MALDI) mass spectra and, in particular, affords the identification of fragments retaining labile substituents present in the native macromolecular LPS structures. The methods were developed and applied using purified LPS of Escherichia coli and Salmonella enterica, as well as more complex LPS of Actnobacillus pleuropneumoniae.