Intact rough- and smooth-form lipopolysaccharides from Escherichia coli separated by preparative gel electrophoresis exhibit differential biologic activity in human macrophages.

We established a new preparative separation procedure, based on DOC/PAGE, to isolate intact lipopolysaccharide (LPS) fractions from natural LPS preparations of Escherichia coli. Analysis of the chemical integrity of LPS fractions by MS showed that no significant chemical modifications were introduced by the procedure. Contamination with toll-like receptor 2 (TLR2)-reactive cell-wall components present in the natural LPS mixture was effectively removed by the procedure, as determined by the absence of reactivity of the purified fractions in a HEK293-TLR2 cell line. Biologic analysis of LPS fractions derived from E. coli O111 in human macrophages demonstrated that the rough (R), semirough (SR) and smooth (S) LPS fractions were highly active at inducing tumor necrosis factor-alpha (TNF-α) in the presence of human serum; however, on a weight basis the R-LPS and SR-LPS fractions were more active, by a factor of 10-100, than was the S-LPS fraction. Under serum-free conditions, the natural LPS mixture, as well as the R-LPS and SR-LPS fractions, showed dose-dependent activation of macrophages, although the response was attenuated by about 10- to 100-fold. In contrast, the S-LPS fraction failed to induce TNF-α. Remarkably, the dose-response of the natural LPS mixture resembled that of the R-LPS and SR-LPS fractions, supporting that short-chain (R and SR) forms of LPS dominate the innate immune response of human macrophages to LPS in vitro. Biologic activity to the S-LPS fraction under serum-free conditions could be restored by the addition of recombinant lipopolysaccharide-binding protein (LBP). In contrast, soluble cluster of differentiation antigen 14 was not able to confer activity of the S-LPS fraction, indicating a crucial role of LBP in the recognition of S-LPS by human macrophages.

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits.

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200 d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.

Monoclonal antibody S60-4-14 reveals diagnostic potential in the identification of Pseudomonas aeruginosa in lung tissues of cystic fibrosis patients.

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa has been identified to contain an inner-core structure expressing a Pseudomonas-specific epitope. This target structure is characterized by a highly phosphorylated and 7-O-carbamoyl-l-glycero-alpha-d-manno-heptopyranose (CmHep) and was found to be present in all human-pathogenic Pseudomonas species of the Palleroni (RNA)-classification I scheme. We raised and selected the monoclonal antibody S60-4-14 (mAb S60-4-14, subtype IgG1) from mice immunized with heat-killed Pseudomonas bacteria. The epitope of this mAb was found to reside in the inner-core structure of P. aeruginosa and, hence, successfully evaluated for the immunohistochemical detection of P. aeruginosa in formalin- or HOPE-fixed (Hepes-glutamic acid buffer-mediated organic solvent protection effect) and paraffin-embedded human lung tissue slices. Lung specimens, mainly from explanted lungs of cystic fibrosis (CF) patients, as well as P. aeruginosa isolates from patients suffering from CF and patients with extrapulmonar Pseudomonas infections were investigated by PCR, immunohistochemistry, and Western blot analysis with mAb S60-4-14. The results revealed an unequivocal coincidence of PCR and immunohistochemistry. Together with the Western blot results mAb S60-4-14 displays a potential diagnostic tool for the specific identification of P. aeruginosa in infected lungs of CF.

Critical role for interleukin-1beta (IL-1beta) during Chlamydia muridarum genital infection and bacterial replication-independent secretion of IL-1beta in mouse macrophages.

Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection. In the present study, IL-1beta(-/-) mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1beta both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1beta secretion in WT mice, the major producers of IL-1beta in vivo are F4/80(+) macrophages and GR-1(+) neutrophils, but not CD45(-) epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1beta, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1beta from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1beta secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1beta secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1beta secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1beta mRNA observed in vivo in this cell type.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Cellular recognition of trimyristoylated peptide or enterobacterial lipopolysaccharide via both TLR2 and TLR4.

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.

The Gram-negative bacterium Chlamydia trachomatis L2 stimulates tumor necrosis factor secretion by innate immune cells independently of its endotoxin.

The endotoxin of Chlamydia trachomatis L(2), the causative agent of lymphogranuloma venerum, has been described as an endotoxin with an atypical structure and weak stimulatory activity. It is, however, unclear whether chlamydial endotoxin plays a role in the stimulation of innate immune cells upon contact with the whole microorganism C. trachomatis L(2). We show here that chlamydial endotoxin and, as expected, Escherichia coli O55:B5 endotoxin depend on Toll-like receptor 4 without depending on Toll-like receptor 2 to stimulate bone marrow-derived dendritic cells to secrete tumor necrosis factor (TNF). In contrast, the whole microorganism C. trachomatis L(2) induces TNF secretion by innate immune cells independently of Toll-like receptor 4, while stimulation by E. coli O55:B5 depends on Toll-like receptor 4. Furthermore, although TNF secretion of the macrophage cell line RAW264.7 with chlamydial or E. coli O55:B5 endotoxin as well as with the bacterium E. coli O55:B5 is inhibited by the endotoxin-neutralizing compound polymyxin B, C. trachomatis L(2)-induced secretion of TNF cannot be reduced. In accordance with the literature, the potential of chlamydial endotoxin is more than 100-fold weaker than E. coli O55:B5 endotoxin on all cell types tested. We conclude that chlamydial endotoxin is unlikely to be involved in C. trachomatis L(2)-induced release of TNF by innate immune cells.

Construction of a deep-rough mutant of Burkholderia cepacia ATCC 25416 and characterization of its chemical and biological properties.

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 --> 8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2 --> 4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor alpha from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.

Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens.

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.

A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides.

We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[alpha-Kdo-(2-->4)-alpha-Kdo-(2-->] and a Kdo trisaccharide [alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies. In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA. Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand. This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives.

Identification of Acinetobacter isolates from species belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with monoclonal antibodies specific for O Antigens of their lipopolysaccharides.

The unambiguous identification of Acinetobacter strains, particularly those belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, is often hindered by their close geno- and phenotypic relationships. In this study, monoclonal antibodies (MAbs) against the O antigens of the lipopolysaccharides from strains belonging to the A. calcoaceticus-A. baumannii complex were generated after the immunization of mice with heat-killed bacteria and shown by enzyme immunoassays and Western blotting to be specific for their homologous antigens. Since the A. calcoaceticus-A. baumannii complex comprises the most clinically relevant species, the MAbs were subsequently tested in dot and Western blots with proteinase K-treated lysates from a large collection of Acinetobacter isolates (n = 631) to determine whether the antibodies could be used for the reliable identification of strains from this complex. Reactivity was observed with 273 of the 504 isolates (54%) from the A. calcoaceticus-A. baumannii complex which were included in this study. Isolates which reacted positively did so with only one antibody; no reactivity was observed with isolates not belonging to the A. calcoaceticus-A. baumannii complex (n = 127). To identify additional putative O serotypes, isolates from the A. calcoaceticus-A. baumannii complex which showed no MAb reactivity were subjected to a method that enables the detection of lipid A moieties in lipopolysaccharides with a specific MAb on Western blots following acidic treatment of the membrane. By this method, additional serotypes were indeed identified, thus indicating which strains to select for future immunizations. This study contributes to the completion of a serotype-based identification scheme for Acinetobacter species, in particular, those which are presently of the most clinical importance.