Lipopolysaccharide biosynthesis: which steps do bacteria need to survive?

A detailed knowledge of LPS biosynthesis is of the utmost importance in understanding the function of the outer membrane of Gram-negative bacteria. The regulation of LPS biosynthesis affects many more compartments of the bacterial cell than the outer membrane and thus contributes to the understanding of the physiology of Gram-negative bacteria in general, on the basis of which only mechanisms of virulence and antibiotic resistance can be studied to find new targets for antibacterial treatment. The study of LPS biosynthesis is also an excellent example to demonstrate the limitations of "genomics" and "proteomics", since secondary gene products can be studied only by the combined tools of molecular genetics, enzymology and analytical structural biochemistry. Thus, the door to the field of "glycomics" is opened.

A monoclonal antibody with specificity for the genus Klebsiella binds to a common epitope located in the core region of Klebsiella lipopolysaccharide.

A mouse monoclonal antibody (mAb) which has been obtained after immunization of mice with heat-killed Klebsiella pneumoniae strain R20/O1(-) followed by standard plasmacytoma cell fusion protocols was investigated for its ability to identify various species of the genus Klebsiella. Based on the published observation that the antibody binds to an epitope located in the core region of lipopolysaccharide (LPS) of strain R20/O1(-), we tested whether this epitope is shared and exposed by other species of the genus Klebsiella. The antibody was able to bind to LPS of clinical isolates of K. pneumoniae (n = 77), K. oxytoca (n = 50), K. terrigena (n = 49) and K. planticola (n = 50) in 93%, 98%, 96% and 100%, respectively, but did not bind to LPS of other Gram-negative genera (n = 159) as tested by Western blots and dot blots using proteinase K-digested whole cell lysates as antigens. Western blot analyses indicated that the antibody bound only to those LPS molecules which did not carry an O-antigen and that the antibody is thus different from those already published.

A monoclonal antibody recognizing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo of Chlamydophila psittaci 6BC lipopolysaccharide.

A monoclonal antibody (mAb) S45-18 was generated against a synthetic neoglycoconjugate containing the trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) which represents a structure of the lipopolysaccharide (LPS) from Chlamydophila psittaci 6BC. The antibody was characterized by binding and inhibition assays in ELISA using: (i) the immunizing antigen and chemically synthesized derivatives thereof; (ii) chlamydial elementary bodies (EB); and (iii) LPS of Chl. psittaci 6BC and Chlamydia trachomatis L2. The specificity was determined in comparison to that of mAb S25-23 recognizing the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide which represents an epitope shared by all species of the family. MAb S45-18 bound to an epitope of the structure alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo, with lower reactivity with the (2-->8)-(2-->4)-linked analog. Using chlamydial EB or LPS, mAb S45-18 bound preferentially to LPS and EB of Chl. psittaci. Therefore, Chl. psittaci LPS contains, in addition to the known genus-specific epitope, a species-specific epitope.

Cloning, sequencing, and functional analysis of three glycosyltransferases involved in the biosynthesis of the inner core region of Klebsiella pneumoniae lipopolysaccharide.

The genes encoding the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (waaA) and heptosyltransferases I (waaC) and II (waaF) in Klebsiella pneumoniae were cloned from a DNA library by functional complementation of corresponding Escherichia coli and Salmonella enterica mutants. Sequence analyses revealed extensive homologies of the deduced proteins to their counterparts in other Enterobacteriaceae. However, differences were evident with regard to the chromosomal organization of the genes. To perform in vitro studies, the waaA, waaC and waaF genes were subcloned and expressed in the Gram-positive host Corynebacterium glutamicum. WaaA was characterized as a bifunctional enzyme capable of transferring two Kdo residues to a synthetic bisphosphorylated tetraacyl-lipid A precursor of E. coli (compound 406). In contrast, waaC and waaF were shown to encode specific glycosyltransferases catalyzing the consecutive transfer of two L-glycero-D-manno-heptose residues to Kdo(2)-406.

Synthesis of neoglycoproteins containing Kdo epitopes specific for Chlamydophila psittaci lipopolysaccharide.

The oligosaccharides alpha-Kdop-(2-->8)-alpha-Kdop-(2-->6)-beta-D- GlcpNAc-(1-->OAll) 4, alpha-Kdop-(2-->4)-alpha- Kdop-(2-->4)-alpha-Kdop-(2-->6)-beta-D-GlcpNAc-(1-->OAll+ ++) 10, and the branched Kdo tetrasaccharide alpha- Kdop-(2-->4)-[alpha-Kdop-(2-->8)]-alpha-Kdop-(2-->4)-a lpha-Kdop-(2-->OAll) 21 have been prepared using en bloc transfer of Kdo oligosaccharide bromide donors to protected mono- or disaccharide acceptors. Radical addition of cysteamine to the anomeric allyl glycosides afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides 5, 11 and 22. The spacer ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 6, 12 and 23 which were used to prepare solid-phase antigens in enzyme immuno-assays for the characterization of monoclonal antibodies against chlamydial LPS. The data showed that the (2-->8)-linked Kdo disaccharide and the (2-->8)-(2-->4)-linked Kdo trisaccharide portion of the neoglycoconjugate 23 were not available for binding of antibodies which recognize these structures as di- and trisaccharide, respectively.

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter strain 96 (DNA group 11).

A polysaccharide containing D-Manp, L-Fucp (6-deoxygalactopyranose, fucose) and D-GlcpNAc was isolated by mild acid hydrolysis, followed by gel-permeation chromatography, from the lipopolysaccharide derived from Acinetobacter strain 96 (DNA group 11). The structure of the O-antigen was determined by compositional analysis and NMR spectroscopy of the polysaccharide as: [carbohydrate structure see text] A monoclonal antibody obtained after immunization of mice with heat-killed bacteria of Acinetobacter strain 96 was shown to bind to the O-antigen and did not cross-react with any Acinetobacter O-antigen of known structure.

Synthesis and immunochemical characterization of neoglycoproteins containing epitopes of the inner core region of Pseudomonas aeruginosa RNA group I lipopolysaccharide.

The disaccharide allyl 2-acetamido-2-deoxy-alpha-D-galactopyranosyl-(1-->3)-7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranoside 5 (GalNAc-cmHep-allyl) was synthesized starting from 1 and 2. Compound 5, cmHep-allyl and the disaccharide cmHep-(1-->3)-Hep-allyl were converted into cysteamine-spacered derivatives and conjugated to bovine serum albumin (BSA) to yield the neoglycoconjugates 7--9, respectively. These conjugates were used to immunize mice and to prepare monoclonal antibodies (mAbs) which were characterized in comparison to mAbs obtained after immunization with heat-killed Pseudomonas aeruginosa strain H4. Two antibodies obtained after immunization with the neoglycoconjugates bound strongly to cmHep-BSA and with lower affinity to cmHep-Hep-BSA but did not bind to GalNAc-cmHep-BSA or to H4 LPS. Another antibody obtained after immunization with heat-killed bacteria bound to LPS and GalNAc-cmHep-BSA but not to cmHep-Hep-BSA or cmHep-BSA

A complement-dependent enzyme immunoassay (C-EIA) with increased sensitivity for IgM-rich rabbit sera.

An enzyme immunoassay involving activation of complement (C-EIA) was developed for rabbit polyclonal IgM antibodies against lipid A and lipopolysaccharide antigens. C-EIA was significantly higher in sensitivity for IgM-rich rabbit sera compared to EIA using anti-immunoglobulin secondary antibodies. Hence, C-EIA should be useful for the detection of weak IgM reactivities in rabbit sera, especially after short-time immunizations. Selective inhibition of both complement pathways indicated that C-EIA measures activation of the classical pathway.

Induction of tumor necrosis factor-alpha release by lipopolysaccharide and defined lipopolysaccharide partial structures.

We have investigated the release of tumor necrosis factor-alpha (TNF-alpha) by human mononuclear cells (MNC) and isolated human monocytes/macrophages stimulated with S- and R-form lipopolysaccharide (LPS), natural lipid A, and natural and synthetic partial structures thereof. We found that LPS of Salmonella minnesota (S. min.) Rb2, which represents a partial structure of wildtype LPS of Salmonella abortus equi (S.a.e.) lacking the O-chain and parts of the outer core region, was the most active inducer of all substances tested, even more active than the wildtype LPS. Lipid A also induced the production of TNF-alpha by monocytes/macrophages but was less active than wildtype LPS. The natural Escherichia coli (E. coli) type hexaacyl lipid A (compound 506) was more active than the natural S. min. type heptaacyl lipid A (compound 516). The 1- and 4'-monodephospho partial structures (compounds 505 and 504) of E. coli lipid A were less active and represented the smallest structures tested that were able to induce TNF-alpha release by monocytes/macrophages. Synthetic tetraacyl lipid A precursor Ia of E. coli lipid A, lacking non-hydroxylated fatty acids (compound 406), and the monosaccharide precursor lipid X did not induce the release of TNF-alpha in MNC or isolated monocytes/macrophages. This might indicate that the ability of a lipid A structure to induce the release of TNF-alpha is closely connected with the conditions to be at least hexaacylated and/or to contain hydroxylated fatty acids. These results demonstrate a structure-dependent hierarchy of LPS and natural or synthetic partial structures in their capacity of inducing TNF-alpha release by monocytes/macrophages.

The antibody reactivity of monoclonal lipid A antibodies is influenced by the acylation pattern of lipid A and the assay system employed.

The influence of the acylation pattern of lipid A on the reactivity of murine monoclonal antibodies (mAb) was tested in different assay systems with synthetic lipid A antigens. Both the number and type of fatty acids had an impact on the antigen amounts needed for optimal sensitization of sheep red blood cells, on the inhibition capacity of compounds and on the reactive antigen amounts in enzyme immunoassay and dot blot assay. Results obtained with two pentaacyl isomers indicated that the location of fatty acids is of no importance. Although all mAbs used recognized epitopes residing in the hydrophilic backbone of lipid A, their reactivities were greatly influenced by the number as well as the type of acyl chains present. In the various assays, the mAbs reacted either similarly or discrepantly suggesting that epitopes are exposed differently in the test systems. We conclude that for the determination of the reactivity of lipid A mAbs it is useful and sometimes necessary to run various assays in parallel and to compare mAbs on the basis of reaction patterns.

The chemical structure of bacterial endotoxin in relation to bioactivity.

Lipopolysaccharides (LPS) constitute the O-antigens and endotoxins of Gram-negative bacteria. Whereas both the polysaccharide and lipid portion of LPS contribute to the pathogenic potential of this class of bacteria, it is the lipid component (lipid A) which determines the endotoxic properties of LPS. The primary structure of lipid A of various bacterial origin has been elucidated and Escherichia coli lipid A has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique structural arrangement and conformation. Such analyses have furthermore provided insight into the determinants required for lipid A binding to and activation of human target cells. Present research efforts aim at the molecular characterization of the specificity, modulation and biomedical consequences of the interaction of lipid A with host cells.

Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation.

Surface structures of bacteria contribute to the microbial pathogenic potential and are capable of causing local and generalized inflammatory reactions. Among these factors, endotoxin and peptidoglycan are of particular medical importance. Both toxic bacterial polymers are now recognized to interact with the same cellular receptor, the CD14 molecule, which is expressed on different types of immune cells, in particular, monocytes/macrophages. The interaction between these bacterial activators and CD14 leads to the production of endogenous mediators such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, which are ultimately responsible for phlogistic responses. The fact that CD14 recognizes not only endotoxin and peptidoglycan but also other glycosyl-based microbial polymers suggests that this cellular surface molecule represents a lectin.

A new lipopolysaccharide antigen identified in Acinetobacter calcoaceticus: occurrence of widespread natural antibody.

Serological investigation of the lipopolysaccharide (LPS) of Acinetobacter calcoaceticus revealed a new antigen to which antibody in high titre is present in the serum of many mammalian species. The passive haemolysis test showed that antibody, in titres ranging from 32-4096, was invariably present in the serum of mice, rats, guinea-pigs, and horses. Rabbits and human beings had lower and more variable titres (less than 2-512). The antigen persisted after prolonged hydrolysis of the LPS in 1% acetic acid at 100 degrees C. Acinetobacter lipid A, which resembled antigenically the lipid A of many gram-negative bacteria, could be distinguished from the new antigen by inhibition and absorption experiments. Antibody to the new antigen could be completely absorbed with acinetobacter lipid A but not with enterobacterial lipid A; moreover, the latter failed to react with the antibody in the passive haemolysis test. Immunisation of rabbits with lipid A-immunogenic acinetobacter cells gave rise to antibodies against the new antigen and to lipid-A antibodies. Absorption of the immune serum with acinetobacter lipid A removed antibody to both antigens, but absorption with enterobacterial lipid A removed only the lipid -A antibodies.

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P-).

Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd1P- mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd1P- LPS bound to the beta 1-->6-linked glucosamine dissacharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificites present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide L-glycero-alpha-D-manno-heptopyranose(1-->3)- L-glycero-alpha-D-manno-heptopyranose(1-->5)3-deoxy-D-manno-oct ulo sonic acid. Antibodies against the 1-->3- and 1-->7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.

Characterization of serum antibody response to chlamydiae in patients with sexually acquired reactive arthritis.

Sera from patients with sexually acquired reactive arthritis (SARA) with antibodies reacting with C. trachomatis and C. pneumoniae (group 1; n = 20) and also with C. psittaci (group 2; n = 19) were analyzed for antibody specificity. Sera from group 2 reacted significantly more often with C. trachomatis serotype E, H and K and had higher antibody titers to serotype E, as tested by microimmunofluorescence tests. Cross-reactivities occurring in microimmunofluorescence tests were related to the presence of antichlamydial lipopolysaccharide antibodies, adsorption of which by recombinant lipopolysaccharide removed microimmunofluorescence reactivity with C. psittaci antigen. In group 2, significantly more sera had antibodies to C. pneumoniae, remaining after lipopolysaccharide adsorption, as proved by adsorption with viable C. trachomatis and C. pneumoniae organisms. None of the sera had antibodies to Yersinia enterocolitica, Shigella flexneri, Sh. sonnei and Salmonella spp. It was observed that the frequency and titer of cross-reacting antibodies to chlamydial serotypes and species were related to the time period between the diagnosis of genital chlamydial infection and of SARA. Cross-reactivities were also related to the presence of lipopolysaccharide, but not heat shock protein 60- or neutralizing antibodies to chlamydiae. Antibody reactivity induced by antichlamydial lipopolysaccharide antibodies can be removed by lipopolysaccharide adsorption.

The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid A for macrophage binding and cytokine induction.

Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of 125I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells and to induce cytokine-release in J774.1 cells. LPS, synthetic Escherichia coli-type lipid A (compound 506) and tetraacyl precursor Ia (compound 406) inhibited the binding of 125I-LPS to macrophage-like J774.1 cells and induced the release of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6). Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release. Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 (alpha-phosphonooxyethyl analogue of 406) > 406 >> 404 (4'-monophosphoryl partial structure of 406) > 405 (1-monophosphoryl partial structure of 406). In the case of hexaacyl preparations, compounds 506, PE-1 (alpha-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a beta-phosphonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction. The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells. These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e. cytokine induction.

Occurrence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen.

An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia-specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occurring in patient sera. These mAbs recognized Chlamydia-specific epitopes [alpha 2-->8-linked disaccharide of 3-deoxy-D-manno-octulosonic acid (Kdo) or the trisaccharide alpha Kdo-(2-->8)-alpha Kdo-(2-->4)-alpha Kdo] or those shared between chlamydial and Re-type LPS (alpha Kdo, alpha 2-->4-linked Kdo disaccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.

Morphological and functional studies on the lateral joints of the first and second cervical vertebrae in man.

The articular surfaces of the lateral atlanto-axial joints in man were analyzed with respect to their biomechanical aspects. The joint surfaces were investigated macroscopically, and the texture of the tangential fiber layer of the articular cartilage was studied with the aid of the split-line method. Furthermore, the thickness of cartilage was measured. From the results obtained the authors draw the conclusion that especially the mode of rotational movement between C1 and C2 changes within a life time. As a consequence, the distribution of stress in the lateral atlanto-axial joint is modified.

Synthesis of a trisaccharide of 3-deoxy-D-manno-2-octulopyranosylonic acid (KDO) residues related to the genus-specific lipopolysaccharide epitope of Chlamydia.

The disaccharides, O-(sodium 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosylonate)-(2----8)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate, were prepared via glycosylation of methyl (allyl 4,5,7-tri-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosid)onat e with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-D-manno-2-octulopyranosyl bromide)onate under Helferich and Koenigs-Knorr conditions, respectively. Based on g.l.c.-m.s. data of the alpha- and beta-(2----8)-linked disaccharide derivatives, obtained after carbonyl- and carboxyl-group reduction, followed by methylation, the alpha-anomeric configuration was assigned to the terminal KDO-residue in the KDO-region of Chlamydial lipopolysaccharide. The trisaccharide O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate was obtained via block synthesis using an alpha-(2----8)-linked disaccharide bromide derivative as the glycosyl donor. Copolymerization of the allyl glycosides with acrylamide gave water-soluble macromolecular antigens, suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydial LPS.

Synthesis of neoglycoproteins containing D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko) ligands corresponding to core units from Burkholderia and Acinetobacter lipopolysaccharide.

Glycal esters of Kdo derivatives were converted into 2,3-anhydro intermediates, which were transformed into D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko), as well as 3-O- and 4-O-p-nitrobenzoyl-Ko derivatives. The exo-allyl orthoester derivative, methyl [5,7,8-tri-O-acetyl-4-O-(4-nitrobenzoyl)-2,3-O-[(1-exo-allyloxy)-ethylidene]-D-glycero-beta-D-talo-oct-2-ulopyranos]onate, prepared from the 4-O-pNBz-protected Ko derivative, was elaborated into the alpha-Ko allyl ketoside, the reducing disaccharide alpha-Kdop-(2-->4)-Ko and the disaccharide alpha-Kdop-(2-->4)-Kop-(2-->OAll). Conversely, methyl[4,5,7,8-tetra-O-acetyl-3-O-(4-nitrobenzoyl)-alpha-D-glycero-D-talo-2-octulopyranosyl bromide]onate [Carbohydr. Res., 244 (1993) 69-84], was coupled with a Kdo acceptor to give the disaccharide alpha-Kop-(2-->4)-Kdop-(2-->OAll) after orthoester rearrangement and deprotection. The allyl glycosides were treated with cysteamine and converted into neoglycoproteins. The ligands correspond to inner core units from Acinetobacter haemolyticus and Burkholderia cepacia lipopolysaccharides.