The core region of Proteus mirabilis R110/1959 lipopolysaccharide

The complete core structure present in the lipopolysaccharide of the R mutant R110/1959 from Proteus mirabilis (Proteus type II core) was investigated using methylation analysis and a number of degradation methods such as Smith degradation and beta-elimination. These studies combined with earlier work on a Rc-type mutant of P. mirabilis O28 (R4/O28) which shares the same inner core region, allowed formulation of the complete core structure of the Proteus type II core as shown in Scheme 1. (formula; see text) A characteristic feature of the Proteus core of type II is the presence of two units of D-galacturonic acid (DGalA); one in terminal, the other one in a chain-linked position. In addition, the presence of the two isomers of glycero-D-manno-heptose (LDHep and DDHep) and the lack of galactose are conspicuous. DDHep occupies a terminal position in the external core region, whereas the three units of LDHep in addition to dOclA form, as in other enterobacterial core types, the internal core region. The taxonomic significance of the presence of DGalA in the Proteus type II core, but also in all R cores of other Proteeae investigated so far, will be discussed.     

The structure of the Escherichia coli O148 lipopolysaccharide core region and its linkage to the O-specific polysaccharide

Recently it was demonstrated that Shigella dysenteriae type 1, a cause of severe dysentery epidemics, gained its O-specific polysaccharide (O-SP) from Escherichia coli O148. The O-SPs of these bacteria differ only by a galactose residue in the repeat unit of S. dysenteriae type 1 in place of a glucose residue in E. coli O148. Herein, we analyzed the core structure and its linkage to the O-SP in E. coli O148 LPS. Both were found to be identical to those of S. dysenteriae type 1 structures, further supporting the relatedness of these two bacteria. The following structure of the core with one repeat unit of the O-SP has been assigned (all have d-configuration except l-Rha):     

The instructor cell for the human procoagulant monocyte response to bacterial lipopolysaccharide is a Leu-3a+ T cell by fluorescence-activated cell sorting

A variety of responses of cells of the lymphoid system are associated with acquisition of the capacity to initiate the coagulation protease pathways. The initiating or procoagulant molecules are produced by the monocyte; however, a number of studies have indicated that lymphocyte collaboration is required. The induction of human monocyte procoagulant activity (PCA) by the model stimulus bacterial lipopolysaccharide (LPS) was examined in the present study by using relatively highly purified monocyte and lymphocyte populations in reconstitution experiments. Consistent with prior studies, the PCA response could not be generated by highly purified monocytes alone after exposure to LPS. The ability to generate PCA was restored to these monocyte populations by the addition of fibronectin-gelatin nonadherent lymphocytes, nylon wool effluent T cells, or Leu-3a+ inducer/helper T cells selected by fluorescence-activated cell sorting. T cells added to monocytes at a ratio of 8:1 or higher, and Leu-3a+ cells added at a ratio of 6:1 or higher, provided a maximal collaboration for monocyte PCA induction by LPS. These results substantiate further previous suggestions of an absolute requirement for collaborating T cells and demonstrate that these instructor cells carry a marker for the inducer/helper subset.     

The structure of the lipopolysaccharide core region of Citrobacter 027

The structure of Citrobacter 027 lipopolysaccharide core has been established using sugar and methylation analyses and 1H-NMR spectroscopy, and was shown to be identical to the core described recently in PCM 1487 strain which represents a separate serotype in Citrobacter genus.     

On the primary structure of the Escherichia coli R4 cell wall lipopolysaccharide core.

From $\text{Escherichia coli}$ R4, and from some deeper rough mutants of it, the cell wall lipopolysaccharides were isolated and subjected to mild acid hydrolysis. By methylation/g.l.c./m.s. and other analyses of the core oligosaccharides thus obtained, the primary structure of the $\text{E. coli}$ R4 core (hexose and heptose region) was elucidated:

     

Structures of the O1B and O1C lipopolysaccharide antigens of Escherichia coli.

The O-specific moieties of the O1B antigen (lipopolysaccharide) from Escherichia coli O1B:K1 and the O1C antigen from E. coli O1C:K- both consist of L-rhamnose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in a molar ratio of 2:1:1:1. By using fragmentation procedures, methylation analysis, and one- and two-dimensional nuclear magnetic resonance spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In the O1B polysaccharide X is 2, and in the O1C polysaccharide X is 3. With the recently published structure of the O1A polysaccharides (B. Jann, A. S. Shashkov, D. S. Gupta, S. M. Panasenko, and K. Jann, Carbohydr. Polym. 18:51-57 1992), three related O1 antigens are now known. Their common (O1-specific) epitope is suggested to be the side-chain N-acetyl-D-mannosamine residue.     

Differential response of B cells in the lymph node and the spleen to bacterial lipopolysaccharide

In vivo polyclonal activation of B cells in the lymph nodes and the spleens of mice injected with bacterial lipopolysaccharide (LPS) was compared. The peak of anti-trinitrophenylated sheep red blood cells plaque-forming cell (PFC) response in the lymph node was reached 6-8 days after the injection of LPS while that in the spleen was reached at 2 days. The maximal increase in the total number of Ig-producing cells in the lymph node also occurred at the later stage. These differences in time courses of polyclonal activation of B cells between the lymph node and the spleen were not due to the absence of B cells in the lymph node, migration of PFC from the spleen to the lymph node, or qualitative differences of B cells. This phenomenon was dependent on the environmental difference between the lymph node and the spleen, because B cells from the lymph node could respond to LPS rapidly in the spleen. Further, the polyclonal activation of B cells was accelerated in the lymph nodes of mice receiving prior injection of LPS. In in vitro cultures of lymph node cells of those mice, a significant amount of interleukin-1 could be detected by stimulation of LPS. It was possible that the delayed activation of B cells in the lymph node was due to the time lag necessary for construction of the environmental condition suitable for activation of B cells, whereas in the spleen this condition can be provided without delay.     

Expression of CD14 corrects the slow response to lipopolysaccharide in the 1B8 mutant of the B cell lymphoma 70Z/3

 B cells and macrophages both activate NF-κB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14⁺ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IκB-α and -β molecules and translocation of the NF-κB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14⁺ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IκB-β was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself. Received: 3 June 1997 / Revised: 26 June 1997     

The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens

The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).     

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

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

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

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.     

The mouse heavy chain variable region marker, J606-GAC, is not restricted to particular B cell isotypes or subsets

The heavy chain variable region (VH) marker J606-GAC, which is expressed on a subset of mouse heavy chain variable region group III antibodies, is expressed at similar frequencies on antibodies with mu, gamma 3, gamma 1, gamma 2, and alpha heavy chains. We have previously shown the J606-GAC determinant to be present on all anti-inulin and on the majority of anti-group-A-carbohydrate (GAC) antibodies examined. The responses to these two antigens are designated thymus-independent type 2 (TI-2) and thymus-dependent type 2 (TD-2), respectively, and have been shown previously to be largely restricted to the mu and gamma 3 heavy chain classes. TI-2 and TD-2 antigens are distinguished from other antigens such as T-independent type 1 (TI-1) and other thymus-dependent (TD) antigens, in part because they are virtually not immunogenic in CBA/N mice which express the x-linked immunodefiency (xid) allele. Surprisingly, we found no difference in the percentage of J606-GAC determinant-bearing plasma cells in the spleens of xid vs normal mice.     

Regulation by interferons of the local inflammatory response to bacterial lipopolysaccharide

Footpad swelling developing in mice after local injection of LPS (S. marcescens) was found to consist of two phases with peaks occurring on days 2 to 3 and 6 to 8, respectively. Histopathologically, the reaction was characterized by edema and mononuclear cell infiltration; the second peak was associated with intravascular thrombosis as is typically described for the Shwartzman reaction to LPS. Recombinant DNA-derived IFN-gamma, administered by i.p. injection, had a suppressive effect on the development of the reaction. The same effect was seen with recombinant DNA-derived IFN-alpha 1 and with the natural mixture of IFN-alpha and -beta. In mice pretreated with neutralizing monoclonal antibodies to IFN-gamma, the footpad response to LPS was modified in that a delayed monophasic rather than a biphasic response occurred. These data indicate that LPS induces local production of IFN-gamma, which acts as a trigger or positive regulator of the reaction. The effect of a single pretreatment with neutralizing anti-IFN-gamma antibody was found to last for as long as 6 wk. Experiments in which antibody administration was delayed till after LPS challenge indicated that endogenous IFN-gamma was also involved in the late phases of the inflammation. The results show that regulation of inflammation by interferons is complex in that local IFN-gamma acts as a positive factor, whereas systemic IFN-alpha 1 and -gamma, probably through indirect mechanisms, downregulate inflammation.     

B cell responses to a peptide epitope. X. Epitope selection in a primary response is thermodynamically regulated

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependent immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependent humoral response is initiated by a step that is under thermodynamic control.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.