Construction and characterization of Haemophilus ducreyi lipooligosaccharide (LOS) mutants defective in expression of heptosyltransferase III and beta1,4-glucosyltransferase: identification of LOS glycoforms containing lactosamine repeats

To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.     

Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide.

We developed a human inhibition monoclonal enzyme-linked immunosorbent assay (HIMELISA) to investigate the human immune response to the lipooligosaccharides (LOS) of Neisseria meningitidis. Monoclonal antibodies (MAb) were used to define seven epitopes on four LOS molecules of a meningococcal strain (126E) previously shown to express immunogenic LOS epitopes. The assay could distinguish epitope-specific antibody within whole sera. Neither the specificity nor the amount of the antibody measured by HIMELISA in sera of vaccinates changed during the immune response to meningococcal capsular polysaccharides, a chemically unrelated antigen. By using the HIMELISA, it was determined that sera from adults convalescing from meningococcal disease strongly inhibited MAb binding to two of the seven defined epitopes. The 3.6-kilodalton LOS of strain 126E expressed both of these epitopes. In addition, one of the inhibited epitopes was also expressed on the 4.0-kilodalton LOS of strain 126E. The convalescent-phase sera inhibited MAb binding to these two epitopes when they were expressed on LOS of diverse meningococcal strains. An acute-phase serum blocked MAb to the two epitopes to a lesser degree than did a convalescent-phase serum from the same patient. Immunoblotting the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated LOS with convalescent-phase sera confirmed the specificity of the human anti-LOS antibody identified by HIMELISA.     

Multiple mechanisms in serum factor-induced resistance of Haemophilus influenzae type b to antibody.

Incubation of Haemophilus influenzae type b at less than or equal to 10(7) CFU/ml with serum ultrafiltrate induces a phenotypic conversion in which complement-mediated bactericidal activity by somatic antibodies decreases while killing by capsular antibody is unchanged. Conversion had been shown to occur in a capsule-deficient (b-) mutant of strain Eag (thus appearing independent of capsulation), to include an increase in lipopolysaccharide content, and to be inhibited by chloramphenicol or puromycin. In the present study, in several strains not previously examined, conversion was not inhibited by the drugs and the corresponding b- mutants did not convert. Incubation in ultrafiltrate was also found to increase capsulation, as detected by radioassay, only 1.6-fold in Eag but 4.5-fold in DL26, the strain with the largest increase in resistance; moreover, complement-mediated opsonization by capsular antibody was greatly decreased. Thus, multiple mechanisms, capsule dependent as well as independent, appear to contribute to the serum factor-induced resistance of H. influenzae type b to antibody.     

Specific antibody filter (SAF) binding capacity enhancement to remove anti-A antibodies

Removal of Anti-A/B antibodies prior to ABO-incompatible transplantation can prevent hyperacute organ rejection. We are developing a specific antibody filter (SAF) device to selectively remove ABO blood group antibodies from the whole blood by utilizing immunoaffinity adsorption. The device consists of ultrafiltration hollow fiber membranes with synthetic antigens specific to bind blood group antibodies immobilized on the inner lumenal walls of the fibers. The aim of this study was to evaluate the effect of antigen molecular weight and surface activation process to increase the antibody binding capacity of the fiber membrane surface. A new higher molecular weight antigen Atri-pNSA-1000 compared with Atri-pNPA-30 (A-trisaccharide (Atri) conjugated to activated polymers of Mol. wt. 1000 kDa and 30 kDa, respectively) was employed to improve accessibility of the antigen to bind antibodies. Also, a cyanogen bromide (CNBr) based surface activation method mediated by TEA in neutral pH medium was used to enhance the number of active sites for antigen binding compared to a strong basic medium of NaOH. Using a CNBr/TEA activation method and by immobilizing Atri-pNSA-1000 antigen, an antibody binding capacity (～0.01 monoclonal anti-A IgM nmol/cm(2)) was achieved on the fiber surface. This binding capacity was sufficient to reduce monoclonal antibody titer from 1:128 to final titer below 1:4 with a surface area to volume ratio that is similar to commercial dialysis device (～1.1 m(2) surface area for an average body blood volume of 5 L).     

Enhanced factor H binding to sialylated Gonococci is restricted to the sialylated lacto-N-neotetraose lipooligosaccharide species: implications for serum resistance and evidence for a bifunctional lipooligosaccharide sialyltransferase in Gonococci

We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Galalpha1 --> 4 [corrected] Galbeta1 --> 4Glcbeta1 --> 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Galbeta1 --> 4GlcNAcbeta1 --> 3Galbeta1 --> 4Glcbeta1 --> 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an alpha2 --> 6 linkage and, as expected, to the terminal Gal of LNT LOS via an alpha2--> 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.     

Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.

The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).     

Response of Haemophilus somnus to iron limitation: expression and identification of a bovine-specific transferrin receptor

Nine clinical isolates of Haemophilus somnus were screened for their ability to use different transferrins as a source of iron growth. All nine strains were capable of using bovine but not porcine, human or chicken transferrin. A screening assay for siderophore production did not show any evidence of siderophore production by these strains. When iron-deficient cells from these strains were screened for their ability to bind peroxidase-conjugated transferrin, binding was detected with conjugated bovine, but not human or porcine transferrin. Competition binding studies demonstrated that the binding of peroxidase-conjugated bovine transferrin was competitively inhibited by unconjugated bovine transferrin but not transferrin from other species. The induction of receptor expression by low iron conditions was inhibited by chloramphenicol and rifampicin but not ampicillin indicating that new protein and mRNA synthesis was required for expression of receptor activity. Affinity isolation of receptor proteins with biotinylated bovine transferrin, but not human or porcine transferrin, yielded three proteins from H. somnus strain H74. Two of the proteins were identified as 105 kDa and 73 kDa iron-regulated outer membrane proteins. A third protein of 85 kDa that was isolated did not co-migrate with any iron-regulated outer membrane protein. Affinity isolation of receptor proteins from other strains of H. somnus yielded a 73 kDa protein from all strains and a 105 kDa and 85 kDa protein in four of the six strains analysed.     

Activation of toll-like receptor 2 (TLR2) and TLR4/MD2 by Neisseria is independent of capsule and lipooligosaccharide (LOS) sialylation but varies widely among LOS from different strains

Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocyte-derived cell line (THP-1) exposed to strains of N. meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, fine-structural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria.     

Epitope expression and partial structural characterization of F62 lipooligosaccharide (LOS) of Neisseria gonorrhoeae: IgM monoclonal antibodies (3F11 and 1-1-M) recognize non-reducing termini of the LOS components.

F62 LOS of Neisseria gonorrhoeae consists of two components. The higher molecular weight (MW) component is recognized by monoclonal antibody (MAb) 1-1-M and the smaller MW component by MAb 3F11. Epitope expression of the two LOS components and their partial structures were investigated by treating the F62 LOS with several glycosidases and then monitoring their antigenicity with the two mouse IgM MAbs. The 1-1-M-defined LOS component was cleaved with both beta-N-acetylhexosaminidase and endo-beta-galactosidase, and each cleavage resulted in the loss of expression of the 1-1-M-defined epitope. The N-acetylhexosamine (HexNAc) released by the hexosaminidase was found to be GalNAc, and the smaller oligosaccharide released by the endo enzyme was identified to be a dimer GalNAc beta----Gal. In contrast, the MAb 3F11-defined LOS component was not digested by the endo galactosidase, but it was cleaved with alpha and beta-galactosidase, and expression of the MAb 3F11-defined LOS epitope expression of the MAb 3F11-defined LOS was abolished by the treatment with each of two exo enzymes. MAb 3F11 bound to the 1-1-M-defined LOS component resulting from the removal of the beta-GalNAc residue, and the resulting LOS was further cleaved with beta-galactosidase, but not with alpha-galactosidase. From these results, we conclude the following: (1) MAbs 1-1-M and 3F11 both recognize the non-reducing termini of the LOS components; (2) the 1-1-M-defined LOS component has the GalNAc beta----Gal beta 1----4-Glc (or GlcNAc) structure, and the GalNAc beta----Gal residue is involved in the MAb 1-1-M-defined epitope; (3) the MAb 3F11-defined LOS component may not have a Gal beta 1----4GlcNAc beta 1----4Gal beta 1----4Glc structure within the molecule. However, it has beta-Gal residue at its non-reducing terminus, and this residue is involved in the MAb 3F11-defined epitope; (4) the two LOS components share a similar antigenic structure, and the 3F11-defined epitope structure is present in the MAb 1-1-M-defined LOS component. Expression of this epitope within the 1-1-M-defined LOS molecule is blocked by the beta-GalNAc residue; however, the beta-GalNAc residue at the non-reducing end may be not the only structural difference between the two components.     

Monoclonal antibody BA-1 to the human B lymphocyte marker CD24 recognizes a sialic acid (N-acetylneuraminic acid) dependent epitope in multi-valent display on peptide.

Evidence is presented that monoclonal antibody BA-1, directed against a marker (CD24) of human lymphocytes of B cell lineage, recognizes a sialic acid-dependent epitope. This conclusion is based on a series of experiments exploiting the reaction of this antibody with bovine and ovine submaxillary mucins. Expression of the epitope was enhanced following alkaline saponification of bovine submaxillary mucin, which converts O-acetylated neuraminic acid residues to N-acetylneuraminic acid. The epitope was destroyed following neuraminidase or mild acid treatment of the mucins, and its expression was diminished following neuraminidase treatment of B lymphoblastoid cells. Glycopeptides obtained by digestion of the bovine mucin with papain, trypsin or pronase were lacking in antigenicity. However, antigenic activity could be regenerated after conjugation of pronase glycopeptides to poly-L-lysine. These results indicate that multivalent display of sialo-oligosaccharide on peptide rather than a protease-susceptible polypeptide domain is required for BA-1 antibody binding.     

Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope.

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.     

Adhesion of leptospires to mouse fibroblasts (L929) and its enhancement by specific antibody

The adhesion of leptospires (Leptospira interrogans serovar. copenhageni L45) to mouse L-cells was studied by microscopic observations. Within 3 h of infection of monolayers many leptospires adhered to 95-100% of the cells, and intracellular leptospires were demonstrated by electron microscopy. No specific site of attachment on the cells or the leptospires was observed. Avirulent or dead leptospires adhered poorly but attachment of the saprophytic leptospire L. biflexa serovar. patoc occurred on cell and glass surfaces. After adhesion, microvilli on the cell surfaces disappeared within 6 h of infection and cell damage was observed after 12 h. The adhesion was greatly enhanced by the presence of specific antiserum at a subagglutinating concentration. No direct penetration by leptospires of the host cells was observed with transmission and scanning electron microscopy. It appears that (1) adhesion of leptospires to L-cells precedes cell damage, and (2) leptospires may enter cells either through damaged membranes, or by a phagocytosis-like mechanism.     

Meningococcal group A lipooligosaccharides (LOS): preliminary structural studies and characterization of serotype-associated and conserved LOS epitopes.

Structural studies indicate that the neisserial lipooligosaccharides (LOS) are composed of an oligosaccharide (OS) portion with a phosphorylated diheptose (Hep) core attached to the toxic lipid A moiety. A conserved meningococcal LOS epitope, defined by monoclonal antibody (MAb) D6A, is expressed on group A and many group B and C meningococci of different LOS serotypes (J. J. Kim, R. E. Mandrell, H. Zhen, M. A. Apicella, J. T. Poolman, and J. M. Griffiss, Infect. Immun. 56:2631-2638, 1988). This MAb-defined D6A epitope is immunogenic in humans (M. M. Estabrook, R. E. Mandrell, M. A. Apicella, and J. M. Griffiss, Infect. Immun. 58:2204-2213, 1990; M. M. Estabrook, C. J. Baker, and J. M. Griffiss, J. Infect. Dis. 197:966-970, 1993). In this study, we characterize this important MAb-defined LOS epitope. Serotype L10 and L11 group A meningococal LOS were chemically modified and used to investigate what portion of the LOS molecule is important for expression of the conserved (D6A) epitope and serotype-associated LOS epitopes by use of immunoblotting techniques and selected MAbs as probes. Preliminary structural characterization of the LOS was also accomplished by electrospray ionization-mass spectrometry. Our results indicate the following. (i) Antibodies that recognize the serotype-associated or conserved LOS epitopes recognize the OS portion of the LOS. (ii) The phosphorylated diheptose core region of the OS is essential for expression of the conserved D6A epitope. (iii) The lipid portion of the molecule is important for optimum expression of the LOS epitopes. (iv) The proposed compositions of the O-deacylated LOS are consistent with the presence of a phosphorylated diheptose core and are as follows: for O-deacylated L10 LOS, 3Hex (hexose), 1HexNAc (N-acetylhexosamine), 2KDO (2-keto-3-deoxy-D-manno-octulosonic acid), 2Hep (heptose), 1PEA or 2PEA (phosphoethanolamine), and O-deacylated lipid A; and for O-deacylated L11 LOS, 2Hex, 1HexNAc, 2KDO, 2Hep, 2PEA, and O-deacylated lipid A. Because the phosphorylated diheptose core region of the LOS is essential for the formation of a conserved LOS epitope (D6A) that is immunogenic in humans, care should be taken to maintain stereochemical requirements for the expression of this conserved epitope in the design of effective, nontoxic LOS vaccines.     

Functional and binding characterization of a single chain Fv antibody to abscisic acid and conjugated abscisic acid

In the present research, we studied the binding characterization of a single chain fragment variable (scFv) antibody S12B8 with abscisic acid (ABA) and conjugated ABA by competitive enzyme-linked immunosorbent assay (ELISA) and in silico molecular docking. The nucleotide sequences of variable light (VL) and variable heavy (VH) were obtained from hybridoma cells (F12B8), which secrete monoclonal antibodies recognizing ABA and conjugated ABA. And then the genes of VL and VH were constructed to a scFvS12B8 with a nucleotide sequence of a flexible linker (G₄S)₃ by splicing overlap extension PCR (SOE-PCR). The fragment of S12B8 was cloned into pMAL-c2x with a maltose binding protein tag and expressed in Escherichia coli with a calculated molecular mass of 68?kDa. The results of ELISA showed dose-dependent inhibition of the purified recombinant protein S12B8 by ABA, (ABA-ME) and ABA glucose ester tetra acetyl (ABAGE tetra acetyl). The detection limits (10% inhibition) of ABA, ABA-ME and ABAGE tetra acetyl were 19.71, 8.06 and 3.95?μg/ml, respectively. On the other hand, we investigated the interactions between S12B8 and ligands by homology modeling using RosettaAntibody and molecular docking by Discovery studio 2.5 (DS 2.5). The binding energy of ABA, ABA-ME and ABAGE when fused to the S12B8 antibody was ?37.1, ?69.6 and ?112.0?kcal/mol, respectively, which agreed well with our competitive ELSIA results.     

Drug sensitivity of Haemophilus sp. and transfer of resistance into E. coli (author's transl)]

From June 1973 to July 1976, 742 strains of Haemophilus influenzae and parainfluenzae, isolated from clinical specimens, were routinely tested for in vitro sensitivity to twelve antibiotics: penicillin, ampicillin, cephalothin, streptomycin, kanamycin, gentamicin, chloramphenicol, tetracyclin, minocyclin, erythromycin, sulfamethoxazol, trimethoprim. 61 strains were found resistant to one or more of these antibiotics (ampicillin, kanamycin, chloramphenicol and tetracyclin). The MICs of 23 antibiotics were determined by the agar dilution method on most of the resistant strains and on 60 sensitive strains isolated during the same period and considered as control. 21 strains transferred their resistance determinants into E. coli K12; 23 plasmids were obtained isolated from these strains: 2 strains contained two different plasmids. 90% of the transconjugants were stable after repeated subcultures.     

Incorporation of rat histocompatibility (AgB) antigens into liposomes, and their susceptibility to immune lysis.

The purification of detergent-solubilized rat histocompatibility antigens is described. The antigen may readily be incorporated into synthetic vesicles (liposomes) which appear to be unilamellar and between 0.1 microbeter and 0.2 micrometer in diameter. The addition of specific antibody leads to the agglutination and precipitation of the liposomes. An enzyme, horseradish peroxidase, may be incorporated into the trapped aqueous phase of the liposome, and its release by antibody and complement can be detected.     

The production of antibody to bovine serum albumin in unresponsive (Sobey) mice

Mice (Sobey) bred to select lack of antibody production against bovine serum albumin have been examined for their responsiveness over a wide range of antigen doses. With low doses of antigen both Sobey mice and an outbred line of Swiss White mice produce similar amounts of antibody. Antigen in amounts greater than 500 μg produce tolerance more effectively in Sobey mice and this is the reason for their unresponsiveness at high antigen dose levels.