Herman Schneider

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.

Experimental Gonococcal Urethritis and Reinfection with Homologous Gonococci in Male Volunteers

Background Reinfection, a common occurrence with gonorrhea, may result from a lack of protective immune response, or from the tremendous gonococcal strain variation. Goal A two-phase study in human volunteers tested whether experimental infection with Neisseria gonorrhoeae MS11mkC would protect against reinfection with the same organisms. Study Design In phase 1, an intraurethral inoculum of 57,000 piliated, transparent (opacity protein–negative [Opa−]) MS11mkC N gonorrhoeae infected 14 of 15 (93%) volunteers. The volunteers were encouraged to delay treatment for at least 5 days. In phase 2, which began 2 weeks after treatment for the initial infection, volunteers were inoculated with 7,100 piliated, Opa− MS11mkC. Results The phase 2 challenge infected 6 of 14 (43%) previously infected volunteers and 5 of 10 (50%) naïve control subjects. Phase 1 volunteers who resisted reinfection were significantly more likely to have had a fourfold or greater increase in lipooligosaccharide immunoglobulin G during phase 1 than those who did not resist reinfection (P = 0.026). Conclusions Although infection did not provide protection from reinfection under the conditions used, the results suggest that immunity to reinfection is more complex than anticipated by the experimental design.

Experimental Shigella Infections. III. Sensitivity of Normal, Starved and Carbon Tetrachloride Treated Guinea Pigs to Endotoxin

Summary Pretreatment of Hartley strain guinea pigs either by subcutaneous injection with carbon tetrachloride or by 4 day period of starvation renders them more susceptible to intravenously administered endotoxin. The LD50 for normal animals is in excess of 1300 μg; for starved animals 52.8 μg and for carbon tetrachloride treated animals 4.2 μg. Distribution of Cr51 label of Cr51 tagged endotoxin in organs of normal animals which survive intravenous dose of 52 μg of endotoxin is the same as that in carbon tetrachloride treated animals which succumb following this challenge.

Neisseria gonorrhoeae MS11mkC opacity protein expression in vitro and during human volunteer infectivity studies.

Background and Objectives: Neisseria gonorrhoeae MS11mkC harbors 11 independently expressed opacity (Opa) protein genes with distinct in vitro expression frequencies. In experimental infections in which human male volunteers were inoculated with transparent (Opa−), piliated (P+) strains, the authors associate onset of symptoms with recovery of opaque (Opa+) gonococci. Goals: In vitro and recovered (Opa) protein expression rates were compared to determine if the human host influences Opa expression. Study Design: Opa expression was determined using Western immunoblot analysis; Opa sizes were determined using a scanning densitometer. Results: Seven of 10 Opa proteins were identified in gonococci recovered from all of the volunteers at frequencies consistent with in vitro results (Opa C, 29.5 kDa; Opa K, 30 kDa; Opa G, 31 kDa; Opa I, 32 kDa; Opa J, 33 kDa; Opa D, 34 kDa; and Opa H, 37 kDa) (P ≥ 0.01, Fisher exact test). Opa B (30.5 kDa) was identified at lower than expected frequencies, whereas Opa E (31.2) and F (31.5) were identified at higher than expected frequencies. When recovered gonococci were reanalyzed for in vitro expression frequencies, they were consistent with preinfection frequencies. Conclusions: The host may influence the prevalence of some Opa proteins.

Structure-Activity of Sulfones and Sulfonamides on Dihydropteroate Synthetase from Neisseria meningitidis

The molecular interaction of various sulfones and sulfonamides with partially purified dihydropteroate synthetase from Neisseria meningitidis M-166 has been examined. The mode of action of the sulfones was similar to that of the sulfonamides. Both groups of drugs were competitive inhibitors of dihydropteroate synthetase with respect to p-aminobenzoate in a partially purified enzyme preparation. 4,4′-Diaminodiphenylsulfone was three times more effective than sulfadiazine and nine times more effective than sulfanilamide as a competitive inhibitor of dihydropteroate synthetase. The inhibitory activity of 4-amino-4′-acetamidodiphenylsulfone and 4-amino-4′-formamidodiphenylsulfone in this system eliminated their prior conversion to 4,4′-diaminodiphenylsulfone as a requirement for activity.

A bacterial microassay for testing serum sensitivity of Neisseria gonorrhoeae

A reproducible gonococcal serum bactericidal microassay is described which has easily determined endpoints and lends itself to large scale tests. The microassay may be used to assess sensitivity to normal human serum among strains of gonococci, and to define the antigens conferring this sensitivity. It may be readily adapted to test for bactericidal activity in hyperimmune antisera and to evaluate antigens which inhibit serum bactericidal activity.

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

Abstract A partition chromatographic procedure utilizing a cationic exchange resin column in the Li + form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy- d - manno -octulosonic acid (KDO) and l - glycero - d - manno -heptose in the lipopolysaccharides (LPS) of R e and R d P − rough mutants of Salmonella minnesota . In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R e or R d P − LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H + form) released more than 80% of the KDO residues within 15 min. The heptose of the R d P − LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R d P − LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.

Phenotypic variation of the antigen expression of the lipooligosaccharide of Neisseria gonorrhoeae

Gonococcal lipooligosaccharides (LOS) are a series of antigenically complex heteropolymers. To investigate whether all members of clonal populations of Neisseria gonorrhoeae express antigenically similar LOS, gonococcal strains 4505 and 220 were studied with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or co-migrating LOS unit(s) on SDS-Page. After sequentially staining organisms on formvar grids with these monoclonal antibodies and then staining with either 5nM or 15 nM colloidal gold spheres conjugated to goat antimurine IgM, 3 populations of cells could be identified among organisms derived from a single clone. Organisms which stained with 6B4 and 3F11 and organisms staining for either 6B4 alone or 3F11 alone. Immunofluorescent microscopy studies using rhodamine and fluorescein goat antimurine IgM conjugates to sequentially stain 3F11 and 6B4 also demonstrated these three populations. Fluorescent activated cell sorting studies of 3F11 coated strain 220 separated the population into two groups corresponding to organisms containing or lacking the 3F11 epitope. These studies indicate that clonally selected strains of N. gonorrhoeae contain members which have different phenotypic expression of different LOS antigens. Previously observed stability of the LOS antigenic and physicochemical structure suggest that this phenotypic variation is under environmental and/or genetic control.

Virulence of a Nutritional Mutant of Vibrio comma.

Summary Mouse virulence of a purine-requiring strain of Vibrio comma is significantly increased if purines are injected at the same time as the challenge suspension. A purine-independent mutant isolated from this strain was significantly more virulent for mice than the parent culture. A concomitant increase in ability to fatally infect guinea pigs by the oral route was not noted in the mutant strain.

Strain-specific direct binding of properdin accounts for variable lysis of Neisseria gonorrhoeae

We studied the interaction of Neisseria gonorrhoeae, (GC) and the human alternative complement pathway (ACP). GC strains with a spectrum of sensitivity to lysis by norman human serum (NHS) depleted ACP activity from NHS by from 13% to 99% residual activity, as determined by lysis of rabbit erythrocytes in the presence of MgEGTA, following absorption at 0°C. The NHS lytic titer for a gonococcal strain was a function of its absorption of ACP activity (ρ = 0.006), which accounted for 70% of the differences in titer among strains. GC variously absorbed properdin (P) from NHS as assessed by antigen-capture solid-phase radioimmunoassay analyses of absorbed sera. Addition of purified P to absorbed sera restored ACP activity to normal levels. Using a fluorescence-activated cell sorter, we found that highly serum-sensitive strain F62, which depleted 79% ACP activity, bound 2 × more purified P in the absence of serum than weakly serum-sensitive strain 56, which depleted only 1.1% ACP activity. These two strains bind NHS IgM to the same lipooligosaccharide epitope. We conclude that classical pathway-initiated NHS lysis of GC is variously augmented by the ACP as a function of strain-specific binding of P, and that the titer at which a strain is lysed is a function of this ACP augmentation.