Inhibitor of bacterial growth released by human cells in culture.

Used medium from cultured human cells contains a factor that inhibits growth of the "less virulent" strains of pathogenic bacteria, but only retards growth of the "more virulent" strains. The factor is heat-stable, dialyzable, and unaffected by change in pH; it chromatographs as material of molecular weight between 700 and 1500. There is evidence that this factor is an alpha-ketoaldehyde attached to a carrier.

Identification of a bacterial growth inhibitors from HeLa cells: a ketoaldehyde.

The growth-inhibiting factor found earlier in human cell cultures appeared to be a ketoaldehyde. An infrared spectral study of 2,4-dinitrophenylhydrazine derivatives suggested that the ketoaldehyde was 4-hydroxy-2-ketobutyraldehyde. The compound, when synthesized, gave the same DNPH derivatives as the natural substance.

Iron as a replacement for mucin in the establishment of meningococcal infection in mice.

Experimental infection of mice with Neisseria meningitidis was established by the injection of the bacteria suspended in solutions of various iron compounds. The progressive and fatal infection caused by otherwise non-lethal doses of organisms was produced in these mice after prior injection with ferrous sulphate or concomitant injection with iron sorbitol citrate or iron dextran. Reduction in LD50 to levels at least comparable to those obtained in the mucin challenge system was achieved; in some serogroups of N. meningitidis the LD50 was decreased more than a million fold. The results suggest that iron, which is a component of hog gastric mucin, is a factor involved in the establishment of meningococcal infection in mice. Use of iron compounds as injection medium offers a more advantageous system than mucin, since controlled administration of chemically defined substances occurs.

Structural elucidation of the 3-deoxy-D-manno-octulosonic acid containing meningococcal 29-e capsular polysaccharide antigen using carbon-13 nuclear magnetic resonance.

The capsular polysaccharide antigen from Neisseria meningitidis serogroup 29-e contains equimolar quantities of 2-acetamido-2-deoxy-D-galactose and 3-deoxy-D-manno-octulosonic acid (KDO), the latter of which is rarely found in biopolymers other than lipopolysaccharides. Carbon-13 nuclear mangetic resonance in conjunction with other chemical data indicated that the polysaccharide is composed of an alternating sequence of these two residues, the linkages being at C-3 of galactosamine and C-7 of KDO in the alpha-D and beta-D configuration, respectively. The native 29-e polysaccharide is O-acetylated, the O-acetyl groups being located at C-4 and C-5 of the KDO residues. Assignments of signals in the 13C nuclear magnetic resonance spectrum of the 29-e polysaccharide were made by consideration of those in the spectra of the monomer models, which necessitated the first recorded syntheses of methyl-alpha- and beta-D-3-deoxy-manno-octulopyranosonic acid. Like the methyl alpha- and beta-D-ketosides of sialic acid (Na+ salts), the equivalent methyl alpha- and beta-D-ketosides of KDO exhibit large chemical shift differences in the exocyclic C-8 position dependent on anomeric configuration. This can again be attributed to hydrogen bonding between the axial carboxylate group of the methyl beta-D anomer of KDO (C1 conformation) and the primary hydroxyl group at C-8. This phenomenon is also exhibited by the beta-D-linked KDO units of the 29-e polysaccharide.

Inhibition of the growth of Neisseria meningitidis by reduced ferritin and other iron-binding agents.

Serogroups of N. meningitidis were characterized as virulent or avirulent according to their capacity to establish meningococcal infection in mice. An agar plate diffusion technique demonstrated that iron had a definite growth-supporting role for both of these meningococcal types. The avirulent strains could use ionic or chelated iron as well as the virulent strains. Iron-reversible growth inhibition occurred to the same extent for both bacterial types in the presence of the synthetic iron-chelating agents Desferal and ethylenediamine-di-orthohydroxy phenylacetic acid. A difference in response was demonstrated for these bacterial types when grown in the presence of various iron-binding proteins from animal body fluids and tissues. The growth of the avirulent strain was inhibited to a greater degree by egg white conalbumin. The humoral iron-binding protein transferrin showed a significant inhibitory capacity only when used in conjunction with bicarbonate. Under conditions of increased iron saturation of this protein, the avirulent strain was inhibited to the furthest extent. In the presence of ferritin, the cellular iron-binding protein, which had been reduced, inhibition of the growth of either strain type did not occur on iron-poor media (less than 5 micrograms/100 ml). However, with the incorporation of iron into the media, the inhibitory effect of the protein became evident. As the concentration of iron increased, the inhibition increased to a certain level and subsequently declined. A substantial difference in the ability of the avirulent type to grow in the presence of reduced horse spleen ferritin was observed. For this microorganism, a correlation appears to exist between the capacity to grow by utilizing the available iron in the presence of reduced ferritin and the ability to establish infection. The host protein ferritin, in the reduced state, apart from simply being a storage protein for iron, can prevent the growth of a procaryotic organism. Our experiments suggest a role for ferritin in the prevention of emningococcal disease. A cehmotherapeutic potential for Desferal is also implied.

Structural determination of the polysaccharide antigens of Neisseria meningitidis serogroups Y, W-135, and BO1.

The purified high molecular weight serogroup Y meningococcal polysaccharide contains equimolar proportions of D-glucose and N-acetylneuraminic acid and is partially O-acetylated. Carbon-13 nuclear magnetic resonance (NMR) studies, together with other chemical data, have indicated that the polysaccharide is linked only at C-6 of the D-glucose and C-4 of the sialic acid residues, all the linkages being in the alpha-configuration. The 13CNMR data also indicated that the Y polysaccharide is composed of an alternating sequence of these two different residues, and this was confirmed by its autohydrolysis where the major product was 4-O-alpha-D-glucopyranosyl-beta-D-N-acetylneuraminic acid. The W-135 polysaccharide differs from that of Y only in the absence of O-acetylation and in the configuration of one hydroxyl group of the disaccharide repeating unit. In this case autohydrolysis yielded 4-O-alpha-D-galactopyranosyl-beta-D-N-acetylneuraminic acid as the major product. Structural evidence indicates that the BO and Y polysaccharides are identical. Methanolysis of the Y polysaccharide yielded in addition to the methyl glycosides of glucose and sialic acid, a 9-O-acetyl derivative of the latter. This derivative was formed during the re-N-acetylation process and its formation was mainly due to the presence of sodium ions in the original polysaccharide.

Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance.

The application of 13-C nuclear magnetic resonance to the analysis of some sialic acid-containing meningococcal polysaccharide antigens is described. Complete assignments of the spectra of both the native serogroup B and the de-O-acetylated serogroup C polysaccharides have been made. These assignments were based on the corresponding data for some related monomers (sialic acid and its alpha-and beta-methylglycosides) and on supportive chemical evidence. The data indicate that the serogroup B polysaccharide is a 2 yields 8-alpha-linked homopolymer of sialic acid, identical in structure with colominic acid from Escherichia coli, whereas the de-O-acetylated serogroup C polysaccharide is a 2 yield 9-alpha-linked homopolymer. The native serogroup C polysaccharide is O-acetylated (1.16 mol of O-acetyl per sialic acid residue), all the O-acetyl substituents being located only at C-7 and C-8 of the sialic acid residues, and in addition contains unacetylated residues (24%). The polysaccharide contains di-O-acetylated residues (O-acetyl on C-7 and C-8), and at least one of the possible monoacetylated residues at C-7 or C-8.

Antibody production in milk serum after antigen instillation of the goat mammary gland. IX. Sham infection studies with Neisseria meningitudis L.C.D.C. 608B.

Specific immune globulins have been prepared in goat milk in response to the intramammary gland instillation of Neisseria meningitidis 608, serogroup B strain. Isolation, purification, and characterization of the goat whey by gel filtration, electrophoresis, and analytical ultracentrifugation demonstrated that the active immune component resided in the IgA class of globulins, specifically 9.2-S IgA. The potential of the lactating mammary gland as a "biological factory" for the large-scale production of diagnostic antiserum to killed bacterial whole cell antigen is described.

The R-type lipopolysaccharides of Neisseria meningitidis.

The lipopolysaccharides of all the different serogroups of Neisseria meningitidis are of the "R" type despite the morphologically smooth appearance and the demonstrated virulence of the organisms from which they were derived. This was confirmed when each of the lipopolysaccharides was found to be devoid of detectable O-antigen side chains, giving only a low "molecular" weight core oligosaccharide when subjected to mild acid hydrolysis. The cores were modified by dephosphorylation and subjected to sugar and methylation analysis by gas-liquid chromatography. Although all the different cores contained identical components (glucose, galactose, glucosamine, heptose, and 2-keto-3-deoxyoctonate) they could be separated into three distinct categories according to their galactose:glucose ratios. These categories are typified by the cores obtained from groups A, C, and 29-e which have galactose:glucose ratios of 1:2, 2:2, and 2:1, respectively. The modified cores were methylated and analyzed by gas chromatography--mass spectrometry and on the basis of differences in the derived methylated sugars the cores could again be divided into the same three categories as above. This structural diversity also results in some serological specificity as demonstrated by the complete serogroup specificity of the group A lipopolysaccharide.

Cross-protective antigens of Neisseria meningitidis obtained from Slaterus group Y.

An extraction of the cells of Neisseria meningitidis serogroup Y with an aqueous solution of calcium choride (0.9 m) has been shown to solubilize a number of antigens. By immunodiffusion, this mixture of antigens has been shown to react with its group-specific antiserum and also to cross-react with a number of other group-specific antisera. The cross-reacting antigen appears to be an antigen common to a number of other serogroups of meningococci, and there is some evidence that it is protein in nature. It has been demonstrated further that the calcium chloride extract contains a strong cross-protective antigen, as shown by its ability to provide good, active immunity in mice to both the homologous and heterologous serogroups of meningococci.

A chemically defined protein-free liquid medium for the cultivation of some species of Neisseria.

Chemically defined, protein-free, liquid culture media are needed for research with pathogenic bacteria and for the preparation of vaccines from them. Variations in growth or yield of organisms should be due only to differences in strain, and the medium itself should have little or no antigenic properties or toxicity. The medium should maintain the integrity of the bacterial species fully and it is particularly important that the complete antigenic complement of the organism should remain unaltered. The medium should be inexpensive and simple to prepare. Such a medium for the cultivation of some species of Neisseria, known to be fastidious, is described.In this new medium all the criteria are satisfied, and its freedom from sensitizing proteins makes it of particular value for use in man. Results of safety tests of vaccines prepared from Neisseria gonorrhoeae and N. meningitidis grown in this medium were encouraging. It should also be useful in studies on the physiological characteristics of the individual species.

Dubos oleic acid agar medium in the differentiation of meningococci and gonococci.

Growth on Dubos oleic acid agar plates, incubated for 48 hr at 37 C in candle jars, provides a method for differentiating meningococci from gonococci. All of our 54 strains of meningococci, but none of the 49 strains of gonococci, grew on this medium.

Strucutres of the capsular polysaccharides of Neisseria meningitidis as determined by 13C-nuclear magnetic resonance spectroscopy.

The application of 13C-nuclear magnetic resonance spectroscopy to the structural determination of the capsular polysaccharide antigens of Neisseria meningitidis is described. Complete assignments of the spectra of the polysaccharides of serogroups A, B, C, W-135, X and Y were made and were based mainly on previous assignments made for the monomer units of the respective polysaccharides. This technique provides information on all structural aspects of the polysaccharides including composition, mode of linkage, location of O-acetyl substituents, sequence, and conformation. In addition, nuclear magnetic resonance is nondestructive, extremely rapid, and has enormous potential in bacteriological research.