Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus.

Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae.

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin-agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.

Isolation of the outer membrane of Branhamella catarrhalis.

The emergence of Branhamella catarrhalis as an important human pathogen has stimulated interest in investigations of the outer membrane (OM) of the bacterium. In this study, the OM of B. catarrhalis was isolated and partially characterized. Radiolabelled cells were lysed and fractionated by isopycnic centrifugation in a continuous sucrose gradient. Five fractions were identified. Fraction A consisted of OM fragments of varying density. Fractions B and C were OM of a discrete density containing some cytoplasmic membrane. Fraction D was cytoplasmic membrane and Fraction E contained smaller less dense fragments of cytoplasmic membrane. The protein composition of the Branhamella OM is typical for that of Gram-negative bacteria in that approximately 10 to 20 proteins were present with six to eight of these proteins predominating. Having isolated and partially characterized the OM by sucrose density centrifugation, five simpler techniques for isolating OM were employed and the preparations compared to OM isolated on the gradient. Techniques that are based on differential detergent solubility of OM and cytoplasmic membrane were ineffective in isolating OM of B. catarrhalis. By contrast, techniques that involved collection of OM vesicles were successful in isolating OM of B. catarrhalis. Collection of vesicles from broth culture supernatants and EDTA-heat-induced vesicles were identified as convenient and reliable methods for isolating OM. Isolating and partially characterizing the OM of B. catarrhalis represents an initial step in a systematic study of outer membrane antigens of the bacterium.

Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes.

Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.

Lipooligosaccharides: the principal glycolipids of the neisserial outer membrane.

The outer-membrane glycolipids of bacteria that colonize mucosal surfaces that are not routinely bathed by bile acids often lack the long, hydrophilic and neutral polysaccharides that protect the lipid membranes of enteric bacteria from dispersal. The glycolipid from these organisms is properly termed a lipooligosaccharide. A Neisseria strain makes from two to six lipooligosaccharide molecules that range in Mr from 3,150 to 7,100. Different species of Neisseria commonly make lipooligosaccharides of identical Mr and epitope content. Differences in oligosaccharides account for most of the observed physical heterogeneity. Oligosaccharides consist of (1) partially conserved and highly substituted basal oligosaccharides that branch at heptose residues; (2) a linear segment consisting of (hexose)n residues that determines the length of the oligosaccharide; and (3) terminal sequences that are similar to those of glycosphingolipids. Epitope expression is linked to physical heterogeneity and is modified by the molecular environment of the outer membrane. Serotype epitopes are present only on lipooligosaccharides of a certain Mr. Certain lipooligosaccharides regulate complement activation onto the bacterial surface and, hence, immune lysis.

Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseriae.

This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.

Detection by gas chromatography of 3-deoxy-D-manno-2-octulosonic acid and L-glycero-D-manno-heptose in whole cells of Neisseria elongata.

Lipopolysaccharide components 3-deoxy-D-manno-2-octulosonic acid and L-glycero-D-manno-heptose were detected in hydrolysates from whole cells of Neisseria elongata by gas-liquid chromatography. Cells from a single plate were hydrolyzed, and carbohydrate components were converted to aldononitrile and O-methyloxime acetate derivatives for subsequent analyses by gas-liquid chromatography. 3-Deoxy-D-manno-2-octulosonic acid was well separated from other cell components as the O-methyloxime acetate derivative. With both derivatives, L-glycero-D-manno-heptose was readily identified by their different retention times. The procedure requires only a relatively small number of cells, and detection is accomplished without prior isolation of the lipopolysaccharide.

Differentiation of Neisseria gonorrhoeae from other Neisseria species by use of the restriction endonuclease HaeIII.

We used the restriction endonuclease HaeIII to differentiate Neisseria gonorrhoeae from other Neisseria species and Branhamella catarrhalis. A total of 16 clinical isolates and four American Type Culture Collection strains of N. gonorrhoeae were resistant to HaeIII digestion, whereas 17 isolates and four American Type Culture Collection strains from eight different bacterial species were susceptible. This resistance was not caused by an enzyme inhibitor. We propose that protection of the HaeIII recognition sequence by methylation is the mechanism of resistance since N. gonorrhoeae DNA became susceptible to digestion when passed in Escherichia coli as part of a plasmid clone.

Isolation of a high molecular weight polyphosphate from Neisseria gonorrhoeae.

Neisseria gonorrhoeae, as well as other Neisseriae, produce polyphosphate. This polyphosphate exists in two forms. Approximately half of it is loosely associated with the cells and can be recovered by washing in neutral buffers under conditions in which no significant lysis of the cells is observed. The other half is either intracellular or tightly associated, because it requires digestion of the cells with perchloric acid or sodium hypochlorite. Polyphosphate obtained by both methods was purified by column chromatography and chemically characterized. In contrast to other organisms, gonococci do not respond with increased polyphosphate synthesis when shifted from phosphate starvation to a phosphate-rich medium. In addition, gonococcal polyphosphate does not serve as a depletable phosphate source during phosphate starvation. All strains of Neisseriae examined produce substantial amounts of polyphosphate.

Structure of the D-glucans produced by Neisseria perflava.

A chemical and enzymic study of the cellular glucan of Neisseria perflava and of the glucan produced from sucrose by a cell-free extract of N. perflava showed from optical rotation, 13C nuclear magnetic resonance, methylation analysis, and alpha- and beta-amylase hydrolysis studies that the glucans had glycogen-like structures. These structures were composed of chains of 1-4-alpha-D-glucopyranosyl units with branch points of 4-O- and 6-O-disubstituted D-glucopyranose units. While the backbone structures of the two glucans were similar, the release of maltose by the action of beta-amylase indicated that the 1-4 linked nonreducing side chains of the cell-free enzymically synthesized glucan were longer (approximately seven units) than those present in the cellular glucan (approximately two to three units), a result in agreement with methylation analyses.

Cellular monosaccharide patterns of Neisseriaceae.

Sixty-four strains of Neisseria, Moraxella, and Acinetobacter were screened for cellular monosaccharides by gas-liquid chromatography and other chromatographic techniques. The four sugars ribose, glucose, glucosamine, and 2-keto-3-deoxyoctonate (KDO) were detected in all strains. Heptose was detected only in "true neisseriae" (Neisseria gonorrhoeae, N. meningitidis, N. sicca, N. cinerea, N. flavescens, and N. elongata) and in the tentaively named species Moraxella urethralis. Some marked interspecies dissimilarities within groups were revealed. Thus, N. ovis and M. atlantae were characterized by the presence of mannose. Intraspecies differences were also encountered. N. meningitidis strains of serogroups B and C were distinguished from strains of serogroup A by their sialic acid content. This sugar was also detected in two out of three examined strains of M. nonliquefaciens. In Acinetobacter, heterogeneity of monosaccharide patterns was rather pronounced. The results show the applicability of gas chromatographic "monosaccharide" profiles fo whole cells or extracted carbohydrate in bacterial classification and identification, including differentiation at the subspecies level. In addition, such profiles may be useful for monitoring during purification of cellular polysaccharides.

Immunochemical study of the peptidoglycan of gram-negative bacteria.

The specificity of antibodies directed against the peptidoglycan of gram-negative bacteria was studied. The peptidoglycans of Proteus vulgaris, Escherichia coli, Moraxella glucidolytica, Neisseria perflava, give identical precipitin reactions. By means of inhibition studies with various peptidoglycan subunits and synthetic peptides, it was shown that the antibodies are essentially directed against the peptide moiety of the peptidoglycan: L-Ala-D-Glu (L)-mesoA2pm-(L)-D-Ala, that the peptide reacts better with antibodies when it is not cross-linked, and that the C-terminal portion-meso-A2pm-D-Ala of the peptide is immunodominant. These results explain the immunological identity of the peptidoglycans of gram-negative bacteria, which possess the same peptide subunit. Only weak cross-reactivity was observed with the peptidoglycans of gram-positive bacteria (Streptococcus faecium, Micrococcus lysodeikticus, Corynebacterium poinsettiae) where meso-diaminopimelic acid is replaced by L-lysine or L-homoserine. However, the peptidoglycan of Bacillus megaterium which possesses the same peptide subunit as gram-negative bacteria, gives only a reaction of partial identity with these bacteria. This result suggests the presence on the peptidoglycan of gram-negative bacteria, of other undefined antigenic determinants.

Studies on the cellular and free lipopolysaccharides from Branhamella catarrhalis.

Cellular and free lipopolysaccharides obtained from Neisseria catarrhalis and Branhamella catarrhalis were found to be essentially identical. Both cellular and free lipopolysaccharides contained core-oligosaccharides of the following composition: D-glucose (4 mol), D-galactose (1 mol), 2-amino-2-deoxy-D-glucose (1 mol), and 3-deoxy-D-manno-octulosonic acid. Aldoheptose and phosphate components were below levels of detection. Several physical methods indicated that all core-oligosaccharide preparations were identical. Lipid A preparations from cellular and free lipopolysaccharides of both organisms were qualitatively and quantitatively similar; they were composed of decanoic acid, dodecanoic acid, 3-hydroxy dodecanoic acid, 2-amino-2-deoxy-D-glucose, phosphate, and ethanolamine. The results tend to justify the transfer of Neisseria catarrhalis to the genus Branhamella.

Studies of the cellular and free lipopolysaccharides form Neisseria canis and N. subflava.

Cellular and free lipopolysaccharides (LPS) obtained from Neisseria canis and N. subflava were essentially identical. Both cellular and free lipopolysaccharides contained O-polysaccharides of the following composition: L-rhamnose (46 mol), D-glucose (16 mol), L-glycero-D-manno-heptose (2 mol), ethanolamine (2 mol), 3-deoxy-D-manno-octulosonic acid (1 mol), and phosphate (1.5 mol). The core oligosaccharide, which was common to the cellular and free LPS of both organisms, contained L-rhamnose (4 mol), D-glucose (2 mol), L-glycero-D-manno-heptose (2 mol), 3-deoxy-D-manno-octulosonic acid (1 mol), ethanolamine (2 mol), and phosphate (1.5 mol). Accumulated results on LPS composition and structure indicated that Neisseria perflava, N. subflava, and N. flava could not be combined into a single species. On the basis of its nutritional requirements and LPS structure, N. canis could be considered a strain of N. subflava.

Comparison of the cell envelope proteins of Micrococcus cryophilus with those of Neisseria dnd Branhamella species.

In an attempt to elucidate the relation between Micrococcus cryophilus, Neisseria caviae, Neisseria ovis, and Branhamella catarrhalis, fractions derived from outer membranes of a strain of each organism were examined for protein composition by SDS - polyacrylamide gel electrophoresis. Micrococcus cryophilus outer membrane protein showed extensive similarities to that of N. ovis and contained a heat-modifiable protein which behaved almost identically with the corresponding bands previously shown to exist in N. caviae and N. ovis. Branhamella catarrhalis protein was distinctly different from those of M. cryophilus and the two 'false neisserias' N. caviae and N. ovis.

Improved techniques for the preparation of bacterial lipopolysaccharides.

Liopolysaccharides were prepared from six organisms by the use of two cell-disruption procedures before conventional phenol-water extraction. Disruption of cells by grinding with glass beads or by digestion with hen egg white lysozyme before phenol extraction facilitated rapid purification and greater yields of lipopolysaccharide. Pretreatment of cells with lysozyme in the presence of ethylenediaminetetraacetic acid was the most efficient method in terms of lipopolysaccharide yield and ease of preparation. Increase in lipopolysaccharide yield achieved by use of the lysozyme method, compared with the conventional phenol extraction, varied from 1.7- to 12.4-fold. Preparations were designated as pure according to several criteria and were judged not to have undergone changes as a result of prephenol extraction procedures.

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.

Cellular and free lipopolysaccharides of some species of Neisseria.

Cultures of eight non-pathogenic species of Neisseria grown in simple defined media released lipopolysaccharide (free lipopolysaccharide) by a process distinct from cellular autolysis. Analyses of the pure cellular and free lipopolysaccharides obtained from six species of Neisseria revealed that they were remarkably similar and were devoid of detectable O-antigen side chains. Three distinct types of core-oligosaccharides were demonstrated. Type I core-oligosaccharide was a branched structure of alpha-D-glucopyranosyl units (7 mol) terminated by a reducing end group of 3-deoxy-D-manno-octulosonic acid. Type II core-oligosaccharide contained D-glucose, 2-deoxy-2-amino-D-glucose, L-rhamnose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, phosphate, and ethanolamine in a molar ratio of 3:2:1:1:1:1:1. Type III coreoligosaccharide was composed of D-glucose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, and phosphate in a molar ratio of 3:3:1:1. Lipopolysaccharides of N. caviae and N. sicca contained type I core-oligosaccharides exclusively, while those of N. flava and N. perflava contained only type II core-oligosaccharide. Cellular lipopolysaccharide from N. cinerea contained core-oligosaccharides of types I and II in a ratio of 27:73, while the analogous preparation from N. flavescens contained core-oligosaccharide types II and III in a ratio of 21:4. Free lipopolysaccharides from these two organisms contained only one type of coreoligosaccharide. Lipid A components of all the lipopolysaccharide preparations were very similar being composed of about 25% by weight of dodecanoic acid, 3-hydroxy-dodecanoic acid, and 3-hydroxy-tetradecanoic acid.

SDS-polyacrylamide gel electrophoresis of lipopolysaccharides.

Lipopolysaccharides (LPS) prepared from four different species of Neisseria have been separated by SDS-polyacrylamide gel electrophoresis. Each LPS possessed a characteristic mobility on gels. Examination of the effect of acrylamide concentration on migration illustrated that the basis of the separation was molecular size and not intrinsic charge.