Genetic structure of Neisseria meningitidis serogroup C epidemic strains in south Brazil

No presente estudo, nos reportamos os resultados de uma analise, baseada na sorotipagem, multilocus enzimatico (MEE) e ribotipagem de N. meningitidis sorogrupo C isoladas de paciente com doenca meningococica no Rio Grande do Sul (RS) e Santa Catarina (SC), onde o Centro de Controle Epidemiologico do Ministerio da Saude detectou um aumento de casos de doenca meningococica (DM) devido a este sorogrupo nos ultimos 2 anos (1992-1993)...

Eight lipooligosaccharides of Neisseria meningitidis react with a monoclonal antibody which binds lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc).

Eight of 12 serologically different lipooligosaccharides (LOS) of Neisseria meningitidis bound a mouse monoclonal antibody (anti-My-28) that recognizes lacto-N-neotetraose (LNnT) (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc). Among the 12 LOS immunotypes, types 2, 3, 4, 7, 8, and 9 exhibited strong binding; types 5 and 10 were moderate; and types 1, 6, 11, and 12 were negative as measured by enzyme-linked immunosorbent assays, immunodot assays, and immunoblot assays. If an LOS showed multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the antibody-reactive epitope was expressed on the larger major component, of which the molecular weight was estimated to be 4,000 for most types. The expression of the reactive epitope on the LOS was influenced by the growth medium, and the epitope could be masked by sialylation when N. meningitidis was grown in tryptic soy broth. N-Acetyllactosamine inhibited the binding of the antibody to all eight reactive LOS. The antibody binding to a representative LOS was best inhibited by LNnT and next by N-acetyllactosamine but was not inhibited by lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc). These results suggest that the LNnT sequence is present in 8 of 12 immunotype LOS. The presence of the LNnT sequence, a structure expressed by a variety of human cells, in the LOS may play a role in the virulence of N. meningitidis by enabling the organism to evade host immune defenses.

Topology of outer membrane porins in pathogenic Neisseria spp.

In Escherichia coli, membrane-spanning amphipathic beta-sheet structures are characteristic of many outer membrane proteins. By applying the principles that have been recognized for them to the four classes of neisserial porins, we have constructed a model for the topology of the porins within the outer membrane. This model predicts eight surface-exposed loops, both in the meningococcal class 1 and 2 proteins and in the gonococcal PIA and PIB proteins. The transmembrane sequences are highly conserved among these porins and are able to form an amphipathic beta-sheet structure. The surface-exposed hydrophilic loops show extensive variation in both length and sequence. Experimental evidence in support of this model has been obtained by using antisera against synthetic peptides which correspond to surface-exposed loops in class 1 and 2 proteins. Thus, binding to the cell surface was observed with antibodies against loops 1, 4, and 5 of class 1 and loops 1 and 5 of class 2. In class 1, these loops are the longest ones and show the highest sequence diversity among strains of different subtypes. Mapping of epitopes recognized by monoclonal antibodies with bactericidal activity has also provided strong support for the model. The epitopes are located in loops 1 and 4 of class 1 protein, loop 5 of PIB, and loop 6 of PIA. A nonbactericidal antibody that binds only weakly to whole cells was shown to recognize loop 3 of PIB. These results suggest that the longest loops are immunodominant, provide the binding sites for bactericidal antibodies, and display the greatest variation among different strains.

Immunization against serogroup B meningococci. Opsonin response in vaccinees as measured by chemiluminescence.

One hundred and thirteen healthy volunteers were immunized twice (six weeks apart) with four different doses (12.5, 25, 50 and 100 micrograms, measured as protein content) of an outer membrane vesicle vaccine from a serogroup B meningococcal strain (44/76, B:15:P1.16) complexed to serogroup C meningococcal polysaccharide and/or Al(OH)3 i.e. 12 different vaccines. Serum opsonic activity against the serogroup B strain was measured using a chemiluminescence method. A significant rise in serum opsonic activity was demonstrated in 84 volunteers (74%) six weeks after the first injection and in 97 (86%) six weeks after the second. All vaccinees with low preimmunization values (less than 25 mVs) experienced a significant increase in opsonic activity. A dose-related response was most evident for the vaccines containing adjuvant, and these vaccines were associated with a maximum response six weeks after the second injection, while the vaccines without Al(OH)3 induced a peak response six weeks after the first injection. The postimmunization opsonic activity was similar to that found in convalescent sera, indicating that the vaccines may protect against serogroup B meningococcal disease.

Control of components of bacterial polysaccharide vaccines by physical methods.

Analysis of polysaccharide components of meningococcal- and pneumococcal vaccines was carried out by proton n.m.r. spectroscopy and gas chromatography. The meningococcal polysaccharides were of high purity but showed differences in degree and position of O-acetylation between manufacturers. The level of contamination of the pneumococcal polysaccharides by C-substance was quantified. These methods provide an alternative to immunological methods for determining serotype and purity.

[The effect of the iron content in the culture medium on the nature of the protein spectrum in Neisseria meningitidis]. / Vliianie soderzhaniia zheleza v srede kul'tivirovaniia na kharakter belkovogo spektra Neisseria meningitidis.

The protein spectra of Neisseria meningitidis, strain A 208, in the process of its cultivation on solid culture medium based on peptone agar under the conditions of the surplus or deficiency of ions of trivalent iron in the medium. The creation of iron deficiency by the introduction of two iron-binding admixtures, desferol and diethylenetriaminepentaacetic acid (DTPA), was found to lead to the production of two additional proteins with molecular weights of 72 kD and 36-37 kD by meningococcal cells. Of these two admixtures, DTPA more readily stimulated the production of low-molecular protein with a molecular weight of 36-37 kD. This protein was found to be noticeably labile, while protein with a molecular weight of 72-73 kD showed no such lability. As the result of the cultivation of meningococci in iron-deficient medium, the content of protein in microbial residue was 2- to 3-fold greater than that obtained by the cultivation of meningococci in culture medium with the surplus of iron in the form of ferric nitrate.

Contraimunoeletroforese no diagnóstico etiológico das meningites bacterianas / Counterimmunoelectrophoresis on etiological diagnosis of bacterial meningitis

Contraimunoeletroforese (CIE) tem sido utilizada para o diagnóstico etiológico de meningites bacterianas, através da detecçäo de antígenos polisacarídicos capsulares no líquido cefalorraquiano. O material é composto por 340 exames de amostras de LCR de 292 pacientes, 171 do sexo masculino e 121 do sexo feminino com idade entre 4 dias e 74 anos. Sendo 91 pacientes com perturbaçöes neurológicas diversas e 146 pacientes com diversas formas de meningites sépticas nas quais o agente etiológico foi identificado pelo exame bacterioscópico direto e provas culturais e 55 casos de meningites sépticas em que o agente etiológico näo foi identificado; em 25 pacientes foram feitos exames em 2 amostras; em 10, em 3 amostras e em 1, em 4 amostras. A primeira amostra foi sempre colhida antes da antibioticoterapia e as seguintes após o início da terapêutica. Este material foi dividido em grupos, para se verificar a especificidade e a sensibilidade da contraimunoeletroforese para Haemophilus influenzae tipo B, Neisseria meningitidis A, B e C e Streptococcus pneumoniae (83 sorotipos). A especificidade da contraimunoeletroforese para o Haemophilus influenzae é de 98, 49 e a sensibilidade é de 94,8%. Para o Neisseria meningitidis a especificidade éde 98,58% e a sensibilidade é de 44,4% e, para o Streptococcus pneumoniae a especificidade éde 93,55% e sensibilidade é de 41,1%.

Dissociation between the interleukin 1-inducing capacity and Limulus reactivity of lipopolysaccharides from gram-negative bacteria.

In this study we compared the interleukin 1 (IL 1)-inducing capacity and the reactivity in the Limulus amoebocyte assay (LAL) of purified lipopolysaccharides (LPSs) from various bacterial strains. LPSs differed greatly in their capacities (on a weight basis) to induce IL 1 release from serum-free cultured human monocytes. LPS species that induced high levels of IL 1 release from human monocytes exhibited a high thiobarbiturate-reactive 2-keto-3-deoxy-octonic acid (KDO) content. No relationship was found between the IL 1-inducing activity and the LAL reactivity of purified LPSs. Filtration experiments in which membranes of decreasing size-exclusion limits were used demonstrated that molecular species of LPS with an apparent Mr below 3,000 may induce IL 1, whereas only species with an apparent Mr above 8,000 are recognized in the LAL assay. The latter observation suggests that the reaction with LAL requires an aggregated form of LPS. These results indicate that biologically active LPS species can cross dialysis membranes in vivo although no LAL reactive material is detected in the blood compartment. The Limulus assay is an insufficient criterion for the absence of LPS in biological fluids.

Two distinct outer membrane serotype subcomplexes of Neisseria meningitidis serogroup A.

A 350-kilodalton serotype outer membrane complex containing the class 1, 3, and 4 outer membrane proteins was isolated from serogroup A Neisseria meningitidis. Partial denaturation yielded two serotype subcomplexes containing the class 3 and 1 proteins (85 kilodaltons) and the class 3 and 4 proteins (94 kilodaltons), respectively.

Neisseria meningitidis group B capsular polysaccharide. Bacterial content and release in relation to categories of infection and filtrability of released endotoxin.

A total of 41 Neissera meningitidis isolates were analyzed for capsular polysaccharide (CP) and lipopolysaccharide (LPS) release and filtrability. Twenty-two of these isolates were serogroup B from blood or cerebrospinal fluid of patients, five were group B isolates from healthy throat carriers, and 14 were nongroupable (acapsular) meningococcal isolates from healthy carriers. Filtration of liquid whole-cell cultures through cellulose acetate-nitrate filters resulted in distinctly lower LPS filtrate activity for acapsular than for capsular meningococci (p less than 0.001). On the other hand, when polysulfone membrane filtration was performed, filtrates from acapsular and capsular meningococci contained LPS in similar amounts. These results indicate that LPS-containing particles released from acapsular isolates are larger or more aggregated than corresponding CP- and LPS-containing particles released from capsular isolates. The LPS released from acapsular isolates apparently are more efficiently retained by adsorption to cellulose acetate-nitrate. From the capsular isolates comparatively more CP than LPS appeared to be released, as related to cell-bound amounts. The total amounts of CP and released, filtrable LPS through cellulose acetate-nitrate filters were both relatively low and had similar values for capsular carrier meningococci and systemic isolates from mild meningococcal disease. The amount of CP released and passing this type of membrane was significantly higher for systemic isolates from severe septicemia than from mild meningococcal disease (p = 0.03).

Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis.

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the larger.

Variation in expression of class 1 and class 5 outer membrane proteins during nasopharyngeal carriage of Neisseria meningitidis.

Accidental nasopharyngeal colonization of a laboratory worker by a human disease isolate of Neisseria meningitidis allowed us to examine the variation in expression of outer membrane components and the host humoral response over time. There were quantitative differences in class 1 outer membrane protein expression in nasopharyngeal isolates obtained at different times. Isolates also showed phase variation of all four class 5 outer membrane proteins produced by the colonizing strain. After colonization, we detected new host serum immunoglobulin G antibodies directed against class 1, class 5, and H.8 outer membrane proteins.

Application of fourth derivative absorption spectroscopy to protein quantitation during purification.

A method for protein quantitation in the presence of nonprotein cellular components is described. The method is based on measurement of two tryptophan-specific signals in the fourth derivative of the protein's ultraviolet absorption spectrum, a peak at 283 nm and a trough at 288 nm. The amplitude between these two extremes is shown to vary linearly with protein concentration for bovine serum albumin and the outer membrane vesicles of Neissera meningitidis even when these protein solutions are supplemented with enough nucleic acid to completely obscure the parent absorption spectrum of the protein. The utility of this method as an in-process assay during isolation of a protein is demonstrated by comparing estimates of protein content from fourth derivative spectroscopy with those from the Lowry assay for samples at several steps along the isolation pathway for outer membrane vesicles of N. meningitidis. The advantages and limitations of the present method are discussed.

Endotoxin liberation studied by biological and chemical methods. Chemical characterization of six meningococcal lipopolysaccharides.

Release of endotoxin (or lipopolysaccharides, LPS) from four meningococcal strains was studied with a chemical and a biological technique. Two strains were endotoxin-liberating (E+; 270E+ and 840E+) and two had no or low endotoxin release E-; 270E- and 840E-). LPS was quantitated by gas chromatography (GC) of LPS-specific hydroxy fatty acid, in parallel with assay of endotoxin by Limulus Amebocyte Lysate (LAL), in cell suspensions of equal O.D. and in filtered samples. The GC and LAL methods showed a reasonably good agreement in the determination of LPS in filtrates, which had distinctly higher levels (approx. 10-100 times) for the E+ strains than the E- strains, in accordance with earlier LAL studies. This difference was not due to overproduction of LPS in the E+ strains, since all four strains had the same level of LPS (by GC) in cell suspensions of equal O.D. Here the agreement between the GC and LAL methods was substantially less, with lower values by LAL for the two E-strains. The chemical composition of purified LPS was determined by methanolysis and GC for the four strains and for two additional strains 247 and 714 with a high degree of genetic similarity with strains 270E- and 840E-, respectively. Amounts of unphosphorylated L-glycero-D-mannoheptose and 2-keto-3-deoxyoctonic acid were the same in all 6 LPS. Otherwise distinct differences were found between LPS of the 6 strains. LPS of the two E+ strains formed one group with about 2.4 mol of galactose (gal), 1.4 mol of glucose (glc) and 2.8 mol of glucosamine (glcN) in the carbohydrate chain. Another group, LPS of all the E- strains except 270E-, had 1.1 mol of gal, 2.8 mol of glc and 1.3 mol of glcN in the LPS chain. LPS 270E- also had 1.3 mol of glcN but deviated strongly form all other LPS by a complete lack of gal and glc. On the basis of genetic evidence strain 270E- is regarded as a "rough" LPS mutant of strain 247. The atypical chemistry of LPS 270E- may explain an observed hydrophobicity of this LPS, and it may be related to the previously described sulfonamide sensitivity. Whether the chemical difference observed for LPS of the E+ and E- strains is a mere coincidence remains to be elucidated by detailed studies of more strains of known tendency of endotoxin liberation.

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.

Purification, cyanogen bromide cleavage, and amino terminus sequencing of class 1 and class 3 outer membrane proteins of meningococci.

Meningococcal class 1 and 3 outer membrane proteins (OMPs) were subjected to cyanogen bromide treatment. The class 3 OMP was found to be resistant to cyanogen bromide, while the class 1 OMP was cut into two main fragments of 25 and 17 kilodaltons. The N-terminal sequences were determined for class 1 and class 3 proteins, which exhibit similarities to one another and to OMP I of gonococci. The C-terminal class 1 OMP fragment bound the bactericidal monoclonal antibodies tested.

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.

Neisseria meningitidis: heterogenicity in the outer membrane proteins released into the growth medium.

Eight strains of Neisseria meningitidis belonging to different serogroups were analysed for their virulence in mice and their release of outer membrane proteins into the medium during growth. All strains released proteins. No detectable lipopolysaccharide was observed. However, SDS-PAGE showed a heterogenicity in the protein number and profile among the different strains of N. meningitidis tested.

Restoration of antibody binding to blotted meningococcal outer membrane proteins using various detergents.

Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.