Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

Methods published for the purification of P.II proteins from Neisseria gonorrhoea have been modified to allow the purification of class 5 proteins from Neisseria meningitidis serogroup A bacteria. The five class 5 protein electrophoretic variants detected within an epidemic in the Gambia (a, b, c, d, and e) and three other variants (f, g, and h) found within other isolates of the same clone in West Africa have been purified with yields of 6-28 mg. The NH2-terminal amino acid sequence for variant c differs from those of the other class 5 proteins, whereas the latter are very similar to the sequence predicted for two class 5 proteins from DNA analyses of serogroup C meningococci and determined for 8 P.II proteins from gonococci. Numerous other regulatory, chemical, and serological differences were found between the c protein and the other class 5 proteins such that we recommend that the class 5 proteins be subdivided into two subclasses. mAbs have been isolated that distinguish between these two protein subclasses and Western blotting with these antibodies enabled us to conclude that both protein subclasses were found in bacteria isolated from different epidemics and pandemics of the last 50 yr.

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and monoclonal antibodies as tools for the subgrouping of Escherichia coli lipopolysaccharide O18 and O23 antigens.

The lipopolysaccharide (LPS) of Escherichia coli O18 isolated from a wide variety of sources was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four different LPS types, designated O18A, O18A1, O18B, and O18B1, were identified. Most O18 strains possess O18A, O18A1, or O18B LPS types, and these types are clonally associated. A reference test strain with the classical O18ab designation possessed O18B LPS, while two reference O18ac strains possessed O18A and O18A1 LPS, respectively. A panel of 15 anti-O18A B-cell hybridomas was isolated. Enzyme-linked immunosorbent assays revealed that some of the monoclonal antibodies produced by these cells recognize different epitopes. Four of these antibodies suffice to distinguish the four O18 types. Numerous strains whose LPS had been typed by SDS-PAGE were tested by agglutination with seven monoclonal antibodies whose specificities had been determined by enzyme-linked immunosorbent assays. The results indicated a perfect correlation between the two methods. Rabbit antisera raised against O18A bacteria agglutinated boiled bacteria of each of the O18 LPS types efficiently. The antisera were adsorbed with bacteria possessing each of the LPS types. The adsorbed sera only distinguished between two groups: O18A and O18A1 versus O18B and O18B1, as shown by agglutination assays and Western blotting. E. coli O4 and O23 and Serratia marcescens O8 antigens, which are reputed to cross-react with O18, were also analyzed. One O4, one O8, and four O23 strains were tested. All made an LPS which was distinguishable from O18 LPS types by SDS-PAGE. Each O23 strain synthesized a different LPS, and three of them synthesized only few short chains. Some of the monoclonal antibodies reacted with O4, O8, and O23A LPSs. The results are interpreted as indicating that numerous E. coli O serogroups will prove to be chemically heterogeneous and that future analyses of subgroup heterogeneity should be guided by results from SDS-PAGE and rely preferentially on monoclonal antibodies as opposed to rabbit hyperimmune sera.

The 5C protein of Neisseria meningitidis is highly immunogenic in humans and induces bactericidal antibodies.

The 5C protein is expressed by the strain of Neisseria meningitidis (44/76) used for production of the Norwegian meningococcal group B outer membrane vesicle vaccine and is included in the final formulation of this vaccine. The immunoglobulin G antibody response to 5C stimulated by vaccination, systemic meningococcal disease, and carriage was measured using ELISAs with synthetic liposomes as antigen and by immunoblotting. Increased levels of IgG were found in paired sera from all three groups. The antibodies were bactericidal to meningococci of serogroups A and B that expressed large amounts of 5C but not to meningococci expressing smaller amounts. There was a linear correlation between bactericidal titer and units of IgG to 5C.

Antibodies against IgA1 protease are stimulated both by clinical disease and asymptomatic carriage of serogroup A Neisseria meningitidis.

IgA1 protease was purified from a strain of serogroup A Neisseria meningitidis subgroup IV-1, representative of bacteria that caused an epidemic of meningococcal meningitis in The Gambia in 1982-1983. ELISAs and immunoblot assays were done using this protease as antigen with paired acute- and convalescent-phase sera from patients from that epidemic and from one in Finland caused by other serogroup A meningococci. Paired sera were also tested from healthy Gambians who were persistent nasopharyngeal carriers, persistent noncarriers, or persons who became carriers after the first serum sample was taken. The results correlated well between the two methods: Antibodies were stimulated by disease or acquisition of carriage, and they remained at a constant level upon continued carriage.

Two-dimensional structure of the Opc invasin from Neisseria meningitidis.

A two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surface-exposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (nabla) from Semliki Forest virus were inserted into BglII restriction sites created by site-directed mutagenesis. The nabla epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while nabla epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

Lipopolysaccharide, capsule, and fimbriae as virulence factors among O1, O7, O16, O18, or O75 and K1, K5, or K100 Escherichia coli.

K1, K5, and K100 Escherichia coli isolates of the lipopolysaccharide antigen types O1, O7, O16, O18, or O75, which had formerly been assigned to clonal groupings were compared with K? E. coli isolates and with laboratory-derived mutants defective in capsule or lipopolysaccharide synthesis. The amount of K1 capsule, the length distribution of the lipopolysaccharide, and the expression of type I and P fimbriae were determined. The clonal groupings were uniform with regard to these properties within each group but different from each other. Many of the K? strains differed from the clonal representatives. The results are interpreted with regard to the different diseases caused by each of these bacterial groups.

Sequence diversity, predicted two-dimensional protein structure, and epitope mapping of neisserial Opa proteins.

The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane beta-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.

Induction of bacteremia in newborn rats by Escherichia coli K1 is correlated with only certain O (lipopolysaccharide) antigen types.

A total of 95 Escherichia coli strains (O1:K1, O7:K1, or O18:K1), obtained from different sources of human infections and from healthy individuals, were analyzed for the ability to cause bacteremia after colonizing the gut of newborn rats. Strains of all three serotypes were able to multiply extensively in the gut after oral inoculation and to translocate (in small numbers) to the mesenteric lymph nodes. With only few exceptions, O7:K1 and O18:K1 strains were able to cause bacteremia, while O1:K1 strains could not. Mixed-infection experiments revealed that the bacteria present in the blood during a case of bacteremia are in most cases the descendants of one cell that has multiplied extraintestinally after translocation to the mesenteric lymph nodes. It appears that virulent O7:K1 and O18:K1, but not avirulent O1:K1, bacteria are able to multiply directly in the bloodstream of the newborn rats. No correlation between virulence and the source of isolation of the different strains was observed. Disease isolates thus do not seem to differ from fecal isolates of the same serotype in special virulence properties. The differences in virulence among different O serotypes of K1 E. coli observed in the rat model were comparable to their relative frequency of isolation from meningitis in newborn children.

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.

Variation in class 5 protein expression by serogroup A meningococci during a meningitis epidemic.

Serogroup A meningococci were isolated from patients and healthy carriers in The Gambia between 1982 and 1988. The class 5 proteins expressed by these bacteria were identified by electrophoretic migration and by serologic tests. Three protein serologic groupings (seroclasses) called A (protein 5a), B (proteins 5b, 5d, or 5e), and C (protein 5c or 5C) were found among 331 bacterial isolates. The number of class 5 proteins expressed per isolate varied from none to four, with a median of two. The class 5 protein composition differed for certain paired isolates obtained from the nasopharynx, blood, and cerebrospinal fluid of diseased patients and for certain pairs of sequential isolates from the nasopharynx of healthy carriers; the medical relevance of this variation remains unclear, although the 5C protein was preferentially isolated from the nasopharynx and the 5a protein from diseased patients. The data show that a large proportion of healthy carriers in The Gambia were exposed to bacteria expressing each of the three seroclasses and that many people were exposed to bacteria expressing each of the three seroclasses and that many people were exposed to two or all three seroclasses during the epidemic of 1982-1983.

Six widespread bacterial clones among Escherichia coli K1 isolates.

Variable properties among Escherichia coli isolates include serotype, electrophoretic migration of major outer membrane proteins, metabolic properties, production of hemolysin or colicin or both, and plasmid content. These characteristics were compared in E. coli strains of capsular types K1, K5, K92, and K100 and in non-encapsulated isolates. The 234 bacterial strains from the United States and Europe which we studied had been isolated from healthy or diseased individuals recently or as long ago as 1941. Regardless of source, most O7:K1, O16:K1, and O75:K100 isolates could be assigned to three unique, serotype-specific groups, which were interpreted as representing three bacterial clones. Two bacterial (sub)clones each were discerned among the O18:K1 and O18:K5 isolates, and two further, distinct clones were discerned among the O1:K1 isolates. The implications of these results for epidemiological analyses and for virulence are discussed.

Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae.

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.

A reexamination of the O1 lipopolysaccharide antigen group of Escherichia coli.

A total of 64 Escherichia coli strains of the O1 serogroup were tested for the migration pattern of their lipopolysaccharides (LPS) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. O1:K1 and O1:K51 strains of the OMP5 outer membrane protein pattern possessed LPS with a doublet pattern (O1A1) or the lowermost band of the O1A1 doublet (O1A2). O1:K1 strains of the OMP9 pattern possessed LPS referred to as O1A, which corresponded to the uppermost band of the O1A1 doublet pattern. A few O1:K? strains possessed LPS of different migration patterns (O1B and O1C). O1A and O1A1 LPS were indistinguishable by chemical techniques, and both reacted with each of 10 different monoclonal antibodies tested. However, O1A1 had an additional epitope within the additional band in each doublet, as demonstrated by adsorption experiments with hyperimmune rabbit sera followed by Western blotting. Furthermore, purified polysaccharide from O1A bacteria was incapable of inhibition in enzyme-linked immunosorbent assays performed with O1A1 LPS as antigen and adsorbed, specific anti-O1A1 antibodies, whereas O1A1 polysaccharide inhibited this reaction. O1B and O1C LPS differed in all respects tested, including chemical composition, from O1A and O1A1 LPS.

Clonal and variable properties of Neisseria meningitidis isolated from cases and carriers during and after an epidemic in The Gambia, West Africa.

A representative collection of meningococci was isolated from cases and healthy carriers in The Gambia between 1982 and 1988, during and after an epidemic of meningococcal meningitis. These bacteria were subjected to a clonal analysis. All serogroup A bacteria from both cases and carriers were of one clone (A IV-1). Several unrelated clones were observed among serogroup 29E and serogroup Y carrier strains. The serogroup A strains were uniform for serotype and subtype antigens (serotype 4, subtype P1.7) and antibiotic sensitivity pattern. Occasional strains varied in their lipopolysaccharide (LPS), DNA fingerprint pattern, and/or the quantitative expression of the class 1 protein. A high degree of strain-specific variation was found for the expression of class 5 proteins, pili, and sulfonamide sensitivity. The frequency of strains expressing reduced amounts of the class 1 protein, altered LPS, and/or increased amounts of capsular polysaccharide rose among case strains obtained after the epidemic had ceased. These strains seem to be generally resistant to antibody-mediated bactericidal activity.

Clonal analysis of Escherichia coli O2:K1 isolated from diseased humans and animals.

Forty-six Escherichia coli isolates of serotype O2:K1 from human urinary tract infections, chicken sepsis, and bovine mastitis were obtained from laboratories in England, Denmark, Sweden, and Finland. The bacteria were compared for outer membrane protein (OMP) pattern, lipopolysaccharide pattern, electrophoretic mobilities of enzymes, and flagellar serotype and were tested for fimbriation, biotype, hydroxamate production, hemolysin production, antibiotic resistance, plasmid content, colicin production, and virulence in neonatal rats. Isolates from humans were assigned to two clonal groups; poultry isolates belonged to one of these clonal groups, whereas bovine isolates belonged to the other. Poultry and human isolates of the same clonal group could be distinguished only by their plasmid content. Strains within this group were heterogeneous with respect to biotype, fimbriation, virulence, and flagellar serotype. Human and bovine isolates of the second clonal group were distinguished by a minor change in OMP pattern and by their plasmid content. It is concluded that meaningful clonal groupings are best recognized by the combination of OMP and electrophoretic enzyme patterns. The O:K serotype can aid in the recognition of important subclones, whereas the other microbiological properties tested can vary widely within clonal groupings. Furthermore, we conclude that certain O:K serotypes can contain very different clonal groupings having little genetic relatedness.

A comparison of the variable antigens expressed by clone IV-1 and subgroup III of Neisseria meningitidis serogroup A.

Serogroup A Neisseria meningitidis of subgroup III has caused two pandemics of meningococcal meningitis since 1966 and recently spread to East Africa. The last epidemics in West Africa in the early 1980s were caused by clone IV-1. Surface antigens of clone IV-1 strains from West Africa and subgroup III strains from both pandemic waves were analyzed. Lipopolysaccharide was stable within clone IV-1 but variable in subgroup III. Pili from clone IV-1 possessed class I epitopes, while those from subgroup III also possessed class IIa epitopes. Certain class 5 protein variants were expressed by both bacterial clones, possibly reflecting either inheritance of primeval genes or horizontal transmission. Exposure of Gambians to clone IV-1 bacteria stimulated production of bactericidal antibodies cross-reactive with subgroup III bacteria in some individuals but of type-specific antibodies in others. Gambians without bactericidal antibodies usually became healthy carriers rather than developing meningococcal disease on exposure to virulent meningococci.

Expression, refolding and crystallization of the OpcA invasin from Neisseria meningitidis.

OpcA is an integral outer membrane from the Gram-negative pathogen Neisseria meningitidis that plays a role in adhesion of meningococci to host cells. The protein was overexpressed in Escherichia coli in an insoluble form and a procedure developed for refolding by rapid dilution from denaturant into detergent solution. The refolded material was identical to native OpcA isolated from meningococci, as judged by overall molecular weight, migration on SDS-PAGE and reaction against monoclonal antibodies. Both native and recombinant OpcA crystallized under similar conditions to give an orthorhombic crystal form (P2(1)2(1)2), with unit-cell parameters a = 96.9, b = 46.3, c = 74.0 A. Complete data sets of reflections were collected from native and refolded OpcA to 2.0 A resolution.

Molecular epidemiology of serogroup a meningitis in Moscow, 1969 to 1997.

Molecular analysis of 103 serogroup A Neisseria meningitidis strains isolated in Moscow from 1969 to 1997 showed that four independent clonal groupings were responsible for successive waves of meningococcal disease. An epidemic from 1969 to the mid-1970s was caused by genocloud 2 of subgroup III, possibly imported from China. Subsequent endemic disease through the early 1990s was caused by subgroup X and then by subgroup VI, which has also caused endemic disease elsewhere in eastern Europe. A 1996 epidemic was part of the pandemic spread from Asia of genocloud 8 of subgroup III. Recent genocloud 8 epidemic disease in Moscow may represent an early warning for spread of these bacteria to other countries in Europe.