Outbreak of meningococcal disease caused by PorA-deficient meningococci.

An outbreak of 7 cases of group C meningococcal disease occurred during the last week of July and the first week of August 2001 in the southwestern part of The Netherlands. Characterization of the 7 patients' isolates by various typing methods showed that the isolates were identical, except for the expression of PorA. Isolates from 5 patients were PorA deficient. These results show that transmission of PorA-deficient meningococci occurs and that PorA-deficient meningococci can cause invasive disease. PorA-based meningococcal vaccines may provide limited protection.

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters.

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.

Multiple mechanisms of phase variation of PorA in Neisseria meningitidis.

Previously, we reported that PorA expression in Neisseria meningitidis is modulated by variation in the length of the homopolymeric tract of guanidine residues between the -35 and -10 regions of the promoter or by deletion of porA. To reveal additional mechanisms of variation in PorA expression, the meningococcal isolates from 41 patients and 19 carriers were studied. In addition, at least 3 meningococcal isolates from different body parts of each of 11 patients were analyzed. Sequence analysis of the porA promoter showed that the spacer between the -35 and -10 regions varies in length between 14 and 24 bp. PorA expression was observed in strains with a porA promoter spacer of 16 to 24 bp. All but one strain with a porA promoter spacer of 16 to 20 bp and undetectable PorA expression have a homopolymeric tract of 8 or 6 instead of 7 adenine residues in the porA coding region. The other PorA-negative strain had a single-base-pair deletion in the coding region. The highest level of PorA expression was observed in strains with a promoter spacer of 17 or 18 bp. PorA expression was reduced twofold in strains with a porA promoter spacer of 16 or 19 bp. Strains with a 16-bp promoter spacer with substitutions in the polyguanidine tract displayed increased levels of PorA expression compared to strains with a homopolymeric tract of guanidine residues in the porA promoter. In conclusion, meningococci display multiple mechanisms for varying PorA expression.

Deletion of porA by recombination between clusters of repetitive extragenic palindromic sequences in Neisseria meningitidis.

PorA is an important component in a vaccine against infection with Neisseria meningitidis. However, porA-negative meningococci were isolated from patients, thereby potentially limiting the role of PorA-mediated immunity. To analyze the mechanism by which the porA deletion occurred, the regions upstream and downstream of porA from three meningococcal strains (H44/76, H355, and 860183) were sequenced. The porA upstream region in strain 860183 contains a cluster of 22 repetitive palindromic RS3 core sequences (ATTCCC-N8-GGGAAT) and 10 RS3 core sequences (ATTCCC) in direct orientation. The cluster is flanked by neisserial repeats, so-called Correia elements, and can be subdivided into three repeats of 518 bp followed by a truncated repeat. The porA upstream region of the other two strains showed deletions, probably caused by a recombination between RS3 core sequences. The porA downstream region of H44/76 and H355 contains the IS1106 element followed by a cluster of 10 palindromic RS3 core sequences, 4 RS3 core sequences, and 1 other RS3 core sequence (GGGAAT) and is followed by a Correia element. This cluster can be subdivided into four direct repeats of 370 bp. Strain 860183 had two such repeats instead of four. Sequence analysis of the porA-negative variants indicated that the deletion of porA occurred via a recombination between two copies of the 116-bp region, containing two palindromic RS3 core sequences and a single RS3 core sequence. This region is homologous in the upstream and downstream clusters.

Comparison of commercial diagnostic tests for identification of serogroup antigens of Neisseria meningitidis.

In the study that is described the sensitivities and specificities of three commercial tests and the standard Reference Laboratory test, used since 1961, to identify Neisseria meningitidis serogroups were compared. The tests marketed by Difco, Murex/Wellcome, and Sanofi/Pasteur showed overall sensitivities of 92, 95, and 100%, respectively, and specificities of 67, 88, and 82%, respectively. When limited to the common serogroups A, B, and C, the three tests yielded sensitivities of 93, 97, and 100%, respectively, and specificities of 98, 100, and 98%, respectively. However, determination of the uncommon serogroups X, W-135, Y, Z, and 29E with these tests is either unreliable or not possible.

Variable expression of class 1 outer membrane protein in Neisseria meningitidis is caused by variation in the spacing between the -10 and -35 regions of the promoter.

The class 1 outer membrane protein encoded by the porA gene of Neisseria meningitidis is a candidate for a vaccine against meningococcal infection. The expression of class 1 outer membrane protein displays phase variation between three expression levels. Northern (RNA) blot and primer extension analysis revealed that this phase variation is regulated at the transcriptional level. The start site for transcription is located 59 bp upstream of the translational initiation codon. Sequence analysis of the promoter region of the porA gene of a variant without class 1 protein expression revealed nine contiguous guanidine residues between the -10 and -35 domains. Comparison of promoter sequences of different phase variants indicated that the length of the polyguanidine stretch correlated with the expression level of the class 1 outer membrane protein; the presence of 11, 10, or 9 contiguous guanidine residues results in high levels, medium levels, or no expression of class 1 mRNA, respectively. These results suggest that the variable porA expression levels seen in different isolates are modulated by guanidine residue insertion and/or deletion due to slipped-strand mispairing on the polyguanidine stretch within the intervening sequence of the -35 and -10 regions of the promoter. The phase variation of class 1 outer membrane protein may provide a molecular mechanism to evade the host immune defense. Therefore, the protective efficacy of a vaccine based on class 1 outer membrane protein may be questioned.

Bacterial entry and intracellular processing of Neisseria gonorrhoeae in epithelial cells: immunomorphological evidence for alterations in the major outer membrane protein P.IB.

The fate of the major outer membrane protein of the gonococcus, P.IB, during the adherence, entry, and intracellular processing of the bacteria in infected epithelial cells was investigated using post-embedding immunoelectron microscopy. Various domains of the P.IB molecule were probed at different stages in the infection. These studies revealed that P.IB epitope exposure remained unaltered during the initial attachment of the bacteria to the host cells. In contrast, upon secondary attachment of the bacteria to the eukaryotic cells, apparent zones of adhesion were formed between the gonococci and the host cell membrane, which were characterized by loss of a defined P.IB epitope. These zones of adhesion with the altered P.IB immunoreactivity continued to exist and increased in number during cellular penetration, suggesting that they were essential to bacterial invasion into the eukaryotic cells. After bacterial entry, two classes of gonococci could be recognized; morphologically intact, P.IB-positive bacteria and disintegrated organisms that showed a change in, and, in a later stage, a complete loss of P.IB immunoreactivity. The intracellular alterations in the P.IB antigen could be prevented by treatment of the host cells with the lysosomotropic agent chloroquine. These observations point to a mechanism by which a subpopulation of intracellular gonococci can escape the epithelial cell defense by preventing or resisting exposure to host cell proteolytic activity.

In situ expression and localization of Neisseria gonorrhoeae opacity proteins in infected epithelial cells: apparent role of Opa proteins in cellular invasion.

During natural infection, gonococcal opacity proteins (Opa) undergo rapid phase variation, but how this phenomenon contributes to the virulence of the bacteria is not well understood. In the present immunomorphological study we examined the actual Opa status of individual gonococci during various stages of gonococcal infection of Chang epithelial cells, by probing ultrathin sections of infected specimens with Opa-specific monoclonal antibodies. Our results demonstrate a heterogeneous Opa expression during the initial interaction of the bacteria, but an almost 100% expression of one of the probed Opas during their secondary attachment and entry into the host cells, suggesting a role for distinct Opas in cellular penetration. The association between Opa expression, tight attachment, and bacterial invasion into the host cells could be confirmed with isogenic variants that expressed different Opa proteins. Once inside the epithelial cells, both morphologically intact, Opa positive and morphologically disintegrated, Opa negative bacteria were observed. The loss of Opa immunoreactivity in intracellular gonococci could not be related to the presence of a particular Opa protein, but could be mimicked by incubating the organisms with extracts of sonicated uninfected epithelial cells, suggesting that it was caused by host cell proteolytic activity. Taken together, our data suggest that Opa phase transitions confer a functional adaptation of the bacteria enabling host cell penetration.

The use of immunogold-silver staining to study antigen variation and bacterial entry into eukaryotic cells by conventional light microscopy.

Immunogold-silver staining is a sensitive staining technique that enables the visualisation of the presence of individual antigens by conventional light microscopy. The application of this method to detect the antigenic heterogeneity of bacterial surface components and also the localisation of intracellular or extracellular bacteria is described. The latter application involved selective immuno-silver staining of the extracellular bacteria and counterstaining of the intracellular bacteria and the eukaryotic cells by crystal violet. The efficacy of the assay was confirmed by transmission electronmicroscopy of the silver-stained specimens. Immunogold-silver staining was shown to be useful for studying bacterial antigen variation and the uptake of bacteria by eukaryotic cells.

Stable expression of lipooligosaccharide antigens during attachment, internalization, and intracellular processing of Neisseria gonorrhoeae in infected epithelial cells.

Immunoelectron microscopy enables the detection and localization of bacterial antigens during in vitro infection (J.F.L. Weel and J.P.M. van Putten, Microb. Pathog. 4:213-222, 1988). In this study, we have used this method to get information on the role of lipooligosaccharides (LOS) in the pathogenesis of neisserial infections at the mucosal level. Ultrathin cryosections of Chang conjunctive epithelial cells infected with Neisseria gonorrhoeae (3 to 18 h) were incubated with LOS-specific monoclonal antibodies and gold-labeled protein A and viewed in the electron microscope. Our results demonstrate that the probed LOS determinants are stably expressed during the adherence, internalization, and intracellular processing of the bacteria. There was no indication of an adaptation of the gonococcal LOS expression to the host cell environment or of a degradation of the probed epitopes. The gold particles, representing LOS molecules, were predominantly located at the bacterial membranes, but sometimes the host cell plasma membrane was labeled as well, suggesting that LOS or LOS-containing membrane fragments interacted with the eucaryotic cells. This was confirmed when purified LOS was added to the cells. Two hours after LOS exposure, gold particles were observed at the plasma membrane of a subpopulation of the cells. After 18 h of LOS exposure, gold particles were also found in large vacuoles inside the cells, suggesting that LOS molecules were internalized by the cells. The function of observed LOS binding and endocytosis in the pathogenesis of neisserial infections remains to be defined.

Comparison of meningococcal outer membrane protein vaccines solubilized with detergent or C polysaccharide.

Outer membrane proteins (OMPs) were isolated from meningococcal strain H44/76 (B:15:P1.16) by detergent extraction of bacteria. A final product containing class 1 (P1.16), 3(15), 4 OMPs and 5% (w/w) lipooligosaccharide was obtained. Two experimental vaccines were prepared: OMP-detergent and OMP-C polysaccharide. The OMP-detergent vaccine tended to show a better bactericidal: ELISA ratio for the antibodies induced as compared to the OMP-C polysaccharide vaccine. The vaccine induced bactericidal antibodies appeared for the greater part to be directed against the class 1 OMP (P1.16). By comparison of cultures grown in Mueller Hinton Broth with and without 0.25% (w/v) glucose, it was found that monoclonal antibodies against the serotype OMP (class 2 or 3) were not bactericidal against meningococci grown in MHB without glucose. Antibodies against class 1 OMP and lipooligosaccharide were not influenced by this. A new major outer membrane protein (appr. 40 kd) is described that may function as a cation-specific porin.

Class 1/3 outer membrane protein vaccine against group B, type 15, subtype 16 meningococci.

Neisseria meningitidis capsular polysaccharides and outer membrane proteins have been incorporated in vaccines and the potential of these vaccines has been evaluated in man. Polysaccharides are the most attractive candidates for a vaccine against group A and C meningococci whereas outer membrane proteins may have a potential for a vaccine against group B meningococci. This paper describes the characteristics of the five classes of outer membrane proteins of group B meningococci and the protective (bactericidal) activity of monoclonal antibodies against class 1 and 2 or 3 outer membrane proteins. Monoclonal antibodies against class 1 outer membrane proteins were bactericidal irrespective of the growth conditions of the bacterium. On the other hand, these conditions influenced the bactericidal activity of monoclonal antibodies against class 2 or 3 outer membrane proteins. These data indicate that class 1 outer membrane protein is an attractive component of a vaccine. The Blake and Gotschlich procedure for the isolation of gonococcal outer membrane protein II (1) was adapted for the isolation of a combination of class 1 and 3 outer membrane proteins from group B, type 15 meningococci. The combination of both outer membrane proteins was adsorbed to ALPO4 in the presence of the detergent Zwittergent 3-14. The vaccine was injected into mice. The antibodies were strongly bactericidal and Western blot analysis indicated that both outer membrane proteins induced antibodies. The vaccine may have a potential to combat an epidemic caused by group B, type 15 meningococci. Such an epidemic was observed in some N.W. - European countries.

Colony variants of Neisseria meningitidis strain 2996 (B:2b:P1.2): influence of class-5 outer membrane proteins and lipopolysaccharides.

Different colonial morphologies were found among colonies of Neisseria meningitidis strain 2996 (B:2b:P1.2). Examination of cultures, selected on the basis of colony transparency or opacity, revealed that both lipopolysaccharides (LPS) and class-5 outer membrane proteins (OMP) are associated with differences in colonial morphology. Among 13 variants, four LPS variants and two class-5 OMP variants were recognised. All variants were non-fimbriate. The LPS variations were confirmed by immunoprecipitation. In addition to these qualitative variations of LPS, meningococci synthesise LPS of different molecular size depending upon growth phase; larger LPS molecules were found after analysis of stationary-phase cultures than with exponential-phase cultures. These changes did not cause a change in serotyping characteristics. The recognition in this study of intra-strain heterogeneity of meningococcal LPS and class-5 OMPs is important for the understanding of meningococcal pathogenicity. This heterogeneity was also detected in simultaneous isolates from different sites of a patient.

Immunogenicity of meningococcal antigens as detected in patient sera.

The immunogenicity of meningococcal surface antigens was tested in acute- and convalescent-phase sera from patients with meningococcal diseases by enzyme-linked immunosorbent assay and gel immunoradioassay. In gel immunoradioassay, the antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before testing their antibody-binding capacity. Both 125I-labeled protein A and 125I-labeled anti-human immunoglobulin G were used to detect antibody binding. It appeared that the variable, low-molecular-weight, heat-modifiable major outer membrane proteins (molecular weights, 25,000 to 32,000) induced strong, strain-specific immunoglobulin G antibody responses. In addition, pili induced strong, cross-reactive antibody responses that could be detected with 125I-labeled protein A, but not with 125I-labeled anti-immunoglobulin G. Antibody responses against capsular polysaccharides, lipopolysaccharides, and minor outer membrane proteins could also be detected by gel immunoradioassay. When tested by enzyme-linked immunosorbent assay against outer membrane complexes, patient sera demonstrated a large amount of cross-reactivity against heterologous meningococcal strains.

Immunochemical characterization of Neisseria meningitidis serotype antigens by immunodiffusion and SDS-polyacrylamide gel electrophoresis immunoperoxidase techniques and the distribution of serotypes among cases and carriers.

The chemical nature of the antigens of the meningococcal serotypes described by Frasch and colleagues was determined by a combination of immunodiffusion and the SDS-polyacrylamide gel electrophoresis immunoperoxidase technique (SGIP). It was confirmed that the serotype antigens of the outer membrane of serotypes 1, 2, 6, 9, 11 and 12 were proteins, whilst those of serotypes 4,5 and 8 were lipopolysaccharides. Serotype 2 can now be divided into three related types, provisionally called 2a (originally serotype 2), 2b and 2c with the specific antigens being proteins having molecular weights of 41,000, 41,500 and 41,500, respectively. A total of 195 strains of meningococci isolated from patients and carriers in the Netherlands and 20 serogroup Y strains from patients in the U.S.A. were serotyped by means of immunodiffusion. Serotype 2a could be demonstrated in some strains belonging to the serogroups B (only those from carriers), C, W-135 and Y (only those from the U.S.A.). The W-135 strains isolated from patients in this series more often belonged to serotype 2a than did the W-135 strains from carriers. Serotype 2b was present in about half of the serogroup B and a few serogroup C strains isolated from patients with meningitis, but absent in serogroup B and C strains from carriers. Serotype 2c could only be demonstrated in serogroup Y strains, both from the Netherlands and the U.S.A. The other serotypes were found only sporadically.