Protein secretion and secreted proteins in pathogenic Neisseriaceae.

Secreted proteins of pathogenic bacteria are often essential virulence factors. They are involved, for example, in the adherence of the bacteria to host cells or required to suppress the host's defence mechanisms. Until recently, only IgA1 protease had been studied in detail in the NeisseriaceaeNeisseria meningitidis and Neisseria gonorrhoeae. The availability of their genome sequences, however, has boosted research in this area. Here, we present a survey of the secretome of the pathogenic Neisseriaceae, based on the available genome sequences, and the current knowledge of the functions and structures of the secreted proteins. Of the six protein-secretion pathways that are widely disseminated among Gram-negative bacteria, three pathways appear to be present among the Neisseriaceae, i.e. the autotransporter-, the two-partner- and the type I-secretion mechanisms. Comparison of the predicted secretomes reveals a considerable flexibility. As compared with N. meningitidis and the nonpathogen N. lactamica, N. gonorrhoeae appears to have a considerably degenerated secretome, which may reflect its altered niche occupancy. The flexibility of the secretome may be enhanced by the presence of ORFs in the genomes potentially encoding fragments of secreted proteins. We hypothesize that these ORFs may substitute for the corresponding fragments in the full-length genes through genetic recombination, thereby changing the host-cell receptor specificity of the secreted protein.

Distribution of the meningococcal insertion sequence IS1301 in clonal lineages of Neisseria meningitidis.

The distribution of the meningococcal insertion sequence IS1301 was analysed in 496 strains of different serogroups and clonal lineages of Neisseria meningitidis, and in 64 neisserial strains other than N. meningitidis. IS1301 was found in meningococci, but not in apathogenic Neisseria sp. and Neisseria gonorrhoeae. The copy numbers of IS1301 varied between 2 and 17 per genome. IS1301 positive strains were mostly found among the serogroups 29E, W135, X, and Y. Clonal lineages of serogroup A, B, and C meningococci associated with epidemic meningococcal disease were rarely positive for IS1301.

An epitope shared by enterobacterial and neisserial porin proteins.

A murine monoclonal antibody (MAb F9-16) raised against a porin protein epitope called Po I of an E. coli 055 strain showed broad cross-reactivity with bacteria within the Enterobacteriaceae, and also recognized neisseriae and moraxellae. In an immunodot assay, the antibody was bound by 32/33 strains of neisseriae and moraxellae after SDS treatment of the bacteria. Testing intact bacteria, 11/33 isolates showed definite MAb binding, including serogroup A and B meningococci. In Western blotting, the anti-Po I MAb targeted the gonococcal porin proteins PIA and PIB, and class 1, class 2, and class 3 porins of meningococci. The MAb showed no reactivity against decapeptides which corresponded to the whole length of a meningococcal class 1 porin protein of the subtype P1, 7, 16. These findings accord with the inference that enterobacterial, neisserial and moraxellae porin proteins share an epitope (Po I) which is determined by the three-dimensional rather than by the primary structure of the proteins and that this epitope is shielded in most isolates but surface-exposed in some isolates, including some strains of meningococci. Since Po I is broadly distributed among commensal and pathogenic bacteria and has demonstrated immunogenicity in humans, this epitope may play a role in elicitation of "normal" antibodies with immunoprotective activity.

Branhamella catarrhalis pneumonia in patients with immunoglobulin abnormalities.

The importance of Branhamella catarrhalis pneumonia has only recently been appreciated. Predisposing underlying illness associated with this organism have not yet been clarified. We report five cases of Branhamella catarrhalis pneumonia occurring in patients with diseases associated with documented quantitative immunoglobulin deficiencies. Normal immunoglobulins appear to be important host defense mechanisms in preventing infection with this organism.

Lack of immunoglobulin A1 protease production by Branhamella catarrhalis.

Clinical isolates of Branhamella catarrhalis from the sputum of 20 patients with acute bronchopulmonary infection were examined for synthesis of immunoglobulin A1 protease by immunoelectrophoresis. Ten strains produced beta-lactamase, and 10 were beta-lactamase negative. None of the strains demonstrated immunoglobulin A1 protease activity despite the fact that three different culture media were used.

Serological identification of Branhamella catarrhalis. Serological evidence for infection.

A protein antigen--P-antigen--characteristic of Branhamella catarrhalis has been described. Precipitating antibodies against this antigen occur in a majority of healthy human sera; serological evidence for a pathogenic role of B. catarrhalis is accumulating. The occurrence of complement-fixing antibodies has been demonstrated during bronchopulmonary infection with B. catarrhalis and in maxillary sinusitis. Increases in antibody titres assessed by an enzyme-linked immunosorbent assay (ELISA) technique have been reported in children with acute otitis media. An ELISA, using purified P-antigen, for the evaluation of IgG, IgM and IgA antibodies against B. catarrhalis is being developed.

Diagnostic value of interactions between members of the family Neisseriaceae and lectins.

The lectin slide agglutination test for Neisseria gonorrhoeae has been modified and improved. Results show that wheat germ agglutinin and soybean lectin agglutinate 100% (193 of 193 tested) of clinical isolates of N. gonorrhoeae. Lectin-reactive meningococci can be readily identified by the hydrolysis of gamma-glutamyl-beta-naphthylamide. Branhamella catarrhalis, Neisseria lactamica, Neisseria sicca, Neisseria subflava, Neisseria perflava, and meningococcal serogroups A, B, C, X, Y, and Z do not interfere with the positive identification of N. gonorrhoeae. The frequently encountered problem of autoagglutination of members of the family Neisseriaceae may be circumvented by a short treatment of cellular suspensions with DNase. Based on agglutination assays, the enzyme treatment did not result in a loss of wheat germ agglutinin receptors from the bacteria. The lectin agglutination test, coupled with the gamma-glutamyl aminopeptidase assay, is proposed as a rapid and accurate means of identifying clinical isolates of gonococci.

The IgG subclass responses of human lymphocytes to B-cell activators.

The induction of IgG synthesis by normal human lymphocytes in response to a range of B-cell activators has been studied by indirect cytoplasmic immunofluorescence using monoclonal antibodies to the four IgG subclasses. Considerable variation was found between cells from different donors tested with the same activator but the following trends were observed:- Epstein-Barr virus and Branhamella catarrhalis induced a predominantly IgG3 response, whereas the most prevalent cells in cultures stimulated with lipopolysaccharide and Staph. aureus were IgG2-positive; the subclass distribution obtained with pokeweed mitogen resembled that found in normal serum (IgG1 greater than 2 greater than 3 greater than 4).

Common lipopolysaccharide specificity: new type of antigen residing in the inner core region of S- and R-form lipopolysaccharides from different families of gram-negative bacteria.

A new antigenic specificity, referred to here as common lipopolysaccharide (LPS) specificity, is described in the LPSs of gram-negative bacteria belonging to various families. The specificity is present in S- and R-form LPS but absent in Re mutants of different enterobacterial genera. By the use of purified LPS and monospecific antibodies obtained by immunoabsorption, the specificity is differentiated from the known core specificities of the genus Salmonella and the lipid A specificity by aid of the passive hemolysis and passive hemolysis inhibition test. In Salmonella minnesota R-form LPS, the specificity may be cryptic (R345, Rb2 mutant) or partly exposed in the intact molecule (R7, Rd1 mutant). The specificity is either demasked or completely exposed after mild acid hydrolysis for a short time, whereas it is destroyed after prolonged hydrolysis. Periodate oxidation, reduction, and hydrolysis under conditions that do not affect the ketosidic linkages of 2-keto-3-deoxyoctulosonic acid destroy the specificity in R4 (Rd2 mutant) LPS, but do not do so in R7 LPS. It is suggested that 2-keto-3-deoxyoctulosonic acid and a following neutral sugar are the compositional requirements for expressing the specificity.

Branhamella catarrhalis as a cause of bacteremic pneumonia.

A new enzyme immunoassay was used to confirm the pathogenetic role of Branhamella catarrhalis in a diabetic young man with pneumonia affecting the left lower lobe with pleural involvement. His temperature rise was only minimal, but his ESR and blood leucocyte count were elevated and one blood culture grew B. catarrhalis. The levels of branhamella antibodies of the IgG and IgA classes were high and rose during the infection.

Formalin-treated bacteria as selective B cell mitogens in the study of lymphocytes from patients with hypogammaglobulinaemia.

Staphylococcus aureus strain Cowan I, Haemophilus influenzae, and Branhamella catarrhalis that stimulate human B lymphocytes, but not T lymphocytes, were used to study the B lymphocyte function in patients with hypogammaglobulinaemia. Lymphocytes from two patients with no circulating B cells were not stimulated to increased DNA synthesis by these bacterial mitogens. The lymphocytes of six patients with hypogammaglobulinaemia and normal numbers of circulating B cells responded to the bacterial mitogens but in five patients the response was decreased in comparison with healthy controls.

A protein antigen characteristic of Branhamella catarrhalis. Serological identification of the genus.

Precipitation patterns of sonicated, acid-extracted and other extracts from Branhamella catarrhalis were examined by double diffusion-in-gel technique, using antiserum to B. catarrhalis. Acid extract gave rise to 4 distinct precipitates. One of these lines was further studied. The bacterial component responsible for this line was trypsin-sensitive, indicating that it was a protein. It was anodally localized by crossed immunoelectrophoresis. By absorption of antiserum with whole bacteria, the precipitating capacity of the serum was diminished, suggesting that the protein antigen (P-antigen) was exposed on the bacterial surface. F(ab')22-fragments of IgG from antiserum, but not from normal rabbit serum, precipitated the P-antigen, indicating that it was a true antigen-antibody reaction. It was possible to make an IgG preparation monospecific for the P-antigen, by absorbing antiserum with trypsinized bacterial extract. 31 strains of B. catarrhalis, 9 strains of N. gonorrhoeae, 10 strains of N. meningitis, 12 other Neisseria spp. and 2 strains of H. influenzae were investigated for presence of cros-reacting surface antigens, using IgG monospecific for the P-antigen and 125I-labelled protein A from Staphylococcus aureus. After antibody exposure, all 31 strains of B. catarrhalis showed abundant uptake of protein A. No significant uptake was detected on any other investigated strain. Hence, the P-antigen appears to be characteristic of B. catarrhalis. The possibility of a serological identification of the species is introduced. Precipitating antibodies against the P-antigen were demonstrated in 69% of normal human sera.

Lectins in diagnostic microbiology: use of wheat germ agglutinin for laboratory identification of Neisseria gonorrhoeae.

A lectin slide agglutination test has been developed for the confirmatory identification of Neisseria gonorrhoeae. With wheat germ lectin as an agglutinin, 164 of 165 clinical isolates of N. gonorrhoeae gave a 3 to 4+ reaction within 6 to 8 min. Four gonococcal isolates, even though negative by the fluoresecent-antibody method, gave strong positive reactions with the wheat germ lectin. Among 23 isolates of Neisseria meningitidis tested, which included representatives of sero-groups A, B, C,D, X, Y, and Z, only one strain in group X gave a false-positive reaction. The nonpathogenic species of Neisseria, as well as Branhamella catarrhalis, all showed negative reactions with the wheat germ agglutinin. The novel method provides a simple, rapid, and inexpensive means for the laboratory diagnosis of gonorrhea and obviates the need for performing second-stage sugar fermentation studies or utilizing the more expensive fluorescent-antibody techniques.

[Immunological characteristics of the protein antigens of the family Neisseriaceae. II. The importance of an immunochemical analysis of the protein complexes for a study of taxonomy problems]. / Immunologicheskaia kharakteristika belkovykh antigenov semeistva Neisseriaceae. Soobshchenie II. Znachenie immunokhimicheskogo analiza belkovykh kompleksov dlia izucheniia voprosov taksonomii.

The study of antigenic interrelations in the family Neisseriaceae resulted in the isolation of 2 main immunologically separated variants of protein complexes: the first variant was characteristic of nonpathogenic and pathogenic species of the genus Neisseria as well as of 7 taxonomically undefined Neisseria species (N. lactamicus, N. cuniculi, N. ellongata, N. ovis, N. animalis, N. cinerea, N. canis) and Gemella haemolysans; the second variant was represented by the genera Branhamella and Acinetobacter. N. caviae and 3 out of 14 Neisseria strains of undefined species had no common antigens with the genera Neisseria and Branhamella. The importance of the immunotyping of protein complexes for studying the problems connected with the taxonomy of the family Neisseriaceae was considered.

IgA protease production as a characteristic distinguishing pathogenic from harmless neisseriaceae.

IgA proteases are extracellular enzymes of bacteria that have human immunoglobulin A of the IgA1 subclass as their only known substrate. The identification of this enzyme in neisseria prompted us to determine whether IgA protease production correlates with pathogenicity within this genus. Multiple clinical isolates of Neisseria gonorrhoeae, N. meningitidis and eight species of non-pathogenic neisseria that commonly colonize the normal human nasopharynx were examined for IgA protease activity. All N. gonorrhoeae and N. meningitidis strains were enzyme positive; all non-pathogenic strains were negative. Among meningococci, the enzyme occurred in strains carried harmlessly in the nasopharynx as well as those isolated from systemic infections. Because mucosal immune defense is largely mediated by antibodies of the IgA isotype, the finding that IgA protease activity is linked specifically to the pathogenic neisseria suggests that the enzyme may be involved in the pathogenesis of neisserial infection.

"Core" glycolipid of enterobacteriaceae: immunofluorescent detection of antigen and antibody.

The Re chemotype mutant of Salmonella minnesota R595 has a cell-wall glycolipid composed principally of 2-keto, 3 deoxyoctonate and Lipid A, which is an antigen widely shared by Enterobacteriaceae. High-titered antiserum against this antigen can be conjugated with fluorescein isothiocyanate for direct detection of this antigen in heterologous bacteria and staining of bacteria in tissue. Alternatively, the indirect immunofluorescence technique can be used for antigen detection on bacterial surfaces and in tissues, and this method can quantitate glycolipid antibody in mammalian sera. The latter may be particularly useful in serologic studies because, although the glycolipid antigen is a surface antigen and purified extracts can be used to coat latex particles, high-titered antisera will not agglutinate bacteria or coated latex particles.