Commensal Neisseria Research Articles

Analysis in Neisseria meningitidis and other Neisseria species of genes homologous to the FKBP immunophilin family

The immunophilin family of FK506-binding proteins (FKBPs), involved in eukaryotic protein-folding and cell regulation, have recently been found to have prokaryotic homologues. Genes with sequences homologous to those encoding human FKBPs were examined in Neisseria species. An FKBP DNA sequence was present, as shown by the polymerase chain reaction and Southern blotting experiments, in the chromosome of Neisseria meningitidis (14 strains) and in all 11 different commensal Neisseria spp. studied, but was not found in Neisseria gonorrhoeae (11 strains tested) or in Moraxella catarrhalis. The nucleotide and predicted protein sequences of the FKBP-encoding domain from five of the meningococcal strains were highly conserved (e.g. > or = 97% homologous). The meningococcal nucleotide sequence was > or = 93% homologous and the consensus meningococcal protein sequence was > or = 97% homologous to FKBP sequences found in seven different commensal Neisseria spp. The meningococcal nucleotide and predicted protein sequences were > or = 59% homologous to the conserved C-terminus of the human FKBP gene family. The FKBP nucleotide sequence was present as a single copy in the chromosome of commensal Neisseria spp. and in most strains of N. meningitidis. The FKBP gene was linked to the silent pilin locus, pilS, in class II-piliated meningococcal strains. In meningococcal strains expressing class I pili, the FKBP gene was linked to one of several pilS loci but not the pilE locus present in these strains. FKBP genes found in commensal Neisseria spp. were not linked to known pilin loci.

Localization of the meningococcal receptors for human transferrin

The interaction between gold-labelled human transferrin (Au-HTF) with live meningococci after growth in vivo or in different in vitro conditions was examined by electron microscopy to localize and quantify the numbers of HTF-binding sites on the cell surface. It was clearly demonstrated that HTF binds to the surface of live meningococci (of different serogroups and serotypes) after growth in either iron-sufficient or iron-restricted cultures, although the degree of labelling was always higher (2- to 35-fold) in the latter case. The commensal Neisseria polysaccharea behaved similarly. Ultrathin sections showed that Au-HTF was localized predominantly on the outer membrane of the cells and vesicles, with hardly any internalization. Au-HTF labelling on meningococci was significantly reduced after incubation with unlabelled HTF or with rabbit antiserum containing antibodies against transferrin-binding proteins (TBPs), demonstrating the specificity of the interaction. These sera also blocked binding between HTF and outer membrane proteins on Western immunoblots. Direct evidence of the expression of the TBPs (Western blots) and localization of the HTF receptor (electron microscopy) on in vivo-grown meningococci was obtained from organisms derived without laboratory culturing from the cerebrospinal fluid of a patient. There was considerable cell-to-cell variation in the amount of labelling present on cells of the same sample (in vitro- or in vivo-grown organisms) and between different strains. The degree of binding varied with time of incubation of the cells with Au-HTF. The gold particles frequently formed discrete circles on the cell surfaces of the in vitro-grown organisms; these circles appear to be associated with outer membrane vesicle formation. The results show that the TBPs, which form part of the active components of the HTF receptor(s), are expressed in vivo and are surface exposed and immunogenic and that antibodies against them can interfere with the HTF binding of the meningococcal cells, which may affect iron utilization. This study further supports the concept of regarding the TBPs as future vaccine candidates.

Neisseria spp. and AIDS.

Neisseria meningitidis from various serogroups and two commensal neisseriae (N. sicca and N. perflava) were isolated from 15 patients at various stages of human immunodeficiency virus infection in this clinical and bacteriological study. The cases were grouped into the following three classes: (i) infections with an N. meningitidis strain of a serogroup known to be pathogenic (A, B, or C) and apparently independent of the human immunodeficiency virus infection, (ii) infections with a N. meningitidis strain of a serogroup which is normally either commensal or poorly pathogenic (serogroups Y, X, Z, and Z,29E), (iii) pulmonary and disseminated infections occurring in the course of the clinical evolutionary stage of AIDS, in two cases of which commensal neisseriae (N. sicca and N. perflava) were isolated from blood cultures.

Expression levels of human transferrin receptors in Neisseria species

All pathogenic Neisseria strains synthesize transferrin receptors. To determine if transferrin receptor expression is quantitatively different in Neisseria meningitidis strains from two separate origins (carrier and invasive) and in commensal Neisseria, we applied an enzyme-linked receptor assay (ELRA) and a radiolabelled receptor assay (RLRA) to evaluate the receptor expression levels after iron-limited growth. There were significant differences in receptor expression in commensal Neisseria and Neisseria meningitidis as determined by both methods and between the two origins (invasive and carrier) when the ELRA method was used, but in the latter case, the biological significance is questionable and the amount of transferrin receptor expressed may not be a useful indicator of the potential pathogenicity of strains. The RLRA method was more suitable for measuring transferrin receptor expression since it provided a lower background in strains lacking transferrin receptor and, consequently, made interpretation of the results easier.

Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species

The two pathogenic species of Neisseria, N. meningitidis and N. gonorrhoeae, have evolved resistance to penicillin by alterations in chromosomal genes encoding the high molecular weight penicillin-binding proteins, or PBPs. The PBP 2 gene (penA) has been sequenced from over 20 Neisseria isolates, including susceptible and resistant strains of the two pathogenic species, and five human commensal species. The genes from penicillin-susceptible strains of N. meningitidis and N. gonorrhoeae are very uniform, whereas those from penicillin-resistant strains consist of a mosaic of regions resembling those in susceptible strains of the same species, interspersed with regions resembling those in one, or in some cases, two of the commensal species. The mosaic structure is interpreted as having arisen from the horizontal transfer, by genetic transformation, of blocks of DNA, usually of a few hundred base pairs. The commensal species identified as donors in these interspecies recombinational events (N. flavescens and N. cinerea) are intrinsically more resistant to penicillin than typical isolates of the pathogenic species. Transformation has apparently provided N. meningitidis and N. gonorrhoeae with a mechanism by which they can obtain increased resistance to penicillin by replacing their penA genes (or the relevant parts of them) with the penA genes of related species that fortuitously produce forms of PBP 2 that are less susceptible to inhibition by the antibiotic. The ends of the diverged blocks of DNA in the penA genes of different penicillin-resistant strains are located at the same position more often than would be the case if they represent independent crossovers at random points along the gene. Some of these common crossover points may represent common ancestry, but reasons are given for thinking that some may represent independent events occurring at recombinational hotspots.

Epidemiology and molecular basis of penicillin-resistant Neisseria meningitidis in Spain: a 5-year history (1985-1989).

Penicillin-resistant (penr) clinical isolates of Neisseria meningitidis, which do not produce beta-lactamase, were first identified in Spain in 1985; the frequency of their recovery, which has been increasing in the past few years, reached 20% in 1989. Serogrouping, determination of serotypes and subtypes, and multilocus enzyme electrophoresis of the penr strains showed an extensive diversity. Resistance is due, at least in part, to a decreased affinity of penicillin-binding protein (PBP) 2 for penicillin. Similar low-affinity forms of PBP 2 are also found in penr isolates of Neisseria lactamica, Neisseria polysaccharea, and Neisseria gonorrhoeae. Genetic transformation of an N. meningitidis type strain to low-level penicillin resistance with DNA from resistant meningococci and other Neisseria species resulted in transformants that possessed low-affinity forms of PBP 2. These altered forms of PBP 2 have been shown to arise from recombinational events that replace parts of the PBP 2 gene with the corresponding regions from the PBP 2 genes of commensal Neisseria species.

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria

Analysis of the molecular mass heterogeneity of the transferrin receptor inNeisseria meningitidisand commensalNeisseria

Peroxidase-conjugated transferrin was used to detect transferrin receptors both in intact outer membrane vesicles (OMVs) from Neisseria species in a dot blot assay, and in SDS-PAGE-separated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction although expression was higher in the latter case, whereas only six N. lactamica and two N. sicca (among 20 commensal species) were able to bind transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa (87.5% of strains), 12.5% had receptors with MM close to 70 kDa, and 5% showed receptors with MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with the 'universal' 98 kDa receptor proposed by some authors, and show a low expression of the receptor in commensal Neisseria.

Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, α-galactosyl-1,4-β-galactose (Galα1, 4Galβ), also found on human cells.

Neisseria lactamica and Neisseria polysaccharea as possible sources of meningococcal beta-lactam resistance by genetic transformation

We studied the susceptibilities of relatively penicillin G-resistant and -susceptible strains of Neisseria meningitidis, as well as Neisseria lactamica and Neisseria polysaccharea, to penicillin, ampicillin, and several cephalosporins. The MICs of penicillin, ampicillin, cephalothin, and cefuroxime for moderately resistant meningococci have increased two- to sixfold in relation to MICs for susceptible strains. For these strains of meningococci, N. lactamica, and N. polysaccharea, penicillin, ampicillin, cephalothin, and cefuroxime MICs for 50 and 90% of strains were similar. By genetic transformation of a penicillin-susceptible strain of N. meningitidis to low-level penicillin resistance with DNA from penicillin-resistant strains of N. meningitidis, N. lactamica, N. polysaccharea, and N. gonorrhoeae, isogenic strains with the same pattern of resistance to beta-lactams were obtained, suggesting that these commensal Neisseria spp. could be the source of meningococcal resistance genes.

The occurrence of Branhamella catarrhalis and other commensal Neisseriaceae in clinical sputum specimens in Lagos, Nigeria

Branhamella catarrhalis and other commensal Neisseria species were isolated from 200 out of 500 sputum samples from patients with lower respiratory tract (LRT) infections at the Lagos University Teaching Hospital (LUTH). B. catarrhalis was isolated from 60 (12%). The isolation rates for other Neisseria species were as follows: N. mucosa from 45 (9%), N. sicca from 40 (8%), N. lactamica from 35 (7%), N. cinerea from 12 (2.4%) and N. subflava from 8 (1.6%). B. catarrhalis occurred in pure cultures in 15 (25%) of the 60 samples positive for this organism. Twenty (33%) out of the 60 N. catarrhalis isolates were beta-lactamase positive.

Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin

Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was nonfimbrial in nature, with aggregates of the adhesin extended out from the cells in a patchy fashion.

Distribution of gonococcal lipopolysaccharide biosynthesis genes among strains of Neisseria gonorrhoeae and other neisserial species

A plasmid, pTME6, containing Neisseria gonorrhoeae lipopolysaccharide biosynthesis genes was used as a probe to analyze DNA from strains of N. gonorrhoeae, N. meningitidis and various commensal Neisseria by Southern blotting. Chromosomal DNA from 26 gonococcal strains probed with 32P-labeled pTME6 produced five different hybridization patterns. No correlation between hybridization pattern and auxotype, serotype, serum sensitivity or SDS-urea-PAGE migration of LPS was observed. DNA from strains of N. meningitidis, N. lactamica and N. cinerea, but not other commensal Neisseria species, hybridized strongly to pTME6.

Characterization of the Tet M determinants in urogenital and respiratory bacteria

Tetracycline-resistant Fusobacterium nucleatum, Haemophilus ducreyi, Mycoplasma hominis, Peptostreptococcus spp., Ureaplasma urealyticum, and Veillonella parvula had DNA sequences which showed homology throughout the length of the Tet M transposon, Tn916. In contrast, Gardnerella vaginalis, commensal Neisseria spp., and the 25.2-megadalton plasmid family lacked the complete transposon.

The significance of Branhamella catarrhalis in bronchopulmonary infection — A case-control study

Data, including clinical features of bronchopulmonary infections, i.e. cough with purulent sputum, fever, peripheral blood leucocytosis, patchy pulmonary shadows on chest radiographs and the presence of crepitations or dullness on examination of the chest, were prospectively recorded in 107 consecutive adult patients whose expectorated sputum yielded Branhamella-like bacterial colonies in routine cultures. Subsequently, isolates from 26 patients were confirmed to be Branhamella catarrhalis. Of 81 patients (unmatched controls) with confirmed commensal Neisseria species, 40 (matched controls) were age and sex matched with the 26 patients (cases) positive for B. catarrhalis. Clinical features of bronchopulmonary infection were significantly more among cases compared to unmatched controls. Compared with matched controls, significantly more of the cases had three or more clinical features of bronchopulmonary infection (P less than 0.05). This is the first study of the clinical significance of B. catarrhalis in expectorated sputum that included controls to circumvent the confounding effects of oropharyngeal and airway colonisation in patients with bronchopulmonary infections. The isolation of this organism in routine sputum culture was found to be significantly related to clinical infection.

Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis.

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria meningitidis produce altered forms of penicillin-binding protein 2 that have decreased affinity for penicillin. The sequence of the penicillin-binding protein 2 gene (penA) from a penicillin-resistant strain of N. meningitidis was compared to the sequence of the same gene from penicillin-sensitive strains and from penicillin-sensitive and penicillin-resistant strains of Neisseria gonorrhoeae. The penA genes from penicillin-sensitive strains of N. gonorrhoeae and N. meningitidis were 98% identical. The gene from the penicillin-resistant strain of N. meningitidis consisted of regions that were almost identical to the corresponding regions in the penicillin-sensitive strains (less than 0.2% divergence) and two regions that were very different from them (approximately 22% divergence). The two blocks of altered sequence have arisen by the replacement of meningococcal sequences with the corresponding regions from the penA gene of Neisseria flavescens and result in an altered form of penicillin-binding protein 2 that contains 44 amino acid substitutions and 1 amino acid insertion compared to penicillin-binding protein 2 of penicillin-sensitive strains of N. meningitidis. A similar introduction of part of the penA gene of N. flavescens, or a very similar commensal Neisseria species, appears to have occurred independently during the development of altered penA genes in non-beta-lactamase-producing penicillin-resistant strains of N. gonorrhoeae.

Detection of antibodies to common antigens of pathogenic and commensal Neisseria species

Sera from 29 children and six adults were used to investigate the nature of antigenic cross-reactivity between Neisseria polysaccharea, N. lactamica and N. meningitidis B,15P1.16 by immunoblotting. Major common antigens of 68-70 Kda, 60-65 Kda and 15-20 Kda were detected. Antibody directed against them uniformly decreased after absorption of the sera with the three different Neisseria species. Antigens of 55 Kda and 35 Kda specific to N. meningitidis, and one of 43 Kda specific to N. lactamica, were also demonstrated. Antibody against all antigens was more prevalent in bactericidal than in non-bactericidal sera, although these differences were statistically not significant. Differences in antibody prevalence between carriers of Neisseria spp. and non-carriers of these organisms were even less marked. Examination of sera by whole-cell enzyme-linked immunosorbent assay against N. meningitidis B,15P1.16 and N. lactamica gave an absorbance ratio of 1:1. Only four sera from children showed no reactivity against the meningococcal strain. These common antigens are likely to be important in vaccine development.

The phenotypic relationship of Neisseria polysaccharea to commensal and pathogenic Neisseria spp.

Eight of 22 non-capsulate strains of Neisseria meningitidis previously isolated from primary school children were re-identified as N. polysaccharea by aminopeptidase reactions and polysaccharide production. N. polysaccharea was not identified amongst 91 non-capsulate strains of N. meningitidis isolated from adults attending the Genito-urinary Medicine clinic, Westminster Hospital, London. The biochemical reactions of N. polysaccharea strains were similar to those of N. lactamica and N. gonorrhoeae, but N. polysaccharea could be distinguished from these organisms by examination of beta-galactosidase activity, carbohydrate reactions and polysaccharide production. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed closer similarity of N. polysaccharea to N. lactamica than to the pathogenic Neisseria spp. An additional finding was variation in the position of one of the major proteins of N. lactamica in the 34-39-Kda region.

Characterization of the neisserial lipid‐modified azurin bearing the H.8 epitope

The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.8 MAb-binding protein with a consensus lipoprotein processing site, an N-terminal domain containing the epitope for H.8 MAb binding, and a C-terminal domain with extensive similarity to the sequences of azurins from other organisms. In the current study, we showed that the product of the cloned gene could be labelled with palmitic acid, that it was subject to globomycin-sensitive processing, and that it was immunologically cross-reactive with azurin from Pseudomonas aeruginosa. All neisserial species tested, both pathogens and commensals, produced a protein recognized by anti-azurin serum. Southern blots with oligonucleotide probes specific for the azurin domain of the gene showed that it was present in a single copy in the chromosome; it was highly conserved in gonococci and meningococci, and less conserved in commensal Neisseria species.

Historical perspectives and identification of Neisseria and related species.

The pathogenic Neisseria spp., N. gonorrhoeae and N. meningitidis, have been studied extensively and rapid identification procedures have been designed to distinguish these species from the commensal Neisseria and related species that are normal flora of the oro- and nasopharynx. The commensal Neisseria spp. have been largely ignored except for isolated studies. It is important that we know about these species, however, because not only may some be misidentified as pathogenic species if identified with inappropriate procedures, but also they may occasionally be isolated from unusual sites and must be correctly identified to the species level for clinical purposes.