Immunologic characteristics of the protein antigens of the Neisseriaceae family. I. Antigenic interrelationships between nonpathogenic representatives of the genus Neisseria]

The immunological characteristics of protein complexes isolated from the filtrates of broth cultures, obtained after prolonged incubation, of 86 strains of N. perflava, N. flava, N. subflava, N. Flaviscens, N. sica and N. mucosa are presented. All these strains, irrespective of their species, had 11--15 common antigens and differed by 1--4 components. The presence of common antigens in all representatives of the family Neisseriaceae has been proved to be important for the study of immunological reactivity to this group of microorganisms.     

Phylogenetic analysis of Alysiella and related genera of Neisseriaceae: proposal of Alysiella crassa comb. nov., Conchiformibium steedae gen. nov., comb. nov., Conchiformibium kuhniae sp. nov. and Bergeriella denitrificans gen. nov., comb. nov

A phylogenetic analysis based on 16S rRNA gene sequences reveals that Alysiella filiformis belongs to the family Neisseriaceae. The genus Simonsiella is phylogenetically separated by the genera Kingella and Neisseria. The species Simonsiella crassa and A. filiformis show a close phylogenetic relationship, with the 16S rDNA sequence similarity and the DNA-DNA hybridization representing 98.7% and 35%, respectively. Therefore, S. crassa should be transferred from the genus Simonsiella to the genus Alysiella as Alysiella crassa comb. nov. Simonsiella steedae and Simonsiella sp. of cat origin show strong genetic affinities and are distantly related with the type species of Simonsiella, S. mulleri. Thus, a new genus, Conchiformibium is proposed; Conchiformibium steedae comb. nov. and Conchiformibium kuhniae sp. nov. are accommodated in this new genus. On the basis of the phylogenetic, phenotypic and chemotaxonomic distinction from the genus Neisseria, N. denitrificans should be reclassified, for which a new genus and new combination Bergeriella denitrificans are proposed.     

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria

Abstract The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp , in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea . The fbp genes from these strains were amplified by the polymerase chain reaction, digested with Stu I or Rsa I, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis , did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electropheris and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp -specific DNA probe.     

Comparison of the cell envelope proteins of Micrococcus cryophilus with those of Neisseria dnd Branhamella species.

In an attempt to elucidate the relation between Micrococcus cryophilus, Neisseria caviae, Neisseria ovis, and Branhamella catarrhalis, fractions derived from outer membranes of a strain of each organism were examined for protein composition by SDS - polyacrylamide gel electrophoresis. Micrococcus cryophilus outer membrane protein showed extensive similarities to that of N. ovis and contained a heat-modifiable protein which behaved almost identically with the corresponding bands previously shown to exist in N. caviae and N. ovis. Branhamella catarrhalis protein was distinctly different from those of M. cryophilus and the two 'false neisserias' N. caviae and N. ovis.     

Structural heterogeneity of the lipopolysaccharides of the Neisseriaceae

Lipopolysaccharides from 5 different genera of the Neisseriaceae were analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and visualized by silver staining. Significant heterogeneity in the banding patterns was observed with some of the strains producing only low molecular mass molecules and others producing O-repeating units. All genera examined except Branhamella contained strains that were able to produce an O-repeating side chain on their lipopolysaccharides. The ability to produce the repeating subunit did not correlate with the presence of plasmids.     

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.     

A note on hydrolysis of tributyrin by Branhamella and Neisseria

Sixty-three strains of Branhamella and Neisseria were tested by two methods for their ability to hydrolyse glycerol tributyrate. After the conventional plate test, gas liquid chromatographical (GLC) analysis of the agar medium was carried out to detect the hydrolysis product, butyric acid, and other volatile fatty acids. All strains of Branhamella catarrhalis, Neisseria caviae, N. cuniculi and N. ovis but no other Neisseria spp. gave positive results with the conventional test. With GLC, however, most strains of Branhamella and Neisseria were shown to liberate butyric acid. In addition, some strains liberated acetic and isovaleric acids. Greater amounts of butyric acid were produced by clinical strains, in particular B. catarrhalis , compared with reference strains. It was concluded that the conventional plate test for tributyrin hydrolysis differentiates B. catarrhalis, N. caviae, N. cuniculi and N. ovis from other Neisseria.     

Identification and characterization of a cross-reactive and a unique B-cell epitope on the hsp60 homologue from Neisseria gonorrhoeae

Two monoclonal antibodies (G6 and 7B), generated against a 63-kDa stress protein (GSP63) from Neisseria gonorrhoeae strain VP1, were used to investigate the antigenic heterogeneity of GSP63 among the Neisseriaceae and its antigenic relationship with the Hsp60 heat-shock protein family. Immunoblotting experiments demonstrated antibody reactivity with all pathogenic Neisseria tested and with some of the commensal strains. One of the antibodies (7B) cross-reacted with the 65-kDa M. bovis BCG heat-shock protein and with 14 out of the 21 similarly sized proteins in other bacterial species. The other antibody (G6) specifically recognized neisserial GSP63 homologues. These results demonstrate that GSP63 is a conserved neisserial antigen bearing both a unique neisserial B-cell epitope and a more widely distributed Hsp60 epitope.     

A note on hydrolysis of tributyrin by Branhamella and Neisseria

Sixty-three strains of Branhamella and Neisseria were tested by two methods for their ability to hydrolyse glycerol tributyrate. After the conventional plate test, gas liquid chromatographical (GLC) analysis of the agar medium was carried out to detect the hydrolysis product, butyric acid, and other volatile fatty acids. All strains of Branhamella catarrhalis, Neisseria caviae, N. cuniculi and N. ovis but no other Neisseria spp. gave positive results with the conventional test. With GLC, however, most strains of Branhamella and Neisseria were shown to liberate butyric acid. In addition, some strains liberated acetic and isovaleric acids. Greater amounts of butyric acid were produced by clinical strains, in particular B. catarrhalis, compared with reference strains. It was concluded that the conventional plate test for tributyrin hydrolysis differentiates B. catarrhalis, N. caviae, N. cuniculi and N. ovis from other Neisseria.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures.     

Identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal Neisseria species

Abstract The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis . In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N, flava, N. mucosa and N. sicca . Using opa -specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae . DNA sequence analysis of one opa -related gene derived from N. flava and N. sicca , respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae .     

Genetic relationships among Neisseria species assessed by comparative enzyme electrophoresis

The electrophoretic mobilities of 12 enzymes from 19 Neisseria species (including 6 strains of N. perflava), Gemella haemolysans, Escherichia coli and Branhamella catarrhalis were characterized by polyacrylamide slab gel electrophoresis. All strains and species tested exhibited qualitatively different zymogram patterns. Species and strain relationships were quantified by pairwise comparisons of all 12 enzyme systems to obtain similarity indices; these data were subjected to numerical clustering methods to obtain groups and a phenogram. The electrophoretic classification compared favourably with those obtained by other criteria. In addition, the quantitative clustering data indicated that N. ovis and N. caviae are sufficiently different from the other Neisseria species to warrant their separation into a distinct group. These two species also lacked the characteristic NADPH-diaphorase zymogram pattern found in all the other Neisseria species. Intra-species similarity indices were generally greater than the inter-species index values. However, certain species such as N. meningitidis and N. gonorrhoeae had similarity index values in the range of inter-strain index values.     

A study by high-resolution two-dimensional polyacrylamide gel electrophoresis of relationships between Neisseria gonorrhoeae and other bacteria

High-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.     

Enzyme electrophoretograms in the analysis of taxon relatedness of Micrococcus cryophilus, Branhamella catarrhalis and atypical Neisserias.

Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.     

Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle

A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.     

The production and characterization of monoclonal antibodies against the protein III of Neisseria gonorrhoeae

Monoclonal antibodies were produced against gonococcal protein III. Antibodies of two different specificities were obtained. One reacted with all Neisseria species tested (N. gonorrhoeae, N. meningitidis and five non-pathogenic species), whereas the other was specific for Neisseria gonorrhoeae and may provide the basis for improved diagnostic reagents.     

Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin.

Hemoglobin complexed to the plasma protein haptoglobin can be used by Neisseria meningitidis as a source of iron to support growth in vitro. An N meningitidis mutant, DNM2E4, was generated by insertion of the mini-Tn3erm transposon into the gene coding for an 85-kDa iron-regulated outer membrane protein. Membrane proteins prepared from DNM2E4 were identical to those of the wild-type strain except that the 85-kDa protein was not produced. This mutant was unable to use hemoglobin-haptoglobin complexes as an iron source to support growth and was also impaired in the utilization of free hemoglobin. The mutant failed to bind free hemoglobin, hemoglobin-haptoglobin complexes, or apo-haptoglobin in a solid-phase dot blot assay. The 85-kDa protein was affinity purified when hemoglobin-haptoglobin complexes were used as a ligand but was not purified when free hemoglobin was used. We hypothesize that the 85-kDa iron-regulated protein is the hemoglobin-haptoglobin receptor and designate this protein Hpu (for hemoglobin-haptoglobin utilization).     

Microflora of the posterior pharyngeal wall, larynx and postoperative wounds in patients after laryngectomy]

The study group were persons with risk factors of colonization by pathogenic strains and included smokers, patients suffering from paradontosis, and patients with visibly neglected oral cavity and teeth. We isolated and classified to the species or genera 488 microorganisms. Of posterior pharyngeal wall flora, 61% of were Gram-negative bacteria, represented predominantly by Haemophilus and anaerobic rods and aerobic cocci belonging to the Neisseria and Moraxella (Branhamella) genera. In the group of Gram-positive cocci (34% of the total number of microorganisms), oral streptococci, Stomatococcus mucilaginosus and Streptococcus pneumoniae were isolated most frequently. The affected by neoplastic lesions larynx, was colonized by similar bacterial groups. However, the incidence of Gram-positive cocci was higher. The main etiologic factor of purulent post-operative wound inflammations were methicillin-resistant Staphylococcus aureus strains (MRSA), which had been absent among the bacteria isolated from patients on admission.     

The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis.

Multilocus sequence typing (MLST) is a recently developed nucleotide sequence-based method for the definitive assignment of isolates within bacterial populations to specific clones. MLST uses the same principles as multilocus enzyme electrophoresis and provides data that can be used to investigate aspects of the population genetics and evolution of bacterial species. We used an MLST data set consisting of the sequences of approximately 450-bp fragments from seven housekeeping loci from a large strain collection of Neisseria meningitidis to estimate the relative impact of recombination compared with point mutation in the diversification of N. meningitidis clonal complexes. 126 meningococcal isolates were assigned to 10 clonal complexes, 9 of which contained minor clonal variants. The allelic variation within each complex was classified as a recombinational exchange or a putative point mutation through a comparison of the sequences of each variant allele with that of the allele typically found in the clonal complex. The nine clonal complexes contained a total of 23 allelic variants, and analysis of the sequences of these variant alleles revealed that a single nucleotide site in a meningococcal housekeeping gene is at least 80-fold more likely to change as a result of recombination than as a result of mutation. This value is estimated to be 10-50-fold for Escherichia coli and approximately 50-fold for Streptococcus pneumoniae.     

Immunological relationships of some non-pathogenic Neisseria.

A collection of nine stock strains of non-pathogenic Neisseria species and one strain of Branhamella catarrhalis has been examined serologically by a simple immunodiffusion technique utilising cultures growing on solid medium.