Development of enterobacterium-specific oligonucleotide probes based on the surface-exposed regions of outer membrane proteins

Outer membrane proteins of members of the family Enterobacteriaceae consist of conserved membrane-spanning segments and hypervariable, surface-exposed regions. We demonstrate that the hypervariable DNA segments corresponding to the surface-exposed regions of these proteins can be used to develop specific DNA probes for the identification of members of the family Enterobacteriaceae.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Proteoglycan receptor binding by Neisseria gonorrhoeae MS11 is determined by the HV-1 region of OpaA

The interaction of the OpaA protein of Neisseria gonorrhoeae MS11mk with heparan sulphate-containing proteoglycan receptors on Chang conjunctiva epithelial cells was examined using isolated receptor binding and cell adherence/internalization assays. OpaA deletion proteins, in which the four surface-exposed regions of the protein were deleted individually, and chimeric OpaA/B proteins, in which the surface-exposed regions of the OpaA and OpaB proteins were exchanged, were expressed in N. gonorrhoeae. The recombinant deletion proteins and the chimeric OpaA/B proteins were surface exposed in the outer membrane of N. gonorrhoeae. Isolated receptor-binding assays and Chang cell infection assays with OpaA deletion variants indicated that hypervariable region 1 was essential for the interaction of N. gonorrhoeae with the proteoglycan receptor. Expression of chimeric OpaA/B proteins confirmed the central role of hypervariable region 1 in receptor binding and demonstrated that this domain alone confers the invasive biological phenotype in a non-heparan sulphate proteoglycan-binding Opa protein. The other variable regions of OpaA enhanced receptor binding in the presence of region 1, but did not constitute binding domains on their own. The results indicate that proteoglycan receptor binding results from a hierarchical interaction between the variable domains of the OpaA protein of MS11mk.     

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.     

Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.     

Canonical structures for the hypervariable regions of T cell alphabeta receptors

T cell alphabeta receptors have binding sites for peptide-MHC complexes formed by six hypervariable regions. Analysis of the six atomic structures known for Valpha and for Vbeta domains shows that their first and second hypervariable regions have one of three or four different main-chain conformations (canonical structures). Six of these canonical structures have the same conformation in complexes with peptide-MHC complexes, the free receptor and/or in an isolated V domain. Thus, for at least the first and second hypervariable regions in the currently known structures, the conformation of the canonical structures is well defined in the free state and is conserved on formation of complexes with peptide-MHC.We identified the key residues that are mainly responsible for the conformation of each canonical structure. The first and second hypervariable regions of Valpha and Vbeta domains are encoded by the germline V segments. Humans have 37 functional Valpha segments and 47 Vbeta segments, and mice have 20 Vbeta segments. Inspection of the size of their hypervariable regions, and of sites that contain key residues, indicates that close to 70 % of Valpha segments and 90 % of Vbeta segments have hypervariable regions with a conformation of one of the known canonical structures. The alpha and beta V gene segments in both humans and mice have only a few combinations of different canonical structure in their first and second hypervariable regions. In human Vbeta domains, the number of different sequences with these canonical structure combinations is larger than in mice, whilst for Valpha domains it is probably smaller.     

Characterization of the repertoire of hypervariable regions in the Protein II (opa) gene family of Neisseria gonorrhoeae

P.II outer membrane proteins of Neisseria gonorrhoeae are encoded by a family of closely related genes. Although the genes are highly conserved, major differences in sequence among them occur in two short regions, designated hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of the hypervariable regions in the P.II genes of strains FA1090. The FA1090 chromosome contained at least eleven P.II loci, having six different versions each of HV1 and HV2 among them. Southern blotting with HV-specific oligonucleotides showed that each version was present in one to three copies, and that there were nine unique combinations of HV1 and HV2 in the P.II genes. Although each of the versions of HV1 or HV2 had a unique DNA sequence, there were some similarities among them, particularly when certain ones were compared. Restriction fragments containing only the HV regions were cloned into an expression vector to demonstrate that the epitopes recognized by a set of monoclonal antibodies specific for different FA1090 P.II proteins were completely encoded by either HV1 or HV2.     

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.     

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons with proteins of the OmpA family

The gene encoding a major outer membrane protein (MopB) of the methanotroph Methylococcus capsulatus (Bath) was cloned and sequenced. The cloned DNA contained an open reading frame of 1044 bp coding for a 348-amino-acid polypeptide with a 21-amino-acid leader peptide. Comparative sequence analysis of the predicted amino acid sequence revealed that the C-terminal part of MopB possessed sequences that are conserved in the OmpA family of proteins. The N-terminal half of the protein had no significant sequence similarity to other proteins in the databases, but the predicted secondary structure showed stretches of amphipathic beta-strands typical of transmembrane segments of outer membrane proteins. A region with four cysteines similar to the cysteine-encompassing region of the OprF of Pseudomonas aeruginosa was found toward the C-terminal part of MopB. Results from whole-cell labeling with the fluorescent thiol-reacting reagent 5-iodoacetamidofluorescein indicated a surface-exposed location for these cysteines. A probe consisting of the 3'-end of the mopB gene hybridized to the type I methanotroph Methylomonas methanica S in Southern blots containing DNA from nine methanotrophic strains representing six different genera.     

Homology of the Gene Coding for Outer Membrane Lipoprotein Within Various Gram-Negative Bacteria

The mRNA for a major outer membrane lipoprotein from Escherichia coli was found to hybridize specifically with one of the EcoRI and one of the HindIII restriction endonuclease-generated fragments of total DNA from nine bacteria in the family Enterobacteriaceae: E. coli, Shigella dysenteriae, Salmonella typhimurium, Citrobacter freundii, Klebsiella aerogenes, Enterobacter aerogenes, Edwardsiella tarda, Serratia marcescens, and Erwinia amylovora. However, among the Enterobacteriaceae, DNA from two species of Proteus (P. mirabilis and P. morganii) did not contain any restriction endonuclease fragments that hybridized with the E. coli lipoprotein mRNA. Furthermore, no hybrid bands were detected in four other gram-negative bacteria outside the family Enterobacteriaceae: Pseudomonas aeruginosa, Acinetobacter sp. HO1-N, Caulobacter crescentus, and Myxococcus xanthus. Envelope fractions from all bacteria in the family Enterobacteriaceae tested above cross-reacted with antiserum against the purified E. coli free-form lipoprotein in the Ouchterlony immunodiffusion test. Both species of Proteus, however, gave considerably weaker precipitation lines, in comparison with the intense lines produced by the other members of the family. All of the above four bacteria outside the family Enterobacteriaceae did not cross-react with anti-E. coli lipoprotein serum. From these results, the rate of evolutionary changes in the lipoprotein gene seems to be closely related to that observed for various soluble enzymes of the Enterobacteriaceae.     

Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.     

Phylogenetic analysis of the family Thermaceae with an emphasis on signature position and secondary structure of 16S rRNA

The sequences of the 16S rRNA genes from 38 strains of the family Thermaceae were compared by alignment analysis. The genus-specific and species-specific base substitutions or base deletions (signature positions) were found in three hypervariable regions (in the helices 6, 10 and 17). The differentiation of secondary structures of the high variable regions in the 5' end (38-497) containing several signature positions further supported the concept. Based on the comparisons of the secondary structures in the segments of 16S rRNAs, a key to the species of the family Thermaceae was proposed.     

Characterization of the opa (class 5) gene family of Neisseria meningitidis

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.     

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.