HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation

Neisseria meningitidis uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the hmbR expression pattern in strains in which the hpuB gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some hpuB mutants is suggestive of phase variation of hmbR. The length of the poly(G) tract starting at position +1164 of hmbR absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring the hmbR reading frame. Inactivation of hmbR in these strains resulted in a loss of Hb utilization, demonstrating that the change in the hmbR gene was responsible for the phenotypic switch. The switching rates from hmbR phase off to phase on were approximately 5 x 10(-4) in four serogroup C strains, 2 x 10(-2) in the serogroup A isolate, and 7 x 10(-6) in the serogroup B isolate.     

Association of iron-regulated outer membrane proteins of Neisseria meningitidis with the RmpM (class 4) protein

The RmpM (class 4) protein of Neisseria meningitidis has previously been shown to be associated with the outer membrane porins. In the present study, we demonstrate that this protein forms complexes with the lactoferrin receptor LbpA, the transferrin receptor TbpA and the siderophore receptor FrpB as well. This complexation apparently resulted in a stabilization of oligomeric forms of these iron-regulated proteins. In vitro experiments further revealed a reduced ability to acquire iron from human lactoferrin in the rmpM mutant. Furthermore, all TonB-dependent receptors investigated here appeared to exist as oligomers (probably dimers), suggesting that this is a general feature of this class of proteins.     

Structural comparison of Neisseria gonorrhoeae outer membrane proteins.

Outer membranes from opaque colonia variants of Neisseria gonorrhoeae P9 contain a major outer membrane protein (protein I) together with one or more of a series of heat-modifiable proteins (proteins II). Proteins I. II, and IIa have been isolated by detergent extraction of outer membranes. Amino acid analysis showed proteins II and IIa to have a very similar composition. Cyanogen bromide cleavage of proteins II and IIa produced a pair of fragments with identical molecular weight and a pari which differed by an amount (0.5K) equivalent to the difference between the intact proteins. Tryptic peptide maps of 125I-labeled proteins II, IIa, and IIb showed many similarities, with only a few peptides unique to any one protein. Peptide maps of protein IIa from cells which had been surface labeled showed that the unique peptides were exposed on the surface. The heat-modifiable proteins thus appear to form a family of proteins with closely related structure probably differing in that part which is exposed on the bacterial surface.     

Cross-linking analysis of Neisseria gonorrhoeae outer membrane proteins.

The arrangement of proteins in the outer membrane of Neisseria gonorrhoeae was investigated through the use of cleavable chemical cross-linking reagents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linking of isolated outer membranes yielded dimers and trimers of the major outer membrane protein. In addition, data were obtained suggesting that a stable interaction exists between the major protein I and protein II, the second most prevalent protein in the gonococcal outer membrane.     

Cloning, sequencing, and characterization of the gene encoding FrpB, a major iron-regulated, outer membrane protein of Neisseria gonorrhoeae.

FrpB (for Fe-regulated protein B) is a 76-kDa outer membrane protein that is part of the iron regulon of Neisseria gonorrhoeae and Neisseria meningitidis. The frpB gene from gonococcal strain FA19 was cloned and sequenced. FrpB was homologous to several TonB-dependent outer membrane receptors of Escherichia coli as well as HemR of Yersinia enterocolitica and CopB of Moraxella catarrhalis. An omga insertion into the frpB coding sequence caused a 60% reduction in 55Fe uptake from heme, but careful analysis suggested that this effect was nonspecific. While FrpB was related to the family of TonB-dependent proteins, a function in iron uptake could not be documented.     

Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida B:2 in mice model

The present study was conducted to investigate the role of iron-regulated outer membrane proteins (IROMP) of Pasteurella multocida B:2 in mice as potential immunogens. Outer membrane proteins extracted from P. multocida B:2 grown under normal (OMP) and iron-deficient (IROMP) conditions were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 85.1 to 16.7 kDa from OMP preparations and two additional polypeptides of MW 95.4 and 89.1 kDa from IROMP preparations were observed with bands of MW 37.2 and 34.7 kDa as major proteins. Mice were immunized twice with OMP, IROMP-enriched fractions and whole cell lysate (WCL) via subcutaneous route at day 0 and 21. Antibody titers were determined from sera collected at weekly interval and protection was studied against challenge using 10(2) cfu of P. multocida two weeks after secondary immunization via intranasal and subcutaneous routes. IROMP and OMP immunized mice provoked significant antibody responses and IROMP induced higher antibody responses. IROMP and OMP immunized mice showed protection (100%) upon intranasal challenge and a protection (84%) following subcutaneous challenge as compared to high mortality (84%) in control mice. These results indicate that OMP enriched with IROMP fractions can be superior means of immunization.     

Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane.

Neisseria meningitidis group B serotype 2 strain M986 contains two predominant outer membrane proteins, with apparent molecular weights of 41,000 (protein b) and 28,000 (protein e). Heating of outer membrane vesicles at 56 degrees C for 20 min caused much of b** to disaggregate and denature into b (41,000 daltons). In contrast, protein e could be rapidly solubilized by SDS at room temperature into its monomeric state (e*), but it was not converted to its final higher apparent molecular weight of 28,000 (e) unless heated at 100 degrees C for 2 min. We propose that protein b exists in the membrane as trimers or tetramers in a transmembrane configuration and that protein e exists as subunits on the exterior surface of the outer membrane and has a highly ordered tertiary structure.     

Identification of iron-regulated outer membrane proteins in uropathogenic Proteus mirabilis and its relationship with heme uptake

The effect of iron deprivation on the expression of outer membrane proteins and the ability to use heme as an iron source by uropathogenic Proteus mirabilis , Pr 6515, was studied. Examination of iron-restricted bacteria showed three outer membrane proteins ranging from 66 to 75 kDa to be affected by iron restriction, as well as a newly expressed 64-kDa protein. These proteins were induced within 15 minutes of iron-deprivation. The strain grew in the presence of ferric citrate, hemin and hemoglobin as iron sources, but could not use transferrin, lactoferrin or siderophores from exogenous sources. The 64- and 66-kDa proteins showed hemin-binding activity by affinity chromatography, and both reacted in Western blots with sera from mice transurethrally infected with the same strain. We suggest that P. mirabilis expresses iron-regulated outer membrane proteins that could be involved in heme uptake and may have a role in pathogenesis.     

Acid stress upregulated outer membrane proteins in clinical isolates of Neisseria gonorrhoeae, but not most commensal Neisseria.

Human immune serum recognition of outer membrane components from commensal and pathogenic Neisseria cultured under neutral and acidic conditions was investigated. Acid stress caused no detectable alterations in lipooligosaccharide migration and (or) staining, in outer membrane protein profiles, or in immune serum recognition of outer membrane components from Neisseria mucosa or Neisseria sicca. There was also no difference in the lipoologosaccharide electrophoretic pattern of acid- and neutral-grown Neisseria lactamica, but there were differences in outer membrane protein expression. The outer membrane protein alterations induced by acid stress in N. lactamica were not the same as those seen in isolates from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection. Many differences were detected in the immune serum recognition of outer membrane components from acid- and neutral-cultured N. lactamica and from the clinical isolates of Neisseria gonorrhoeae, and these should be considered in vaccine design.     

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).     

Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp

As an approach to understanding the molecular basis of the reduction in plant yield depression by root-colonizing Pseudomonas spp. and especially of the role of the bacterial cell surfaces in this process, we characterized 30 plant-root-colonizing Pseudomonas spp. with respect to siderophore production, antagonistic activity, plasmid content, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of their cell envelope proteins. The results showed that all strains produce hydroxamate-type siderophores which, because of the correlation with Fe3+ limitation, are thought to be the major factor responsible for antagonistic activity. Siderophore-negative mutants of two strains had a strongly decreased antagonistic activity. Five strains maintained their antagonistic activity under conditions of iron excess. Analysis of cell envelope protein patterns of cells grown in excess Fe3+ showed that most strains differed from each other, although two classes of similar or identical strains were found. In one case such a class was subdivided on the basis of the patterns of proteins derepressed by iron limitation. Small plasmids were not detected in any of the strains, and only one of the four tested strains contained a large plasmid. Therefore, it is unlikely that the Fe3+ uptake system of the antagonistic strains is usually plasmid encoded.     

Siderophore-mediated iron transport correlates with the presence of specific iron-regulated proteins in the outer membrane of Rhizobium meliloti.

A universal chemical assay used to detect the production of siderophores in a range of Rhizobium strains showed that production is strain specific. Iron nutrition bioassays carried out on Rhizobium meliloti strains to determine cross-utilization of their siderophores showed that R. meliloti 2011, 220-5, and 220-3 could each use the siderophores produced by the other two but not the siderophore produced by R. meliloti DM4 (and vice versa). Mutants of R. meliloti 2011 and 220-5 defective in siderophore production were isolated by Tn5-mob mutagenesis. The Tn5-mob-containing EcoRI fragment of mutant R. meliloti 220-5-1 was cloned into pUC19. By using this fragment as a probe, the presence of a homologous region was observed in R. meliloti 2011 and 220-3 but not in R. meliloti DM4. A complementing cosmid from a gene bank of R. meliloti 2011 was identified by using the same probe. Introduction of this cosmid into R. meliloti 102F34, a strain not producing a siderophore, resulted in the ability of this strain to produce a siderophore and also in the ability to utilize the siderophores produced by R. meliloti 2011, 220-5, and 220-3 but not the siderophore produced by R. meliloti DM4. A comparative analysis of the outer membrane proteins prepared from iron-deficient cultures of R. meliloti 102F34 and 102F34 harboring the cosmid revealed the presence, in the latter, of a low-iron-induced outer membrane protein corresponding to a low-iron-induced protein in R. meliloti 2011, 220-5, and 220-3. This protein is not present in R. meliloti DM4. The results suggest that R. meliloti 2011, 220-5, and 220-3 produce siderophores that are identical or sufficiently similar in structure to be transported by the membrane transport system of each strain while also indicating that utilization of a particular siderophore is correlated with the presence of specific outer membrane proteins.     

In vivo production of A-protein, lipopolysaccharide, iron-regulated outer membrane proteins and 70-kDa serine protease by Aeromonas salmonicida subsp. salmonicida

Using specific immunostaining of Western blots, the in vivo expression of several putative virulence factors of Aeromonas salmonicida subsp. salmonicida was demonstrated in infected muscle tissue of Atlantic salmon and rainbow trout. Three virulent isolates of A. salmonicida were used. One isolate was chosen because in vitro it was apparently a non-producer of the 70-kDa serine protease. Infected furuncle tissue was centrifuged and samples of the pellet and supernatant probed for evidence that the components of interest were bacterial cell-associated or secreted. The A-protein was detected in pelleted furuncle material but not in the supernatant. Lipopolysaccharide, both high and low molecular mass, was present in the pellet but only high molecular mass lipopolysaccharide was detected in the furuncle supernatant. Iron-regulated outer membrane proteins were detected in the furuncle pellet. The 70-kDa serine protease was detected in the furuncle supernatant of both protease-producing strains. However, whilst the protease-deficient isolate was demonstrated to produce low levels of the 70-kDa protease when grown in vitro under iron restricted conditions, none could be detected in vivo.     

In silico local structure approach: a case study on outer membrane proteins

The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results.     

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.     

Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis

The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 microM and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.