TonB-dependent Outer Membrane Proteins Research Articles

Attenuation of Yersinia pestis fyuA Mutants Caused by Iron Uptake Inhibition and Decreased Survivability in Macrophages.

Yersinia pestis is the etiological agent of plague, a deadly infectious disease that has caused millions of deaths throughout history. Obtaining iron from the host is very important for bacterial pathogenicity. Y. pestis possesses many iron uptake systems. Yersiniabactin (Ybt) plays a major role in iron uptake in vivo and in vitro, and in virulence toward mice as well. FyuA, a β-barrel TonB-dependent outer membrane protein, serves as the receptor for Ybt. In this study, we examined the role of the fyuA gene in Y. pestis virulence using different challenging ways and explored the underlying mechanisms. The BALB/c mouse infection assay showed that the virulence of the mutant strains (ΔfyuA and ΔfyuA GCAdel) was lower when compared with that of the wild-type (WT) strain 201. Furthermore, the attenuation of virulence of the mutant strains via subcutaneous and intraperitoneal challenges was far greater than that via intravenous injection. Iron supplementation restored lethality during subcutaneous challenge with the two mutants. Thus, we speculated that the attenuated virulence of the mutant strains toward the mice may be caused by dysfunctional iron uptake. Moreover, ΔfyuA and ΔfyuA GCAdel strains exhibited lower survival rates in murine RAW264.7 macrophages, which might be another reason for the attenuation. We further explored the transcriptomic differences between the WT and mutant strains at different temperatures and found that the expressions of genes related to Ybt synthesis and its regulation were significantly downregulated in the mutant strains. This finding indicates that fyuA might exert a regulatory effect on Ybt. Additionally, the expressions of the components of the type III secretion system were unexpectedly upregulated in the mutants, which is inconsistent with the conventional view that the upregulation of the virulence genes enhances the virulence of the pathogens.

Growth of Pseudomonas aeruginosa in zinc poor environments is promoted by a nicotianamine-related metallophore.

Previous studies have suggested that P. aeruginosa possesses redundant zinc uptake systems. To identify uncharacterized zinc transporters, we analyzed the genome-wide transcriptional responses of P. aeruginosa PA14 to zinc restriction. This approach led to the identification of an operon (zrmABCD) regulated by the zinc uptake regulator Zur, that encodes for a metallophore-mediated zinc import system. This operon includes the genes for an uncharacterized TonB-dependent Outer Membrane Protein (ZrmA) and for a putative nicotianamine synthase (ZrmB). The simultaneous inactivation of the ZnuABC transporter and of one of these two genes markedly decreases the ability of P. aeruginosa to grow in zinc-poor media and compromises intracellular zinc accumulation. Our data demonstrate that ZrmB is involved in the synthesis of a metallophore which is released outside the cell and mediates zinc uptake through the ZrmA receptor. We also show that alterations in zinc homeostasis severely affect the ability of P. aeruginosa to cause acute lung and systemic infections in C57BL/6 mice, likely due to the involvement of zinc in the expression of several virulence traits. These findings disclose a hitherto unappreciated role of zinc in P. aeruginosa pathogenicity and reveal that this microorganism can obtain zinc through a strategy resembling siderophore-mediated iron uptake.

High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8–1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.

Synthesis of nickel-iron hydrogenase in Cupriavidus metallidurans is controlled by metal-dependent silencing and un-silencing of genomic islands.

Cupriavidus metallidurans CH34 is able to grow autotrophically as a hydrogen-oxidizing bacterium and produces nickel-dependent hydrogenases, even under heterotrophic conditions. Loss of its two native plasmids resulted in inability of the resulting strain AE104 to synthesize the hydrogenases and to grow autotrophically in phosphate-poor, Tris-buffered mineral salts medium (TMM). Three of eleven previously identified catabolic genomic islands (CMGIs; Van Houdt et al., 2009), two of which harbor the genes for the membrane-bound (CMGI-2) and the soluble hydrogenase (CMGI-3), were silenced in strain AE104 when cultivated in phosphate-poor TMM, explaining its inability to produce hydrogenases. Production of the soluble hydrogenase from the aut region 1 of CMGI-3, and concomitant autotrophic growth, was recovered when the gene for the zinc importer ZupT was deleted in strain AE104. The transcriptome of the ΔzupT mutant exhibited two up-regulated gene regions compared to its parent strain AE104. Expression of the genes in the aut region 1 increased independently of the presence of added zinc. A second gene region was expressed only under metal starvation conditions. This region encoded a TonB-dependent outer membrane protein, a putative metal chaperone plus paralogs of essential zinc-dependent proteins, indicating the presence of a zinc allocation pathway in C. metallidurans. Thus, expression of the genes for the soluble hydrogenase and the Calvin cycle enzymes on aut region 1 of CMGI-3 of C. metallidurans is under global control and needs efficient ZupT-dependent zinc allocation for a regulatory role, which might be discrimination of nickel.

Analysis of the Caulobacter crescentus Zur regulon reveals novel insights in zinc acquisition by TonB-dependent outer membrane proteins.

BackgroundIntracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane.ResultsIn this work we determined the Caulobacter crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur in response to zinc availability are those encoding a putative high affinity ABC uptake system (znuGHI), three TonB-dependent receptors (znuK, znuL and znuM) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by β-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuK and znuL mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems.ConclusionsThe characterization of the C. crescentus Zur regulon showed that this regulator coordinates not only uptake, but also the extrusion of zinc. The uptake of zinc by C. crescentus in conditions of scarcity of this metal is highly dependent on TonB-dependent receptors, and the extrusion is mediated by an RND and P-type ATPase transport systems. The absence of Zur causes a disturbance in the dynamic equilibrium of zinc intracellular concentration, which in turn can interfere with other regulatory networks as seen for the Fur regulon.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-734) contains supplementary material, which is available to authorized users.

Inactivation of a fibronectin-binding TonB-dependent protein increases adhesion properties of Bacteroides fragilis

Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp.

The Vibrio parahaemolyticuspvuA1 gene (formerly termed psuA) encodes a second ferric vibrioferrin receptor that requires tonB2

We previously reported that the Vibrio parahaemolyticus pvsABCDE and psuA-pvuABCDE operons are involved in the biosynthesis and transport of its own siderophore, vibrioferrin (VF). Of these, psuA and pvuA encode TonB-dependent outer-membrane proteins (OMPs). Although pvuA was characterized as the ferric vibrioferrin receptor gene, the role of the psuA product remains unknown. In this study, a growth assay of isogenic psuA, pvuA, and psuA-pvuA double-deletion mutants followed by complementation of the double-deletion mutant with psuA or pvuA was used to identify psuA as a gene encoding an OMP involved in the uptake of ferric VF. Thus, psuA and pvuA were renamed pvuA1 and pvuA2, respectively. Moreover, we clarified the TonB specificities of PvuA1 and PvuA2, because V. parahaemolyticus has three sets of the TonB systems. The triple deletion of pvuA1, tonB1, and tonB2, and the double deletion of pvuA2 and tonB2 resulted in the complete loss of growth promotion by VF. This finding indicates that the energy required for PvuA1 and PvuA2 to transport ferric VF across the outer membrane is provided by the TonB2 system and by both the TonB1 and TonB2 systems, respectively.

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved β-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

Characterization of NikR-responsive promoters of urease and metal transport genes of Helicobacter mustelae

The NikR protein is a nickel-responsive regulator, which in the gastric pathogen Helicobacter pylori controls expression of nickel-transporters and the nickel-cofactored urease acid resistance determinant. Although NikR-DNA interaction has been well studied, the Helicobacter NikR operator site remains poorly defined. In this study we have identified the NikR operators in the promoters of two inversely nickel-regulated urease operons (ureAB and ureA2B2) in the ferret pathogen Helicobacter mustelae, and have used bioinformatic approaches for the prediction of putative NikR operators in the genomes of four urease-positive Helicobacter species. Helicobacter mustelae NikR bound to the ureA2 promoter to a sequence overlapping with the -35 promoter region, leading to repression. In contrast, NikR binding to a site far upstream of the canonical sigma(80) promoter in the H. mustelae ureA promoter resulted in transcriptional induction, similar to the situation in H. pylori. Using H. pylori NikR operators and the newly identified H. mustelae NikR operators a new consensus sequence was generated (TRWYA-N(15)-TRWYA), which was used to screen the genomes of four urease-positive Helicobacter species (H. mustelae, H. pylori, H. acinonychis and H. hepaticus) for putative NikR-regulated promoters. One of these novel putative NikR-regulated promoters in H. mustelae is located upstream of a putative TonB-dependent outer membrane protein designated NikH, which displayed nickel-responsive expression. Insertional inactivation of the nikH gene in H. mustelae resulted in a significant decrease in urease activity, and this phenotype was complemented by nickel-supplementation of the growth medium, suggesting a function for NikH in nickel transport across the outer membrane. In conclusion, the H. mustelae NikR regulator directly controls nickel-responsive regulation of ureases and metal transporters. The improved consensus NikR operator sequence allows the prediction of additional NikR targets in Helicobacter genomes, as demonstrated by the identification of a new nickel-repressed outer membrane protein in H. mustelae.

A TonB-dependent outer membrane protein as aBacteroides fragilisfibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation-reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c. 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c. 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.

Unravelling the role of the ToxR-like transcriptional regulator WmpR in the marine antifouling bacterium Pseudoalteromonas tunicata

The dark-green-pigmented marine bacterium Pseudoalteromonas tunicata produces several target-specific compounds that act against a range of common fouling organisms, including bacteria, fungi, protozoa, invertebrate larvae and algal spores. The ToxR-like regulator WmpR has previously been shown to regulate expression of bioactive compounds, type IV pili and biofilm formation phenotypes which all appear at the onset of stationary phase. In this study a comparison of survival under starvation or stress between the wild-type P. tunicata strain and a wmpR mutant (D2W2) does not suggest a role for WmpR in regulating starvation- and stress-resistant phenotypes such as those that may be required in stationary phase. Both proteomic [2-dimensional PAGE (2D-PAGE)] and transcriptomic (RNA arbitrarily primed PCR) studies were used to discover members of the WmpR regulon. 2D-PAGE identified 11 proteins that were differentially expressed by WmpR. Peptide sequence data were obtained for six of these proteins and identified using the draft P. tunicata genome as being involved in protein synthesis, amino acid transamination and ubiquinone biosynthesis, as well as hypothetical proteins. The transcriptomic analysis identified three genes significantly up-regulated by WmpR, including a TonB-dependent outer-membrane protein, a non-ribosomal peptide synthetase and a hypothetical protein. Under iron-limitation the wild-type showed greater survival than D2W2, indicating the importance of WmpR under these conditions. Results from these studies show that WmpR controls the expression of genes encoding proteins involved in iron acquisition and uptake, amino acid metabolism and ubiquinone biosynthesis in addition to a number of proteins with as yet unknown functions.

ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane ofCaulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.

Siderophore Transport through Escherichia coli Outer Membrane Receptor FhuA with Disulfide-tethered Cork and Barrel Domains

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.

The plug domain of a neisserial TonB-dependent transporter retains structural integrity in the absence of its transmembrane β-barrel

Transferrin binding protein A (TbpA) is a TonB-dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the β-barrel, but extracellular loops of the β-barrel are required for ligand binding.

The bacterial receptor protein, transferrin-binding protein B, does not independently facilitate the release of metal ion from human transferrin.

Pathogenic Gram-negative bacteria of the Pasteurellaceae and Neisseriaceae acquire iron for growth from host transferrin through the action of specific surface receptors. Iron is removed from transferrin by the receptor at the cell surface and is transported across the outer membrane to the periplasm. A periplasmic binding protein-dependent pathway subsequently transports iron into the cell. The transferrin receptor is composed of a largely surface-exposed lipoprotein, transferrin binding protein B, and a TonB-dependent integral outer membrane protein, transferrin binding protein A. To examine the role of transferrin binding protein B in the iron removal process, complexes of recombinant transferrin binding protein B and transferrin were prepared and compared with transferrin in metal-binding and -removal experiments. A polyhistidine-tagged form of recombinant transferrin binding protein B was able to purify a complex with transferrin that was largely monodisperse by dynamic light scattering analysis. Gallium was used instead of iron in the metal-binding studies, since it resulted in increased stability of recombinant transferrin binding protein B in the complex. Difference absorption spectra were used to monitor removal of gallium by nitrilotriacetic acid. Kinetic and equilibrium binding studies indicated that transferrin binds gallium more tightly in the presence of transferrin binding protein B. Thus, transferrin binding protein B does not facilitate metal ion removal and additional components are required for this process.

Specific ligand binding attributable to individual epitopes of gonococcal transferrin binding protein A.

The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

Neisserial TonB-dependent outer-membrane proteins: detection, regulation and distribution of three putative candidates identified from the genome sequences.

Computer searches were carried out of the gonococcal and meningococcal genome databases for previously unknown members of the TonB-dependent family (Tdf) of outer-membrane receptor proteins. Seven putative non-contiguous genes were found and three of these (identified in gonococcal strain FA1090) were chosen for further study. Consensus motif analysis of the peptide sequences was consistent with the three genes encoding TonB-dependent receptors. In view of the five previously characterized TonB-dependent proteins of pathogenic neisseriae, the putative genes were labelled tdfF, tdfG and tdfH. TdfF had homology with the siderophore receptors FpvA of Pseudomonas aeruginosa and FhuE of Escherichia coli, whereas TdfG and TdfH had homology with the haemophore receptor HasR of Serratia marcescens. The aim of this project was to characterize these proteins and determine their expression, regulation, distribution and surface exposure. Strain surveys of iron-stressed commensal and pathogenic neisseriae revealed that TdfF is unlikely to be expressed, TdfG is expressed by gonococci only and that TdfH is expressed by both meningococci and gonococci. Expression of TdfH was unaffected by iron availability. Susceptibility of TdfH to cleavage by proteases in live gonococci was consistent with surface exposure of this protein. TdfH may function as a TonB-dependent receptor for a non-iron nutrient source. Furthermore, TdfH is worthy of future investigation as a potential meningococcal vaccine candidate as it is a highly conserved, widely distributed and surface-exposed outer-membrane protein.

Involvement of HxuC outer membrane protein in utilization of hemoglobin by Haemophilus influenzae.

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.