Iron-repressible Outer Membrane Proteins Research Articles

Comparative study of iron acquisition by biotype 1 and biotype 2 strains of Actinobacillus pleuropneumoniae

Four strains of the swine pathogen, Actinobacillus pleuropneumoniae, namely, the type strain (ATCC 27088; biotype 1), the ‘reference’ strain of biotype 2 (Bertschinger 2008 76 ), and two additional biotype 1 strains, strain BC181, which is less virulent than the type strain, and strain K17, which was isolated from a lamb, were investigated with respect to iron acquisition. All strains produced iron-repressible outer membrane proteins. However, only the type and biotype 2 strains could acquire iron from procine transferrin and no organism could utilize human, bovine or ovine transferrin, or ovine or porcine lactoferrin; haemoglobin supported good growth of all strains except strain K17. In all cases, iron acquisition from transferrin and haemoglobin required direct contact between the organisms and the proteins indicating the existence of specific receptors. An affinity isolation technique, using biotinylated porcine transferrin plus streptavidin-agarose, allowed the isolation of the following polypeptides from total membranes of organisms grown under iron-restricted conditions: 99 kDa and 64 kDa from ATCC 27088; 93 kDa from Bertschinger 2008 76 ; 95 kDa (trace amounts) and 60 kDa from BC181; none from K17. These results indicate that the 93–99 kDa polypeptides are involved in the acquisition of iron from porcine transferrin and that the inability of strain K17 to use transferrin as an iron source is due, probably, to the lack of, or a defect in, an analogous component.

Siderophore production and outer membrane protein synthesis of vibrio parahaemolyticus strain with different serotypes under iron-limited condition.

Iron uptake mechanisms were investigated in strains of Vibrio parahaemolyticus with different serotypes. Although the production of siderophore was detected in all the strains tested, they were not able to grow in the presence of high concentrations of the iron chelator EDDHA but were able to grow in the presence of only low concentrations of EDDHA (ranging from lower than 8 to 64 μM). Two iron-repressible outer membrane proteins (IROMPs) with molecular weights of 71,000 (71 K protein) and 72,000 (72 K protein) were produced in the strain 3283-61 grown in iron-deficient medium. Kinetic analysis revealed that the 71K and 72K proteins occurred 1.5 and 2.5h, respectively, after downshift from chemically defined medium to iron-deficient medium. Analysis of IROMPs from V. parahaemolyticus strains with different serotypes showed that the patterns of IROMPs from these strains were very similar to that of strain 3283-61: all the strains produced the two IROMPs of 71K and 72K proteins, except one strain, which produced only the 71K protein.

Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis

Two strains of Haemophilus parasuis, namely, the type strain (ATCC 19417) and strain E751, were investigated with respect to iron acquisition. Both strains produced iron-repressible outer membrane proteins and could acquire iron from porcine transferrin but not from porcine lactoferrin. Neither strain used bovine transferrin, and human transferrin was used to only a very limited extent, if at all. In all cases, iron acquisition from transferrin required direct contact between the organisms and the protein. An affinity isolation technique based on biotinylated porcine transferrin plus streptavidin-agarose, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine transferrin binding polypeptides (94 and 60 kDa) from total membranes derived from the type strain grown under iron-restricted conditions but only one (96 kDa) from strain E751. Each of these polypeptides was iron repressible and was not isolated when biotinylated human or bovine transferrin was used instead of biotinylated porcine transferrin. It is concluded that both strains acquire transferrin-bound iron by means of siderophore-independent mechanisms and that the isolated polypeptides represent porcine transferrin receptor components.

Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis

Possession of specialized iron acquisition systems is a prerequisite for the survival of pathogenic bacteria in their host. The purpose of this study was to determine whether Bacteroides fragilis, a clinically important Gram-negative anaerobic bacterium, possesses a specific haem-uptake system. Growth studies indicated that this microorganism can utilize haem from either haemoglobin or haptoglobin-haemoglobin as its sole source of iron. Iron-repressible haem-binding protein complexes ( HBP complexes), involved in the uptake of haem from haptoglobin-haemoglobin were detected by means of lithium dodecyl sulfate polyacrylamide gel electrophoresis ( LDS-PAGE). Four polypeptides of approximately 60, 58, 49 and 35 kDa, which are part of these HBP complexes, were identified as haem-binding proteins. A 44 kDa iron-repressible outer-membrane protein is needed for a functional HBP complex, but the exact role of this protein in the uptake of haem is still unknown.

The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function

The iron-repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacteriocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse-virulent Y. enterocolitica strains of biogroup IB. Cloning of the FyuA-encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin-sensitive and mouse-virulent Y. enterocolitica O:8 strain WA into the pesticin-resistant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse virulence). The reisolated transconjugants which survived in mice for 3 d harboured a unique cosmid and phenotypically were pesticin sensitive. From this cosmid a 2650 bp SalI-PstI fragment conferring pesticin sensitivity was subcloned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73,677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71,368 Da. The open reading frame is preceded by a sequence which shares homology with the postulated consensus Fur iron-repressor protein-binding site. FyuA shows homology to other iron-regulated TonB-dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, IutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB-dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB-dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cloned fyuA gene, yersiniabactin uptake and mouse virulence were restored. These studies demonstrate that the cloned pesticin/yersiniabactin receptor FyuA of Y. enterocolitica has the typical features of iron-regulated TonB-dependent outer membrane receptors for siderophores and bacteriocins and is required for mouse virulence.

Iron Chelation in Plants and Soil Microorganisms

Part 1 Production and characteristics of metal chelator: classes of microbial siderophores, M. Hofte biochemical and genetic analysis of siderophores produced by plant-associated Pseudomonas spp. and Erwinia spp, C.A. Ischimaru and J.E. Loper growth conditions for the demonstration of siderophores and iron repressible outer membrane proteins in soil bacteria, with an emphasis on free-living diazotrophs, W.J. Page production of phytosiderophores, S.-I Takagi plant and microbial ferritins, E.C. Theil and T. Hase glutathione-derived metal-binding polypeptides and metallothioneins, P.J. Jackson and C.R. Kuske. Part 2 Enzymes and interactive systems: overview of bacterial iron transport and siderophore systems in Rhizobia, J.B. Neilands iron and the nodule, M.L. Guerinot kinetics, energetics, and mechanisms of siderophore-iron transport in fungi, G. Winkelmann enzymatic reduction of iron in siderophores, J.S. Lodge the role of iron in fungal phytopathologies, I, Barash, et al the role of iron in the suppression of bacterial-plant pathogens by fluorescent Pseudomonas, A.H.M. Bakker and B. Schippers ferrochelatase and related enzymes, L.L. Barton three genetically distinct nitrogenase systems in Asotobacter vinelandii, P.E. Bishop. Part 3 Physiological and chemical characteristics of the iron stress response: iron and plant pigments, J. Abadia and A. Abadia plant iron uptake physiology by non-siderophore systems, G.W. Welkie and G.W. Miller selected physiological responses associated with Fe(III) and Fe(II) metabolism, G.V. Johnson and L.L. Barton a case study with soy-beans - iron-efficiency evaluations in field tests versus controlled conditions, S. Rodriquez de Cianzio assays for microbial siderophores, F.A. Fekete Mossbauer spectroscopy, E.V. Mielczarek sample preparation and determination of iron in biological material, Q. Wallace and J.B. Jones, Jr evaluation of iron in soil samples, R.C. Hartwig and R.H. Loeppert.

Role of siderophore in iron uptake in cowpea Rhizobium GN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.

Role of siderophore in iron uptake in cowpeaRhizobiumGN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.

Virulence of Yersinia enterocolitica is closely associated with siderophore production, expression of an iron-repressible outer membrane polypeptide of 65,000 Da and pesticin sensitivity.

Iron-repressible outer membrane proteins (Irp) and siderophore production of Yersinia enterocolitica, serotype 08, were subjected to analysis. Here four Irps of apparent molecular weights of 62,000, 65,000, 74,000 and 75,000 could be identified which were expressed constitutively by a fur mutant. Production of a novel catechol-containing siderophore (denoted yersiniabactin) was detected by siderophore-indicator agar (chrome azurol S) and feeding experiments. Growth support by yersiniabactin under iron-restricted conditions was TonB- and Irp65-dependent and correlated with pesticin-sensitivity of Yersinia enterocolitica and Escherichia coli O. From these results we conclude that Irp65 of Y. enterocolitica functions as yersiniabactin receptor (FyuA) and as pesticin receptor. By immunoblotting using rabbit antibodies against Irp65 and chrome azurol S-agar, we were able to demonstrate that all tested mouse-lethal Y. enterocolitica and Yersinia pseudotuberculosis strains of different serotypes express siderophores and Irp65. Moreover, the anti-Irp65 rabbit serum did not cross-react with the known iron-repressible high-molecular-weight proteins (HMWPs). Evidently, the mouse lethality trait in enteropathogenic Yersinia spp. is closely associated with a novel iron-uptake system, comprising the production of a siderophore and a siderophore receptor of apparent molecular mass 65,000 Da.

Virulence factors in anaerobic bacteria.

Various surface structures can be expressed in Bacteroides fragilis, but little is known about capsular structures in other non-spore-forming anaerobes. Fimbriae have been isolated from Bacteroides fragilis and Porphyromonas gingivalis. The importance of iron-repressible outer membrane proteins as virulence factors in Bacteroides fragilis is under study. The low endotoxic activity of Bacteroides fragilis lipopolysaccharide can be attributed to the chemical composition of this organism's lipid A. A tissue culture system for the demonstration of Bacteroides fragilis enterotoxin has recently been described. The toxins A and B of Clostridium difficile are immunologically distinct. The importance of IgA proteases and other enzymes as virulence factors in anaerobic bacteria remains unclear.

Iron acquisition in Haemophilus influenzae: receptors for human transferrin.

As an adaptation to the iron-limited environment of the host, Haemophilus influenzae has a transferrin receptor-mediated mechanism of iron acquisition such that it can acquire iron directly from human transferrin. The absence of detectable siderophore production and the presence of transferrin binding in a collection of type b and nontypeable H. influenzae strains indicate that the mechanism is widespread in this species. Growth and binding studies have consistently shown that the receptor is specific for human transferrin, which correlates with the host range of this pathogen. Inhibitor experiments indicate that iron regulation of receptor activity is mediated at the gene level. Affinity isolation experiments indicate that, as observed with other bacterial pathogens, the receptor is composed of two iron-repressible outer membrane proteins, transferrin binding proteins 1 and 2.

Human immune response to an iron-repressible outer membrane protein of Bacteroides fragilis

Under conditions of iron starvation, Bacteroides fragilis expresses various iron-repressible outer membrane proteins (IROMPs). A 44-kDa protein appears to be one of the major outer membrane proteins (OMPs) in B. fragilis under iron stress and plays a role in heme uptake by this bacterium. To determine whether the 44-kDa IROMP of B. fragilis is expressed in vivo and whether this protein is immunogenic, we used Western immunoblotting to examine serum samples from patients with an infection caused by Bacteroides species. All the serum samples from patients and from normal controls showed reactivity with several proteins of B. fragilis. Only serum samples from patients infected with B. fragilis showed immunoreactivity with the 44-kDa protein. We also used a rat infection model to study the immune response against this protein during the process of an intra-abdominal infection in these animals. During the first 8 days of infection a gradual increase of antibodies to the 44-kDa protein in the rat was detected. These results suggest that the 44-kDa IROMP is expressed in vivo, since it induces an antibody response in patients and animals. We also analyzed 85 strains of the B. fragilis group for the presence of proteins antigenically related to the B. fragilis 44-kDa protein. The data indicate that this protein was conserved in B. fragilis strains and was absent in the other bacterial strains tested.

Siderophore production by Salmonella species isolated from different sources

A total of 230 Salmonella strains were screened for enterobactin and aerobactin production, sensitivity to bacteriocins and resistance to antibiotics. All the isolates produced the phenolate siderophore enterobactin. Amongst these, 74 strains, most belonging to S. enteritidis, were sensitive to colicin B. Only 26 isolates, all belonging to S. wien, produced an additional iron chelator, i.e. the siderophore aerobactin, and 22 out of these were sensitive to cloacin DF13. Analysis of iron repressible outer membrane proteins and plasmid profiles in S. wien strains showed that the expression of a 74-kDa iron-repressible outer membrane protein and the presence of large plasmids were associated with multiple antibiotic resistance, aerobactin production and sensitivity to cloacin DF13. The incidence of aerobactin-producing strains among S. wien isolates was higher during years 1974-1985; the epidemiological implications of these results are discussed.

Siderophore production bySalmonellaspecies isolated from different sources

A total of 230 Salmonella strains were screened for enterobactin and aerobactin production, sensitivity to bacteriocins and resistance to antibiotics. All the isolates produced the phenolate siderophore enterobactin. Amongst these, 74 strains, most belonging to S. enteritidis, were sensitive to colicin B. Only 26 isolates, all belonging to S. wien, produced an additional iron chelator, i.e. the siderophore aerobactin, and 22 out of these were sensitive to cloacin DF13. Analysis of iron repressible outer membrane proteins and plasmid profiles in S. wien strains showed that the expression of a 74-kDa iron-repressible outer membrane protein and the presence of large plasmids were associated with multiple antibiotic resistance, aerobactin production and sensitivity to cloacin DF13. The incidence of aerobactin-producing strains among S. wien isolates was higher during years 1974–1985; the epidemiological implications of these results are discussed.

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature-sensitive production of azoverdin, the pyoverdin-like sid rophore of Azomonas macrocytogenes ATCC 12334

SUMMARY: Azoverdin, a visible yellow, blue--white fluorescent compound produced by iron-limited Azomonas macrocytogenes ATCC 12334, was isolated from cell-free culture supernatant fluid and purified in the ferrated form to 98% purity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography, thereby separating it from several related iron-binding fluorescent compounds. Purified ferrated azoverdin exhibited a pH-independent absorption spectrum which became pH-dependent following deferration, typical of a pyoverdin-like siderophore. Azoverdin enhanced 55Fe3+ assimilation by iron-limited A. macrocytogenes and therefore functioned as a siderophore. Iron-limited cells were unable to produce azoverdin when grown at 34 °C rather than 28 °C. However, these cells still expressed a 74 kDa and a 70 kDa iron-repressible outer membrane protein and were capable of azoverdin-mediated iron transport. The use of A. macrocytogenes cells grown at 34 °C eliminated endogenous azoverdin production during iron uptake assays, which made it possible to accurately determine azoverdin-mediated 55Fe3+ transport rates. Azoverdin-mediated 55Fe-uptake proceeded with an apparent K m of 0·2 μM and a V of 0·46ng Fe3+ (108 cells)−1 min−1.

Identification and characterization of a porcine‐specific transferrin receptor in Actinobacillus pleuropneumoniae

All six strains of Actinobacillus pleuropneumoniae screened for the ability to use different transferrins as a source of iron for growth were capable of using porcine but not human, bovine, or avian transferrins. A specific binding activity for porcine transferrin (pTf) was expressed in cells grown in the presence of specific iron-chelators and was repressed by addition of excess iron. Two iron-repressible outer-membrane proteins of 105 and 56 kD were specifically isolated from serotype 1, 2 and 7 strains of A. pleuropneumoniae by an affinity-isolation method using biotinylated porcine transferrin and streptavidin-agarose.

Characterisation of a siderophore from Acinetobacter calcoaceticus

The Gram-negatice bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant bu 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.

Characterisation of a siderophore fromAcinetobacter calcoaceticus

The Gram-negative bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant bu 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.

Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups

Recently, beta-lactam agents containing iron-chelating moieties, such as E0702, which contains catechol, and pirazmonam and U-78,608, which contain 3-hydroxypyridone, have been developed. By determining the susceptibility to these agents of Escherichia coli mutants lacking various iron-repressible outer membrane proteins, we showed that two of these proteins with hitherto unknown functions, Fiu and Cir, were apparently involved in the transport of monomeric catechol and its analogs. These results confirm the conclusion of Curtis and co-workers, which was obtained by using a different set of catechol-containing antibiotics (N. A. C. Curtis, R. L. Eisenstadt, S. J. East, R. J. Cornford, L. A. Walker, and A. J. White, Antimicrob. Agents Chemother. 32:1879-1886, 1988). E0702 was shown to enhance the uptake of radioactive ferric iron into wild-type cells but not into cir fiu double mutants. By combining the influx of E0702 with its hydrolysis by a periplasmic beta-lactamase, we showed that the wild-type cells transported unliganded E0702 at a rate comparable to or even higher than the rate of transport of the E0702-Fe3+ complex. We postulate that the main function of Cir and Fiu may be to recapture the hydrolytic products of enterobactin, such as 2,3-dihydroxybenzoic acid and 2,3-dihydroxybenzoylserine.