Haemin Binding Research Articles

Transcriptional organization, regulation and role of the Porphyromonas gingivalis W83 hmu haemin-uptake locus

Porphyromonas gingivalis, an oral bacterium associated with periodontal disease, requires haemin for growth. Although several multigenic clusters encoding haemin-uptake systems are present on the genome of P. gingivalis, little is known regarding their transcriptional organization and expression. This study identified a 23 kDa iron-regulated haemin-binding protein encoded by a larger than previously reported variant of hmuY. It was shown that the hmu locus is larger than previously reported and is composed of six genes, hmuYRSTUV, encoding a novel hybrid haemin-uptake system. The locus has an operonic organization and the transcriptional start site is located 292 bp upstream of hmuY. The data indicate that the regulation of the operon is iron-dependent. Interestingly, differential regulation within the operon was demonstrated, resulting in excess of the hmuYR message encoding the outer-membrane proteins when compared to the full-length transcript. In addition, the hmuY transcript is more prevalent than the hmuR transcript. Secondary structure analysis of the hmuYRSTUV mRNA predicted the formation of several potential stem-loops in the 5' ends of hmuR- and hmuS-specific mRNAs, consistent with the differential regulation observed. Finally, it was demonstrated that haemin binding and uptake are elevated in iron-depleted conditions and are reduced 45 % and 70 %, respectively, in an hmu-deficient strain when compared to the parental strain, indicating that the hmu locus plays a major role in haemin acquisition in P. gingivalis. Since homologues of the hmu locus were also found in Bacteroides fragilis, Bacteroides thetaiotaomicron and Prevotella intermedia, these findings may have implications for a better understanding of haemin acquisition in those organisms as well.

HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms‐dependent biofilm formation in Yersinia pestis

The Hms(+) phenotype of Yersinia pestis promotes the binding of haemin or Congo red (CR) to the cell surface at temperatures below 34 degrees C. We previously demonstrated that temperature regulation of the Hms(+) phenotype is not controlled at the level of transcription. Instead, HmsH, HmsR and HmsT are degraded upon a temperature shift from 26 degrees C to 37 degrees C. We used random transposon mutagenesis to identify new genes involved in the temperature-regulated expression of the Hms phenotype. One of these genes, which we designated hmsP, encodes a putative phosphodiesterase with a conserved EAL motif. Mutations in hmsP caused formation of red colonies on CR plates at 26 degrees C and 37 degrees C. Strains complemented with hmsP(+) on a plasmid form white colonies at both temperatures. We used a crystal violet assay and confocal laser scanning microscopy to demonstrate Hms-dependent biofilm formation by Y. pestis cells. Y. pestis Hms(+) strains grown at 26 degrees C but not at 37 degrees C form a biofilm on borosilicate glass surfaces. Strains that either overexpress HmsT (a GGDEF domain protein) or have a mutation in hmsP produced an extremely thick biofilm. Alanine substitutions for each of the GGEE residues (amino acids 296-299) of HmsT as well as the E506 and L508 residues of HmsP caused a loss of function. We propose that HmsT and HmsP together control the amount of biofilm produced in Y. pestis. Degradation of HmsT at 37 degrees C may be a critical factor in controlling the temperature-dependent expression of the Hms biofilm.

Haemin uptake and use as an iron source by Candida albicans: role of CaHMX1-encoded haem oxygenase.

Candida albicans, unlike Saccharomyces cerevisiae, was able to use extracellular haemin as an iron source. Haemin uptake kinetics by C. albicans cells showed two phases: a rapid phase of haemin binding (with a K(d) of about 0.2 microM) followed by a slower uptake phase. Both phases were strongly induced in iron-deficient cells compared to iron-rich cells. Haemin uptake did not depend on the previously characterized reductive iron uptake system and siderophore uptake system. CaHMX1, encoding a putative haem oxygenase, was shown to be required for iron assimilation from haemin. A double DeltaCahmx1 mutant was constructed. This mutant could not grow with haemin as the sole iron source, although haemin uptake was not affected. The three different iron uptake systems (reductive, siderophore and haemin) were regulated independently and in a complex manner. CaHMX1 expression was induced by iron deprivation, by haemin and by a shift of temperature from 30 to 37 degrees C. CaHMX1 expression was strongly deregulated in a Deltaefg1 mutant but not in a Deltatup1 mutant. C. albicans colonies forming on agar plates with haemin as the sole iron source showed a very unusual morphology. Colonies were made up of tubular structures that were organized into a complex network. The effect of haemin on filamentation was increased in the double DeltaCahmx1 mutant. This study provides the first experimental evidence that haem oxygenase is required for iron assimilation from haem by a pathogenic fungus.

Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants.

Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) < 40 microM), 16 (84%) showed >40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

Binding of haemin by the fish pathogen Photobacterium damselae subsp. piscicida.

Whole cells of virulent (DI 21 and B 51) and avirulent (ATCC 29690 and EPOY 8803-II) strains of Photobacterium damselae subsp. piscicida, grown under iron-supplemented or iron-restricted conditions, were able to bind haemin. Iron limitation resulted in an increased binding of haemin by DI 21, B 51 and ATCC 29690 cells but did not affect the haemin-binding ability of the EPOY 8803-II cells. Proteinase K treatment of whole cells markedly reduced the binding of haemin, indicating that protein receptors located at the cell surface are involved in the binding. This was confirmed by the observation that isolated total as well as outer membrane proteins from all the strains, regardless of the iron levels of the media, were able to bind haemin, with the outer membranes showing the strongest binding. Haemin binding by membrane protein extracts was not affected by heat treatment but was almost completely abolished by Proteinase K treatment, suggesting the presence of thermostable protein receptors for haemin. The capsular polysaccharide also appears to play a minor role in binding of haemin. It was concluded that constitutive as well as inducible mechanisms of haemin binding occur in P. damselae subsp. piscicida. These mechanisms would rely mainly upon the direct interaction between the haemin molecules and surface-exposed outer membrane protein receptors.

Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association.

The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown. The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles. If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles. [14C]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism. The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA. However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle. Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [14C]haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm. Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.

Effect of environmental haemin upon the physiology and biochemistry of Prevotella intermedia R78.

The effect of environmental haemin on the physiology and biochemistry of Prevotella intermedia R78 grown in batch culture was assessed. Extent and rate of growth increased as the environmental haemin concentration was raised. In addition, cell morphology was predominantly cocco-bacillary when cultured in high haemin environments, while bacillary forms were prevalent in low haemin conditions (< 2.5 mumol l-1). Cells harvested from low haemin environments produced greater numbers of extracellular vesicles and greater amounts of peptidolytic activity, haemagglutinating potential and haemin binding activity when compared with cells harvested from high haemin conditions. The results of the present study indicate that aspects of the biochemistry and physiology of P. intermedia are influenced by changes in environmental haemin levels.

Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens

The antimicrobial effect of lactoferrin (apoLf) on the oral, black-pigmented anaerobes Porphyromonas gingivalis, Prevotella intermedia and P. nigrescens has been studied. ApoLf did not kill any of these species but it did inhibit the growth of P. gingivalis, while iron-saturated Lf (FeLf) had no effect. The other two species were unaffected by apoLf. This growth inhibitory effect of apoLf could not be explained on the basis of chelation of inorganic iron, since growth of P. gingivalis occurred in the presence of ethylenediamine di- o-hydroxyphenylacetic acid provided haemin was added. Both apoLf and FeLf reduced haemin uptake by all three species and caused the release of cell-bound haemin in a dose-dependent manner. In addition, haemin reduced the binding of both apoLf and FeLf to P. intermedia and P. nigrescens but stimulated the binding of Lf by P. gingivalis. These data suggest that Lf forms complexes with haemin in solution and competes for the binding of haemin to certain cell receptors, possibly lipopolysaccharides, but this is not sufficient to inhibit growth of the bacteria. P. gingivalis appears to bind Lf-haemin complexes, as well as haemin alone, which may facilitate access of the Lf to the outer and cytoplasmic membranes of P. gingivalis, so disrupting function.

Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

The antimicrobial effect of lactoferrin (apoLf) on the oral, black-pigmented anaerobes Porphyromonas gingivalis, Prevotella intermedia and P. nitrescens has been studied. ApoLf did not kill any of these species but it did inhibit the growth of P. gingivalis, while iron-saturated Lf (FeLf) had no effect. The other two species were unaffected by apoLf. This growth inhibitory effect of apoLf could not be explained on the basis of chelation of inorganic iron, since growth of P. gingivalis occurred in the presence of ethylenediamine di-o-hydroxyphenylacetic acid provided haemin was added. Both apoLf and FeLf reduced haemin uptake by all three species and caused the release of cell-bound haemin in a dose-dependent manner. In addition, haemin reduced the binding of both apoLf and FeLf to P. intermedia and P. nigrescens but stimulated the binding of Lf by P. gingivalis. These data suggest that Lf forms complexes with haemin in solution and competes for the binding of haemin to certain cell receptors, possibly lipopolysaccharides, but this is not sufficient to inhibit growth of the bacteria. P. gingivalis appears to bind Lf-haemin complexes, as well as haemin alone, which may facilitate access of the Lf to the outer and cytoplasmic membranes of P. gingivalis, so disrupting function.

Haemin binding as a factor in the virulence of Porphyromonas gingivalis

Haemin (iron protoporphyrin IX) is an essential growth factor for the periodontal pathogen, Porphyromonas gingivalis. Iron protoporphyrin IX (IPP IX) binding to the avirulent P. gingivalis beige variant (W50/BE1) and the black-pigmenting parent wild-type strain W50 was quantified. W50/BE1 grown in a chemostat under haemin excess-bound IPP IX under both oxidising and reducing conditions but with both lower capacity and avidity than either the haemin-limited- and haemin-excess-grown parent strain W50. Rosenthal plots for W50/BE1 indicated cooperative binding. W50/BE1 cells expressed a 32 kDa outer membrane haemin-binding protein when grown under conditions of haemin excess, and this strain might serve as a useful source from which to isolate this protein. The reduced IPP IX binding ability of W50/BE1 may be the rate-limiting factor for haem uptake and explain the reduced virulence and slower rate of pigmentation of this strain.

Haemin binding as a factor in the virulence of Porphyromonas gingivalis.

Haemin (iron protoporphyrin IX) is an essential growth factor for the periodontal pathogen. Porphyromonas gingivalis. Iron protoporphyrin IX (IPP IX) binding to the avirulent P. gingivalis beige variant (W50/BE1) and the black-pigmenting parent wild-type strain W50 was quantified. W50/BE1 grown in a chemostat under haemin excess-bound IPP IX under both oxidising and reducing conditions but with both lower capacity and avidity than either the haemin-limited- and haemin-excess-grown parent strain W50. Rosenthal plots for W50/BE1 indicated cooperative binding. W50/BE1 cells expressed a 32 kDa outer membrane haemin-binding protein when grown under conditions of haemin excess, and this strain might serve as a useful source from which to isolate this protein. The reduced IPP IX binding ability of W50/BE1 may be the rate-limiting factor for haem uptake and explain the reduced virulence and slower rate of pigmentation of this strain.

Kinetics of Congo-red Binding by Haemin-limited and Haemin-excess Cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.

Multiple effects of haemin binding on protease susceptibility of bovine serum albumin and a novel isolation procedure for its large fragment

Multiple effects of haemin binding on protease susceptibility of bovine serum albumin and a novel isolation procedure for its large fragment

Multiple effects of haemin binding on protease susceptibility of bovine serum albumin and a novel isolation procedure for its large fragment.

The effects of ligand binding on the proteolytic susceptibility of BSA were investigated. The rate for proteolytic digestion with either trypsin or chymotrypsin decreased in the presence of bilirubin and fatty acids, suggesting that overall albumin conformation is stabilized by these ligands. In contrast, haemin showed multiple effects on a proteolytic digestion pattern: the rate for the degradation of intact albumin greatly increased, but a large 45 kDa fragment accumulated during proteolytic digestion in the presence of this ligand. This unique fragmentation pattern allowed us to isolate the 45 kDa fragment at a high yield (about 30% on a molar basis) by one-step purification. Sequence analyses indicated that this fragment lies between residues Thr190 and Ala583, which constitutes domains II and III of the albumin molecule. Far-u.v. c.d. spectra strongly suggested that the secondary structure in the intact albumin is almost retained in the 45 kDa fragment. The isolated 45 kDa fragment showed haemin-binding ability, as evaluated by spectroscopic titration; upon re-digestion of the 45 kDa fragment, haemin showed strong protective effects. These results were consistent with the idea that haemin binding to BSA induces an increased protease susceptibility in the loop region between domains I and II, but in the overall conformation of domains II and III, a protease-resistant property.

Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-α- benzoyl- l- arginine-p- nitroanilide ( l-BAPNA) compared to haemin-limited cells. The specific activities against l-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.

Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.

Influence of the virulence plasmid and the Congo red reaction on the antimicrobial susceptibility of Yersinia species.

Seventeen strains of Yersinia enterocolitica and related species were examined for their susceptibility to a variety of antimicrobial agents. Twelve of the strains were clinical isolates which carried a virulence plasmid; five were food isolates which lacked plasmids. When grown on agar containing Congo red or haemin, each strain yielded pigmented (CR+) and non-pigmented (CR-) variants. CR+ bacteria of clinical origin were virulent, whereas CR+ food isolates and all CR- bacteria were avirulent. The susceptibility of CR+ and CR- bacteria to the following agents was compared: ampicillin, chloramphenicol, latamoxef, nalidixic acid, novobiocin, polymyxin B, rifampicin, streptomycin, tetracycline and tobramycin. The results showed that bacteria which bound Congo red were more susceptible to each drug examined than their CR- counterparts. This relationship held regardless of whether the CR+ bacteria possessed a virulence plasmid or not. The findings suggest that binding of antimicrobial agents by yersiniae may parallel binding of haemin and Congo red.

A spectroscopic study of the haemin--human-serum-albumin system.

The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron‐free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM−1. From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long‐chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open‐chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.

Immunoelectrophoretic studies of the beta1-haem-binding globulin (haemopexin) in hereditary haemolytic disorders.

NEALE et al.1 have demonstrated by paper electrophoresis a haem-binding globulin in the β-globulin fraction of human serum. Since then, several investigators examined this protein2–6, which was identified by immunoelectrophoresis with β1-B-globulin and called haemopexin7. On starch-gel electrophoresis the haem-binding protein was found to migrate with the fast α2-globulins8,9. Haemopexin is present in the perchloric acid-soluble fraction of serum7,10 from which Schultze et al.11 obtained a highly purified preparation by combined ammonium sulphate precipitation and zone electrophoresis. Haemopexin is a glycoprotein containing approximately 20 per cent carbohydrate and has a sedimentation rate of 4.8S corresponding to a molecular weight of approximately 80,000. The purified protein has a binding capacity of 15–20 mols haemin per mol haemopexin; evidence for binding of haemoglobin or cytochrome c could not be obtained. The haemin binding capacity of normal human serum is approximately 10 mg/100 ml. (refs. 2 and 9).