Heme Utilization Research Articles

Bacterial heme-transport proteins and their heme-coordination modes

Efficient iron acquisition is critical for an invading microbe’s survival and virulence. Most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. Utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. Once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in order to release the iron. Some Gram-negative bacteria also secrete extracellular heme-binding proteins called hemophores, which function to sequester heme from the environment. The heme-transport genes are often genetically linked as gene clusters under Fur (ferric uptake regulator) regulation. This review discusses the gene clusters and proteins involved in bacterial heme acquisition, transport and processing processes, with special focus on the heme-coordination, protein structures and mechanisms underlying heme-transport.

Differential expression of Bordetella pertussis iron transport system genes during infection

Temporal expression patterns of the Bordetella pertussis alcaligin, enterobactin and haem iron acquisition systems were examined using alcA-, bfeA- and bhuR-tnpR recombinase fusion strains in a mouse respiratory infection model. The iron systems were differentially expressed in vivo, showing early induction of the alcaligin and enterobactin siderophore systems, and delayed induction of the haem system in a manner consistent with predicted changes in host iron source availability during infection. Previous mixed infection competition studies established the importance of alcaligin and haem utilization for B. pertussis in vivo growth and survival. In this study, the contribution of the enterobactin system to the fitness of B. pertussis was confirmed using wild-type and enterobactin receptor mutant strains in similar competition infection experiments. As a correlate to the in vivo expression studies of B. pertussis iron systems in mice, sera from uninfected and B. pertussis-infected human donors were screened for antibody reactivity with Bordetella iron-repressible cell envelope proteins. Pertussis patient sera recognized multiple iron-repressible proteins including the known outer membrane receptors for alcaligin, enterobactin and haem, supporting the hypothesis that B. pertussis is iron-starved and responds to the presence of diverse iron sources during natural infection.

ShmR Is Essential for Utilization of Heme as a Nutritional Iron Source inSinorhizobium meliloti

The bacterium Sinorhizobium meliloti is able to use heme as a nutritional iron source. Here, we show that the iron-regulated shmR gene encodes an outer membrane protein required for growth on heme. Furthermore, an shmR mutant is resistant to the toxic heme analog gallium protoporphyrin. Thus, the receptor protein of the heme transport system has been identified in S. meliloti.

Identification of iron regulated genes in the fish pathogen Aeromonas salmonicida subsp. salmonicida: Genetic diversity and evidence of conserved iron uptake systems

In this study we applied the Fur titration assay (FURTA) to isolate iron regulated genes in Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish. We have identified genes for siderophore biosynthesis and for ferri-siderophore transport, some of which have been previously described in this species. A gene for a hitherto uncharacterized functional hemin receptor HutE was identified, but its inactivation did not affect significantly the use of hemin as sole iron source, suggesting that redundant genes encoding hemin receptors exist in the A. salmonicida genome. Additional FURTA positive clones contained genes encoding a Flavoprotein, a transcriptional regulator protein, a DNA binding protein Fis, as well as genes encoding putative DapD and Flp/Fap-pilin proteins. A screening of gene distribution demonstrated that all the analyzed strains shared genes for siderophore biosynthesis and transport and for heme utilization, indicating that these two systems of iron acquisition are a conserved trait in this subspecies, whereas other genes are not common to all the isolates. Thus, the Fur regulon of A. salmonicida subsp. salmonicida includes both conserved and differentially occurring genes, suggesting that the genetic diversity within this fish pathogen might be higher than expected.

Role of PUG1 in Inducible Porphyrin and Heme Transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.

Plesiomonas shigelloides hugZ encodes an iron-regulated heme binding protein required for heme iron utilization

Plesiomonas shigelloides is an intestinal pathogen that uses heme as an iron source. The P. shigelloides heme utilization system consists of 10 genes, 7 of which permit heme transport and 3 of which are associated with utilization of heme as an iron source once it is inside the cell. The goal of this study was to examine hugZ, 1 of the 3 genes associated with utilization of heme iron. DPH8, a hugZ mutant, failed to grow to full cell density in media containing heme as the iron source, indicating that hugZ is required for heme iron utilization. Western blots using antibodies against Vibrio cholerae HutZ to detect the P. shigelloides HugZ indicated that hugZ encodes an iron-regulated cytoplasmic protein, which is absent in DPH8. A heme affinity bead assay performed on soluble protein fractions from P. shigelloides DPH8/pHUG24.5 (pHUG24.5 encodes hugZ) indicated that HugZ binds heme. Heme utilization was restored in DPH8 by hox1, which encodes the alpha-heme oxygenase from Synechocystis sp. strain PCC6803. However, HugZ did not exhibit alpha-heme oxygenase activity in an assay that detects the conversion of heme to the bilin functional group present in phycobiliproteins. These results do not rule out that HugZ exhibits another type of heme oxygenase activity not detected in the assay.

Functional Differences between Heme Permeases: Serratia marcescens HemTUV Permease Exhibits a Narrower Substrate Specificity (Restricted to Heme) Than the Escherichia coli DppABCDF Peptide-Heme Permease

Serratia marcescens hemTUV genes encoding a potential heme permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide permease responsible for heme, dipeptide, and delta-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp permease for ALA uptake, indicating that the HemTUV permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp permease, HemTUV permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific permeases different from the Dpp peptide/heme permease characterized in E. coli.

Porphyromonas gingivalis HmuY and HmuR: further characterization of a novel mechanism of heme utilization

Porphyromonas gingivalis HmuY is a putative heme-binding lipoprotein associated with the outer membrane. It is part of an operon together with a gene encoding an outer-membrane hemin utilization receptor (HmuR) and four uncharacterized genes. A similar operon organization was found in Bacteroides fragilis and B. thetaiotaomicron, with the former containing an additional HmuY homologue encoded upstream of the hmuR-like gene. In P. gingivalis cultured under heme-limited conditions, a approximately 1-kb hmuY transcript was produced at high levels along with some approximately 3.5 and approximately 9-kb transcripts. Compared with the parental strain, mutants deficient in hmuY or hmuR or hmuY-hmuR gene function grew more slowly and bound lower amounts of hemin and hemoglobin. Significantly, they grew more slowly or were unable to grow when human serum was used as the sole iron/heme source. Analysis of the hmu promoter showed that it is regulated by iron. The HmuY protein normally occurs as a homodimer, but in the presence of hemin it may form tetramers. These results show that HmuY may be the first reported member of a new class of proteins in Porphyromonas and Bacteroides species involved in heme utilization, a function being exerted in conjunction with HmuR, an outer-membrane heme transporter.

Inhibition of the Bacterial Heme Oxygenases from Pseudomonas aeruginosa and Neisseria meningitidis: Novel Antimicrobial Targets

The final step in heme utilization and iron acquisition in many pathogens is the oxidative cleavage of heme by heme oxygenase (HO), yielding iron, biliverdin, and carbon monoxide. Thus, the essential requirement for iron suggests that HO may provide a potential therapeutic target for antimicrobial drug development. Computer-aided drug design (CADD) combined with experimental assays identified small-molecule inhibitors of the Neisseria meningitidis HO (nm-HO). CADD virtual screening applied to 800 000 compounds identified 153 for biological assay. Several of the compounds were shown to have KD values in the micromolar range for nm-HO and the Pseudomonas aeruginosa HO (pa-HO). The compounds also inhibited the growth of P. aeruginosa as well as biliverdin formation in E. coli cells overexpressing nm-HO. Thus, CADD combined with experimental analysis has been used to identify novel inhibitors of the bacterial heme oxygenases that can cross the cell membrane and specifically inhibit HO activity.

Expression of hurP, a gene encoding a prospective site 2 protease, is essential for heme-dependent induction of bhuR in Bordetella bronchiseptica.

Expression of the hurIR bhuRSTUV heme utilization locus in Bordetella bronchiseptica is coordinately controlled by the global iron-dependent regulator Fur and the extracytoplasmic function sigma factor HurI. Activation of HurI requires transduction of a heme-dependent signal via HurI, HurR, and BhuR, a three-component heme-dependent regulatory system. In silico searches of the B. bronchiseptica genome to identify other genes that encode additional participants in this heme-dependent regulatory cascade revealed hurP, an open reading frame encoding a polypeptide with homology to (i) RseP, a site 2 protease (S2P) of Escherichia coli required for modifying the cytoplasmic membrane protein RseA, and (ii) YaeL, an S2P of Vibrio cholerae required for modification of the cytoplasmic membrane protein TcpP. A mutant of B. bronchiseptica defective for hurP was incapable of regulating expression of BhuR in a heme-dependent manner. Furthermore, the hurP mutant was unable to utilize hemin as a sole source of nutrient Fe. These defects in hemin utilization and heme-dependent induction of BhuR were restored when recombinant hurP (or recombinant rseP) was introduced into the mutant. Introduction of hurP into a yaeL mutant of V. cholerae also complemented its S2P defect. These data provided strong evidence that protease activity and cleavage site recognition was conserved in HurP, RseP, and YaeL. The data are consistent with a model in which HurP functionally modifies HurR, a sigma factor regulator that is essential for heme-dependent induction of bhuR.

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae: SUBSTRATE SPECIFICITY AND IDENTIFICATION OF THE HEME PROTEIN LIGANDS

Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.

Purification and Characterization of a New Heme‐Binding Protein (HBP59) from the Mutant Strain DJ35 of Azotobacter vinelandii

AbstractA new protein, an approximately 59‐kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Lipmann. After analysis by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry, the protein was identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra (ABS) in the reduced state exhibited three peaks at 421, 517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichroism (CD) spectrum of HBP59 in the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the Δε (CD extinction coefficient) values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, −0.65 and −1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully interacted with heme, its maximal ABS value and Soret CD intensity were increased by approximately 10‐fold compared with values before interaction. Therefore, it seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be involved in heme utilization or adhesion.

Crystal structure of AGR_C_4470p from Agrobacterium tumefaciens

We report here the crystal structure at 2.0 A resolution of the AGR_C_4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in addition to the ChuS protein.

Heme Inhibits the DNA Binding Properties of the Cytoplasmic Heme Binding Protein ofShigella dysenteriae(ShuS)

Heme uptake and utilization by pathogenic bacteria are critical for virulence and disease, since heme and heme proteins are a major source of iron within the host. Although the role of outer membrane heme receptors in this process has been extensively characterized at the genetic and biochemical level, the role of the cytoplasmic heme binding proteins is not yet clear. The Shigella dysenteriae cytoplasmic heme binding protein, ShuS, has previously been shown to promote utilization of heme as an iron source at low to moderate heme concentrations and to protect against heme toxicity at high heme concentrations. Herein, we provide evidence that ShuS of S. dysenteriae sequesters DNA non-sequence-specifically with a binding affinity of 3.6 microM as determined by fluorescence anisotropy studies. The ability to bind DNA was observed to be restricted to the apoprotein only. The molecular mass of the apo-ShuS-DNA complex was estimated to be approximately 700 kDa by size exclusion chromatography. Atomic force microscopy (AFM) revealed that apo-ShuS forms aggregates in the presence of DNA and provides a scaffolding matrix from which DNA is observed to loop outward. The AFM images of apo-ShuS-DNA complexes were strikingly similar to the AFM images of the stress-induced Escherichia coli protein, Dps, when complexed with DNA; however, unlike the Dps protein, ShuS failed to protect DNA against oxidative stress in vitro and in vivo. Since free heme can generate reactive oxygen species which are damaging to cellular DNA, the ability of ShuS to physically sequester DNA may provide a molecular basis for its role in preventing toxicity associated with high heme concentrations.

Comparative Analysis of hmuO Function and Expression in Corynebacterium Species

We have constructed defined deletions in the hmuO gene from Corynebacterium diphtheriae and Corynebacterium ulcerans and show that the C. ulcerans hmuO mutation results in a significant reduction in hemoglobin-iron utilization, whereas in C. diphtheriae strains, deletion of hmuO caused no or only partial reduction in the utilization of heme as an iron source. We also show that expression from the C. ulcerans hmuO promoter exhibits minimal regulation by iron and heme whereas transcription from the C. diphtheriae hmuO promoter shows both significant iron repression and heme-dependent activation. These findings indicate that variability in HmuO function and expression exists among Corynebacterium species.

Heme, an iron supply for vibrios pathogenic for fish

One of the main mechanisms present in gram-negative bacterial pathogens to obtain iron is the utilization of free heme or heme proteins from the host tissues. Vibrio anguillarum, the etiological agent of vibriosis in fish, and Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis, can acquire iron from free heme or heme-containing proteins present in the host tissues by a siderophore-independent mechanism. Similarly to other animal and human pathogens, the general mechanism for heme uptake in these two species consists in the presence of an outer membrane receptor that transport the heme molecule into the periplasm via a TonB-dependent process, and additional proteins that complete the transport of heme from the periplasm into the cell cytoplasm. Expression of heme uptake genes is iron-regulated at the transcriptional level by the repressor protein Fur. The heme uptake mechanisms are believed to contribute to virulence for fish. The existence of variability in the distribution of heme transport genes among strains suggests that gene inactivation and/or horizontal transfer might play a significant role in generating intraspecific genetic diversity.

The haem–haemopexin utilization gene cluster (hxuCBA) as a virulence factor of Haemophilus influenzae

Haemophilus influenzae has an absolute growth requirement for a porphyrin source, which can be supplied in vitro by haem, haemoglobin, or the haemoglobin-haptoglobin, haem-haemopexin and haem-albumin complexes. Utilization of the haem-haemopexin complex is known to be mediated by the products of the hxuCBA gene cluster. It was demonstrated that hxuC, but not hxuA or hxuB, is also essential for the utilization of haem from haem-albumin complexes. Mutants of the type b strain E1a lacking genes in the hxuCBA gene cluster were examined for their ability to cause bacteraemia in rat models of invasive disease. In 5-day-old rats, mutants in the hxuCBA genes yielded a significantly reduced bacteraemic titre compared to the wild-type strain. In addition, 5-day-old rats infected with the hxuCBA mutant strains exhibited significantly improved survival rates compared to those infected with the wild-type strain. Mutations in the haemoglobin/haemoglobin-haptoglobin-binding protein genes (hgps), either alone or in combination with the hxuCBA mutations, had no impact on virulence in 5-day-old rats. In 30-day-old rats infected with either the hxuCBA mutants or the wild-type strains, there was no significant difference in the ability to establish bacteraemia although bacterial titres were lower in rats infected with the hxuCBA mutants than in those infected with the wild-type strain. These age-related differences in the impact of mutations in the hxuCBA gene cluster may be related to changes in levels of host haem-binding proteins during development of the rat.

EspC, an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC), displays protease activity on human hemoglobin

Some enteropathogenic Escherichia coli (EPEC) strains, which are an important cause of diarrhea among infants, secrete a serine protease autotransporter protein called EspC. The pathogenic role of EspC upon EPEC infection is unknown. In this study, we demonstrated that purified EspC protein, obtained from supernatants of EPEC cultures, interacted with hemoglobin and degraded it. Moreover, we have shown that EspC is a hemin-binding protein. We hypothesized that hemoglobin proteolysis by EspC may contribute to the utilization of heme and hemoglobin iron for bacterial growth.

Heme Utilization in Campylobacter jejuni

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.

The hmuQ and hmuD genes from Bradyrhizobium japonicum encode heme-degrading enzymes.

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a K(d) value of 0.8 microM and showed spectral properties consistent with a heme oxygenase. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. The findings show that hmuQ and hmuD encode heme oxygenases and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to gram-positive pathogenic bacteria.