Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment.     

Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin

The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe²⁺ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.     

Metabolic flux of extracellular heme uptake in Pseudomonas aeruginosa is driven by the iron-regulated heme oxygenase (HemO)

Heme utilization by Pseudomonas aeruginosa involves several proteins required for internalization and degradation of heme. In the following report we provide the first direct in vivo evidence for the specific degradation of extracellular heme to biliverdin (BV) by the iron-regulated HemO. Moreover, through isotopic labeling ((13)C-heme) and electrospray ionization-MS analysis we have confirmed the regioselectivity and ratio of (13)C-δ and β-BV IX (70:30) is identical in vivo to that previously observed for the purified protein. Furthermore, the (13)C-BV IXδ and BV IXβ products are effluxed from the cell by an as yet unidentified transporter. Conversion of extracellular heme to BV is dependent solely on the iron-regulated HemO as evidenced by the lack of BV production in the P. aeruginosa hemO deletion strain. Complementation of P. aeruginosa ΔhemO with a plasmid expressing either the wild type HemO or α-regioselective HemO mutant restored extracellular heme uptake and degradation. In contrast deletion of the gene encoding the cytoplasmic heme-binding protein, PhuS, homologs of which have been proposed to be heme oxygenases, did not eliminate (13)C-BV IXδ and IXβ production. In conclusion the metabolic flux of extracellular heme as a source of iron is driven by the catalytic action of HemO.     

Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production

5-Aminolevulinic acid (ALA) synthase (ALAS) HemA from non-sulfur photosynthetic bacteria has been used for the ALA bioproduction, whereas the isoenzyme HemT/HemO is less studied and not used for ALA production. Two ALAS-encoding genes, hemA and hemO from Rhodopseudomonas palustris were cloned, purified and characterized. The ALASs had very high specific activity, 3.6 and 2.7 U/mg, respectively, and strong affinity for one of its substrates, succinyl-CoA, K _m with values of 11 and 4.4 μM, respectively. HemO retained up to 60 % maximum activity within a broad range of concentrations of hemin, while HemA kept only 20 % at 10 μM hemin. Escherichia coli overexpressing HemA or HemO produced 5.7 and 6.3 g ALA/l, respectively, in a 5 l bioreactor.     

Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa

The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.     

ShmR Is Essential for Utilization of Heme as a Nutritional Iron Source in Sinorhizobium 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.     

Degradation of heme in gram-negative bacteria: the product of the hemO gene of Neisseriae is a heme oxygenase

A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXalpha and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica, Neisseria subflava, Neisseria flava, Neisseria polysacchareae, Neisseria kochii, and Neisseria cinerea. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.     

Functional identification of HugZ, a heme oxygenase from Helicobacter pylori

Background

Iron is recognized as an important trace element, essential for most organisms including pathogenic bacteria. HugZ, a protein related to heme iron utilization, is involved in bacterial acquisition of iron from the host. We previously observed that a hugZ homologue is correlated with the adaptive colonization of Helicobacter pylori (H. pylori), a major gastro-enteric pathogen. However, its exact physiological role remains unclear.

Results

A gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits 66% similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6 × His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity analysis indicated that the recombinant HugZ protein can bind to hemin. Absorption spectroscopy at 411 nm further revealed a heme:HugZ binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. The biochemical and enzymatic characteristics agreed closely with those of Campylobacter jejuni Cj1613c protein, implying that hp0318 is a functional member of the HugZ family. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels.

Conclusion

These findings provide biochemical and genetic evidence that hugZ (hp0318) encodes a heme oxygenase involved in iron release/uptake in H. pylori.     

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.     

Role of clathrin‐mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non‐iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin‐mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin‐containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.     

Utilization of Heme as an Iron Source by Marine Alphaproteobacteria in the Roseobacter Clade

The bioavailability and utilization of porphyrin-bound iron, specifically heme, by marine microorganisms have rarely been examined. This study used Ruegeria sp. strain TrichCH4B as a model organism to study heme acquisition by a member of the Roseobacter clade. Analogs of known heme transporter proteins were found within the Ruegeria sp. TrichCH4B genome. The identified heme uptake and utilization system appears to be functional, as the heme genes were upregulated under iron stress, the bacterium could grow on ferric-porphyrin complexes as the sole iron source, and internalization of ⁵⁵ Fe from ferric protoporphyrin IX was observed. The potential ability to utilize heme in the Roseobacter clade appears to be common, as half of the isolates in the RoseoBase database were found to have a complete heme uptake system. A degenerate primer set was designed and successfully used to identify the putative heme oxygenase gene (hmus) in the roseobacter heme uptake system from diverse nonenriched marine environments. This study found that members of the Roseobacter clade are capable of utilizing heme as an iron source and that this capability may be present in all types of marine environments. The results of this study add a new perspective to the current picture of iron cycling in marine systems, whereby relatively refractory intracellular pools of heme-bound iron may be taken up quickly and directly reincorporated into living bacteria without previous degradation or the necessity of a siderophore intermediate.     

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.     

Hal Is a Bacillus anthracis Heme Acquisition Protein

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development.     

A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host''s body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7^−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.