Heme acquisition by hemophores.

Bacterial hemophores are secreted to the extracellular medium, where they scavenge heme from various hemoproteins due to their higher affinity for this compound, and return it to their specific outer membrane receptor. HasR, the outer membrane receptor of the HasA hemophore, assumes multiple functions which require various energy levels. Binding of heme and, of heme-free or heme-loaded hemophores is energy-independent. Heme transfer from the holo-hemophore to the outer membrane receptor is also energy-independent. In contrast, heme transport and hemophore release require basal or high levels of TonB and proton motive force, respectively. In addition, HasR is a component of a signaling cascade, regulating expression of the has operon via specific sigma and anti-sigma factors encoded by genes clustered at the has operon. The signal is the heme landing on HasR in the presence of the hemophore in its apo form. The has system is the only system thus far characterized in which the anti-sigma factor is submitted to the same signaling cascade as the target operon. Specific autoregulation of the has system, combined with negative regulation by the Fur protein, permits bacterial adaptation to the available iron source. In the presence of a heme-loaded hemophore, inactive anti-sigma factor is accumulated and can be activated as soon as the heme source dries up. Hence, the has system, instead of being submitted to amplification like other systems regulated by sigma anti-sigma factors, functions by pulses triggered by heme availability.

Characterization of HasB, a Serratia marcescens TonB-like protein specifically involved in the haemophore-dependent haem acquisition system.

In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC-ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.

Folded HasA inhibits its own secretion through its ABC exporter.

Gram-negative bacterial proteins secreted by ABC exporters carry a secretion signal in their carboxylic extremities. This characteristic suggests that the polypeptide needs to be fully synthesized before it can be secreted and, therefore, presumably may fold at least in part before its secretion. We investigated the relationship between folding and secretion using HasA, a hemoprotein of Serratia marcescens secreted into the extracellular medium by a dedicated Has ABC exporter. We first demonstrated that when HasA is sequestered in the cytoplasm it can acquire its tertiary structure, as assessed from its capacity to bind heme. The cytoplasmic pool of HasA cannot be secreted and inhibits the secretion of newly synthesized molecules. HasA folding in the cytoplasm was independent of either its capacity to bind heme or the presence of SecB, although SecB is essential for HasA secretion. Our findings indicate a strong coupling between synthesis and secretion in the type I secretion pathway.

Haemophore-mediated bacterial haem transport: evidence for a common or overlapping site for haem-free and haem-loaded haemophore on its specific outer membrane receptor.

Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore-dependent haem acquisition pathway and alone allow free or haemoglobin-bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three-dimensional structure of the Serratia marcescens holo-haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Ndelta atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem-binding constants of the wild-type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore-dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem-free and haem-loaded HasASM proteins to HasRSM-producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A-Y75A competitively inhibits binding to the receptor of both holo-HasASM and apo-HasASM, showing that there is a unique or overlapping site on HasRSM for the apo- and holo-haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem-loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.

Functional characterization of the HasA(PF) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor.

Hemophores are secreted by several gram-negative bacteria (Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Yersinia pestis) and form a family of homologous proteins. Unlike the S. marcescens hemophore (HasA(SM)), the P. fluorescens hemophore HasA(PF) has an additional region of 12 residues located immediately upstream from the C-terminal secretion signal. We show that HasA(PF) undergoes a C-terminal cleavage which removes the last 21 residues when secreted from P. fluorescens and that only the processed form is able to deliver heme to the S. marcescens outer membrane hemophore-specific receptor, HasR(SM). Functional analysis of variants including those with an internal deletion of the extra C-terminal domain show that the secretion signal does not inhibit the biological activity, whereas the 12-amino-acid region located upstream does. This extra domain may inhibit the interaction of the hemophore with HasR(SM). To localize the hemophore regions involved in binding to HasR, chimeric HasA(PF)-HasA(SM) proteins were tested for biological activity. We show that residues 153 to 180 of HasA(PF) are necessary for its interaction with the receptor.

Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores.

The major mechanisms by which Gram-negative bacteria acquire heme from host heme-carrier proteins involve either direct binding to specific outer membrane receptors or release of bacterial hemophores that take up heme from host heme carriers and shuttle it back to specific receptors. The ability to interact with and remove heme from carrier proteins distinguishes heme from conceptually similar siderophore and vitamin B12 receptors. Recent genetic, biochemical and crystallization studies have started to unravel the mechanism and molecular interactions between heme-carrier proteins and components of bacterial heme assimilation systems.

Evidence for a novel heme-binding protein, HasAh, in Alzheimer disease.

Multiple lines of evidence indicate that oxidative stress is an integral component of the pathogenesis of Alzheimer disease (AD). The precipitating cause of such oxidative stress may be misregulated iron homeostasis because there are profound alterations in heme oxygenase-1 (HO-1), redox-active iron, and iron regulatory proteins. In this regard, HasA, a recently characterized bacterial protein involved in heme acquisition and iron metabolism, may also be important in the generation of reactive oxygen species (ROS) given its ability to bind heme and render iron available for free radical generation through the Fenton reaction. To study further the role of heme binding and iron metabolism in AD, we show an abnormal localization of anti-HasA to the neurofibrillary pathology of AD, but not in normal-appearing neurons in the brains of cases of AD or in age-matched controls. These results suggest the increased presence in AD of a HasA homologue or protein sharing a common epitope with HasA, which we term HasAh. We conclude that heme binding of HasAh is a potential source of free soluble iron and therefore toxic free radicals in AD and in aging. This furthers the evidence that redox-active iron and subsequent Fenton reaction generating reactive oxygen are critical factors in the pathogenesis of AD.

Interactions of HasA, a bacterial haemophore, with haemoglobin and with its outer membrane receptor HasR.

The major mechanism by which bacteria acquire free or haemoglobin-bound haem involves direct binding of haem to specific outer membrane receptors. Serratia marcescens and Pseudomonas aeruginosa have an alternative system, which involves an extracellular haemophore, HasA, that captures free or haemoglobin-bound haem and shuttles it to a specific cell surface outer membrane receptor, HasR. Both haem-free (apoprotein) and haem-loaded (holoprotein) HasA bind to HasR, evidence for direct protein-protein interactions between HasA and HasR. HasA binding to HasR takes place in a tonB mutant. TonB is thus required for a step subsequent to HasA binding.

The crystal structure of HasA, a hemophore secreted by Serratia marcescens.

Free iron availability is strongly limited in vertebrate hosts, making the iron acquisition by siderophores inappropriate. Pathogenic bacteria have developed various ways to use the host's iron from iron-containing proteins. Serratia marcescens can use the iron from hemoglobin through the secretion of a hemophore called HasA, which takes up the heme from hemoglobin and shuttles it to the receptor HasR, which in turn, releases heme into the bacterium. We report here the first crystal structure of such a hemophore, bound to a heme group at two different pH values and at a resolution of 1.9 A. The structure reveals a new original fold and suggests a hypothetical mechanism for both heme uptake and release.

NMR studies of the C-terminal secretion signal of the haem-binding protein, HasA.

HasA is a haem-binding protein which is secreted under iron-deficiency conditions by the gram-negative bacterium Serratia marcescens. It is a monomer of 19 kDa (187 residues) able to bind free haem as well as to capture it from haemoglobin. HasA delivers haem to a specific outer-membrane receptor HasR and allows the bacteria to grow in the absence of any other source of iron. It is secreted by a signal peptide-independent pathway which involves a C-terminal secretion signal and an ABC (ATP-binding cassette) transporter. The C-terminal region of the secretion signal containing the essential secretion motif is cleaved during or after the secretion process by proteases secreted by the bacteria. In this work, we study by 1H NMR the conformation of the C-terminal extremity of HasA in the whole protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environment. We identify a helical region followed by a random-coil C-terminus in the peptide-micelle complex and we show that in both the whole protein and the complex, the last 15 residues containing the motif essential for secretion are highly flexible and unstructured. This flexibility may be a prerequisite to the recognition of HasA by its ABC transporter. We determine the cleavage site of the C-terminal extremity of the protein and analyse the effect of the cleavage on the haem acquisition process.

Isolation and characterization of an extracellular haem-binding protein from Pseudomonas aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore.

The major mechanism by which bacteria acquire free or haemoglobin-bound haem involves direct binding to specific outer membrane receptors. Serratia marcescens also secretes a haem-binding protein, HasA, which functions as a haemophore that catches haem and shuttles it to a cell surface specific outer membrane receptor, HasR. We report the isolation and characterization of hasAp, a gene from Pseudomonas aeruginosa. HasAp is an iron-regulated extracellular haem-binding protein that shares about 50% identity with HasA. HasAp is required for P. aeruginosa utilization of haemoglobin iron. It can replace HasA for HasR-dependent haemoblobin acquisition in a system reconstituted in Escherichia coli. HasAp, like HasA, lacks a signal peptide and is secreted by an ABC transporter. These findings show that haemophore-dependent haem acquisition is not unique to S. marcescens.

The SecB chaperone is involved in the secretion of the Serratia marcescens HasA protein through an ABC transporter.

The secretion pathways of the heme-binding protein HasA from Serratia marcescens and of the metalloproteases A, B, C and G from Erwinia chrysanthemi have been reconstituted in Escherichia coli. They are secreted in a single step from the cytoplasm across both membranes of the Gram-negative envelope, after recognition of their specific C-terminal secretion signal by their cognate ABC transporter. We report strong evidence that both HasA and the metalloproteases bind the SecB chaperone involved in the export of several envelope proteins via the Sec pathway. We also show that the secretion of the HasA protein is strongly dependent upon SecB in the reconstituted system, whereas that of the proteases is not. HasA secretion in the original host is strongly inhibited by a protein known to interfere with E.coli SecB function. We propose that the proteins secreted by the ABC pathway may have to be unfolded for efficient secretion.

Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters.

Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters.

A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli.

The utilization by Serratia marcescens of heme bound to hemoglobin requires HasA, an extracellular heme-binding protein. This unique heme acquisition system was studied in an Escherichia coli hemA mutant that was a heme auxotroph. We identified a 92-kDa iron-regulated S. marcescens outer membrane protein, HasR, which alone enabled the E. coli hemA mutant to grow on heme or hemoglobin as a porphyrin source. The concomitant secretion of HasA by the HasR-producing hemA mutant greatly facilitates the acquisition of heme from hemoglobin. This is the first report of a synergy between an outer membrane protein and an extracellular heme-binding protein, HasA, acting as a heme carrier, which we termed a hemophore.

Purification and characterization of an extracellular heme-binding protein, HasA, involved in heme iron acquisition.

Many bacterial hemoproteins involved in heme acquisition have been isolated recently, comprising outer membrane receptors and extracellular heme-binding protein. The mechanisms by which these proteins extract heme have not been described up to now. One such protein, HasA, which can bind free heme as well as capture it from hemoglobin, is secreted by the Gram-negative bacteria Serratia marcescens under iron deficiency conditions. The fact that HasA does not present sequence similarities with other known hemoproteins suggests that it possesses a new type of heme binding site. This work describes the main physicochemical properties of HasA, essential for understanding its function. HasA is a monomer of 19 kDa that binds one b heme per molecule with high affinity. The electron paramagnetic resonance spectra indicate that the heme iron is in a low-spin ferric state and that the two iron axial ligands are His and His-. The low oxidation-reduction potential value (-550 mV vs standard hydrogen electrode) of the heme bound to HasA suggests that heme could be exposed to the solvent. According to circular dichroism data, the binding of heme does not seem to modify the conformation of HasA.

Protein secretion by Gram-negative bacterial ABC exporters--a review.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells is sec-independent and dedicated to proteins lacking an N-terminal signal peptide. Most of these proteins display a C-terminal secretion signal located in the last 60 amino acids (aa). Using one Erwinia chrysanthemi protease, PrtG, secreted by such a pathway it was shown that the smallest C-terminal sequence allowing efficient secretion contains the last 29 aa of PrtG and that low but significant secretion can be promoted by the last 15 aa of PrtG. Moreover, the extreme C-terminal motif, consisting of a negatively charged aa followed by several hydrophobic aa must be exposed and is conserved amongst many proteins following this pathway. This secretion system depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. These Gram-negative bacterial protein exporters are dedicated to the secretion of one or several closely related proteins belonging to the toxin, protease and lipase families. The genes encoding the three secretion proteins and the exoproteins are usually all linked, consistent with the specificity of the systems. Er. chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on protease and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three components' association was ordered and substrate binding was required for the formation of this multiprotein complex.

Protein secretion by gram-negative bacterial ABC exporters.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins: an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteinases B and C, and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on proteinase and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter/secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three component association was ordered and substrate binding was required for the formation of this multiprotein complex.