Philippe Delepelaire

Heme uptake across the outer membrane as revealed by crystal structures of the receptor-hemophore complex.

Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 Å from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor.

Bacteria capture iron from heme by keeping tetrapyrrol skeleton intact

Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.

The housekeeping dipeptide permease is the Escherichia coli heme transporter and functions with two optional peptide binding proteins.

Heme, a major iron source, is transported through the outer membrane of Gram-negative bacteria by specific heme/hemoprotein receptors and through the inner membrane by heme-specific, periplasmic, binding protein-dependent, ATP-binding cassette permeases. Escherichia coli K12 does not use exogenous heme, and no heme uptake genes have been identified. Nevertheless, a recombinant E. coli strain expressing just one foreign heme outer membrane receptor can use exogenous heme as an iron source. This result suggests either that heme might be able to cross the cytoplasmic membrane in the absence of specific carrier or that there is a functional inner membrane heme transporter. Here, we show that to use heme iron E. coli requires the dipeptide inner membrane ATP-binding cassette transporter (DppBCDF) and either of two periplasmic binding proteins: MppA, the l-alanyl-γ-d-glutamyl-meso-diaminopimelate binding protein, or DppA, the dipeptide binding protein. Thus, wild-type E. coli has a peptide/heme permease despite being unable to use exogenous heme. DppA, which shares sequence similarity with the Haemophilus influenzae heme-binding protein HbpA, and MppA are functional heme-binding proteins. Peptides compete with heme for binding both “in vitro” and “in vivo.”

The heme transfer from the soluble HasA hemophore to its membrane-bound receptor HasR is driven by protein-protein interaction from a high to a lower affinity binding site.

HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 × 1010 m-1). Heme is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 × 106 m-1 and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA·HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.

Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the gram-negative bacterium Erwinia chrysanthemi.

Erwinia chrysanthemi, a Gram‐negative phythopathogenic bacterium, secretes two related extracellular metalloproteases, B and C, which do not have N‐terminal signal sequences. The specific pathway by which they are secreted, which has been reconstituted in Escherichia coli, comprises three proteins — PrtD, PrtE and PrtF. Hybrid proteins containing segments of these proteins fused to the C‐terminus of protease B were purified and used to immunize rabbits. The antisera thus obtained were used to study the location and membrane topology of the three proteins. PrtD and PrtE were found to cofractionate almost exclusively with the cytoplasmic membrane, whereas PrtF was found to co‐fractionate mostly with the outer membrane. Proteinase K accessibility experiments as well as sequence data lead us to propose that PrtF has one or both ends exposed to the periplasm, that PrtE has one transmembrane segment with its amino‐terminus facing the cytoplasm and its C‐terminal hydrophilic domain exposed to the periplasm, and that PrtD has six transmembrane segments with its N‐terminus and its C‐terminal hydrophilic domain in the cytoplasm.

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 Nδ 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.

Free and Hemophore-Bound Heme Acquisitions through the Outer Membrane Receptor HasR Have Different Requirements for the TonB-ExbB-ExbD Complex

Many gram-negative bacteria have specific outer membrane receptors for free heme, hemoproteins, and hemophores. Heme is a major iron source and is taken up intact, whereas hemoproteins and hemophores are not transported: the iron-containing molecule has to be stripped off at the cell surface, with only the heme moiety being taken up. The Serratia marcescens hemophore-specific outer membrane receptor HasR can transport either heme itself or heme bound to the hemophore HasA. This second mechanism is much more efficient and requires a higher TonB-ExbB-ExbD (TonB complex) concentration than does free or hemoglobin-bound heme uptake. This requirement for more of the TonB complex is associated with a higher energy requirement. Indeed, the sensitivity of heme-hemophore uptake to the protonophore carbonyl cyanide m-chlorophenyl hydrazone is higher than that of heme uptake from hemoglobin. We show that a higher TonB complex concentration is required for hemophore dissociation from the receptor. This dissociation is concomitant with heme uptake. We propose that increasing the TonB complex concentration drives more energy to the outer membrane receptor and speeds up the release of empty hemophores, which, if they remained on receptors, would inhibit heme transport.

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low‐molecu‐lar‐weight, heat‐stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellu‐lar protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escher‐ichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 codons. The N‐terminal amino acid sequence of the purified inhibi‐tor was also determined. It indicated the existence of an amino‐terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.

Aerosolized beclomethasone in chronic bronchitis. Improved pulmonary function and diminished airway inflammation.

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 Nδ 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.

Haem release from haemopexin by HxuA allows Haemophilus influenzae to escape host nutritional immunity

Haemophilus influenzae is an obligate human commensal/pathogen. This haem auxotroph must acquire haem from its host to sustain aerobic growth. Haem–haemopexin complexes are one of the potential sources of haem for this microorganism. Haemopexin is a glycoprotein that binds haem with high affinity (subpicomolar Kd) and involved in haem recycling. HxuA, a cell surface protein, is the key to haem acquisition from haemopexin. In this study, we reconstituted a functional Hxu system from H. influenzae in Escherichia coli K‐12 that mediated active haem transport across the outer membrane from haem–haemopexin, in the presence of the inner membrane energy‐transducing TonB–ExbB–ExbD complex from H. influenzae. A secreted variant of HxuA, HxuAdm, was produced in E. coli. HxuAdm functionally complemented an hxuA mutant of H. influenzae for haem–haemopexin acquisition. HxuAdm interacted with haemopexin and haem–haemopexin, with which it formed high‐affinity, stoichiometric complexes. Following the interaction between haem–haemopexin and HxuAdm, haem was no longer bound to its initial high‐affinity site and became accessible to its cognate haem receptor, HxuC. HxuAdm and the HxuAdm–haemopexin complex do not appear to bind haem at detectable levels (affinities below 106 M−1). HxuA thus appears to ‘release’ haem from haem–haemopexin complexes and to prevent haem sequestering by haemopexin.