Characterization of a hemophore-like protein from Porphyromonas gingivalis

The porphyrin auxotrophic pathogen Porphyromonas gingivalis obtains the majority of essential iron and porphyrin from host hemoproteins. To achieve this, the organism expresses outer membrane gingipains containing cysteine proteinase domains linked to hemagglutinin domains. Heme mobilized in this way is taken up by P. gingivalis through a variety of potential portals where HmuY/HmuR of the hmu locus are best described. These receptors have relatively low binding affinities for heme. In this report, we describe a novel P. gingivalis protein, HusA, the product of PG2227, which rapidly bound heme with a high binding constant at equilibrium of 7 × 10(-10) M. HusA is both expressed on the outer membrane and released from the organism. Spectral analysis indicated an unusual pattern of binding where heme was ligated preferentially as a dimer. Further, the presence of dimeric heme induced protein dimer formation. Deletional inactivation of husA showed that expression of this moiety was essential for growth of P. gingivalis under conditions of heme limitation. This finding was in accord with the pronounced increase in gene expression levels for husA with progressive reduction of heme supplementation. Antibodies reactive against HusA were detected in patients with chronic periodontitis, suggesting that the protein is expressed during the course of infection by P. gingivalis. It is predicted that HusA efficiently sequesters heme from gingipains and fulfills the function of a high affinity hemophore-like protein to meet the heme requirement for growth of P. gingivalis during establishment of infection.     

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.     

The five near-iron transporter (NEAT) domain anthrax hemophore, IsdX2, scavenges heme from hemoglobin and transfers heme to the surface protein IsdC

Pathogenic bacteria require iron to replicate inside mammalian hosts. Recent studies indicate that heme acquisition in Gram-positive bacteria is mediated by proteins containing one or more near-iron transporter (NEAT) domains. Bacillus anthracis is a spore-forming, Gram-positive pathogen and the causative agent of anthrax disease. The rapid, extensive, and efficient replication of B. anthracis in host tissues makes this pathogen an excellent model organism for the study of bacterial heme acquisition. B. anthracis secretes two NEAT hemophores, IsdX1 and IsdX2. IsdX1 contains a single NEAT domain, whereas IsdX2 has five, a novel property among hemophores. To understand the functional significance of harboring multiple, non-identical NEAT domains, we purified each individual NEAT domain of IsdX2 as a GST fusion and analyzed the specific function of each domain as it relates to heme acquisition and transport. NEAT domains 1, 3, 4, and 5 all bind heme, with domain 5 having the highest affinity. All NEATs associate with hemoglobin, but only NEAT1 and -5 can extract heme from hemoglobin, seemingly by a specific and active process. NEAT1, -3, and -4 transfer heme to IsdC, a cell wall-anchored anthrax NEAT protein. These results indicate that IsdX2 has all the features required to acquire heme from the host and transport heme to the bacterial cell wall. Additionally, these results suggest that IsdX2 may accelerate iron import rates by acting as a "heme sponge" that enhances B. anthracis replication in iron-starved environments.     

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Several Gram-positive pathogenic bacteria employ near-iron transporter (NEAT) domains to acquire heme from hemoglobin during infection. However, the structural requirements and mechanism of action for NEAT-mediated heme extraction remains unknown. Bacillus anthracis exhibits a rapid growth rate during systemic infection, suggesting that the bacterium expresses efficient iron acquisition systems. To understand how B. anthracis acquires iron from heme sources, which account for 80% of mammalian iron stores, we investigated the properties of the five-NEAT domain hemophore IsdX2. Using a combination of bioinformatics and site-directed mutagenesis, we determined that the heme extraction properties of IsdX2 are dependent on an amino acid with an amide side chain within the 3₁₀-helix of the NEAT domain. Additionally, we used a spectroscopic analysis to show that IsdX2 NEAT domains only scavenge heme from methemoglobin (metHb) and that autoxidation of oxyhemoglobin to metHb must occur prior to extraction. We also report the crystal structures of NEAT5 wild type and a Q29T mutant and present surface plasmon resonance data that indicate that the loss of this amide side chain reduces the affinity of the NEAT domain for metHb. We propose a model whereby the amide side chain is first required to drive an interaction with metHb that destabilizes heme, which is subsequently extracted and coordinated in the aliphatic heme-binding environment of the NEAT domain. Because an amino acid with an amide side chain in this position is observed in NEAT domains of several genera of Gram-positive pathogenic bacteria, these results suggest that specific targeting of this or nearby residues may be an entry point for inhibitor development aimed at blocking bacterial iron acquisition during infection.     

An investigation of hemopexin redox properties by spectroelectrochemistry: biological relevance for heme uptake

Hemopexin (HPX) has two principal roles: it sequesters free heme in vivo for the purpose of preventing the toxic effects of this moiety, which is largely due to heme’s ability to catalyze free radical formation, and it transports heme intracellularly thus limiting its availability as an iron source for pathogens. Spectroelectrochemistry was used to determine the redox potential for heme and meso-heme (mH) when bound by HPX. At pH 7.2, the heme-HPX assembly exhibits E _1/2 values in the range 45–90 mV and the mH-HPX assembly in the range 5–55 mV, depending on environmental electrolyte identity. The E _1/2 value exhibits a 100 mV positive shift with a change in pH from 7.2 to 5.5 for mH-HPX, suggesting a single proton dependent equilibrium. The E _1/2 values for heme-HPX are more positive in the presence of NaCl than KCl indicating that Na⁺, as well as low pH (5.5) stabilizes ferro-heme-HPX. Furthermore, comparing KCl with K₂HPO₄, the chloride salt containing system has a lower potential, indicating that heme-HPX is easier to oxidize. These physical properties related to ferri-/ferro-heme reduction are both structurally and biologically relevant for heme release from HPX for transport and regulation of heme oxygenase expression. Consistent with this, when the acidification of endosomes is prevented by bafilomycin then heme oxygenase-1 induction by heme-HPX no longer occurs.     

革兰氏阴性菌血红素载体蛋白Hemophore的结构及作用机制

血红素作为宿主体内最丰富的铁离子来源,是致病菌营养竞争的主要目标,尤其对于血红素自身合成途径部分丧失的细菌。革兰氏阴性菌血红素转运系统由血红素载体蛋白(Hemophore)、外膜血红素受体、TonB-ExbB-ExbD复合物、ABC转运体等组成。Hemophore是存在于细菌细胞膜上或分泌到胞外环境中的一种蛋白。它能从宿主血红素结合蛋白中捕获血红素并将其传递给外膜受体。目前,在不同革兰氏阴性菌中已发现3种类型的Hemophore,分别是HasA、HxuA和HmuY型。本文将详细描述这3种Hemophore捕获血红素及与外膜受体相互作用的机制,以期为进一步研究其他细菌血红素载体蛋白的功能及作用机制奠定基础。     

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.