Iron acquisition systems in the pathogenic Neisseria

Pathogenic neisseriae have a repertoire of high-affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron-binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobin-haptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron-siderophore) across the outer membrane is a TonB-dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor-based vaccines may be protective in humans.     

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism.

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.     

Intracellular distribution of haem after uptake by different receptors. Haem-haemopexin and haem-asialo-haemopexin.

How the interaction of haemopexin with two different receptors affects its subsequent metabolism and 'intracellular' haem transport was examined by using mesohaem-haemopexin and mesohaem-asialo-haemopexin. The physical properties of the two haem proteins, including their absorption and c.d. spectra, are similar. Binding studies in vitro showed that haem-asialo-haemopexin interacts with both the haemopexin-specific and galactose-specific receptors on liver plasma membranes, but that haem-haemopexin interacts only with the haemopexin receptor. In vivo haem-asialo-haemopexin rapidly interacts with the liver via the galactose-specific receptor, since the protein is extensively catabolized and uptake is blocked by asialofetuin. Haem iron from haem-asialo-haemopexin is not accumulated in the liver to the same extent as from intact haem-haemopexin, and the native sialylated protein is not proteolysed. Moreover, after fractionation of homogenized liver by using colloidal-silica gradients, liver-associated haem-haemopexin and haem-asialo-haemopexin produced distinctly different patterns for both protein and ligand, consistent with their uptake by two distinct receptors. These results demonstrate that the interaction of haemopexin with different receptors influences its subsequent metabolic fate and that haem iron from haem-haemopexin is efficiently conserved only if it enters the liver cell via the specific haemopexin receptor.     

Quelling the red menace: haem capture by bacteria

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed-function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface-exposed receptors dedicated to binding haem and haem-proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB-dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is presented.     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

Haem utilization in Vibrio cholerae involves multiple TonB-dependent haem receptors

Vibrio cholerae has multiple iron transport systems, one of which involves haem uptake through the outer membrane receptor HutA. A hutA mutant had only a slight defect in growth using haemin as the iron source, and we show here that V. cholerae encodes two additional TonB-dependent haem receptors, HutR and HasR. HutR has significant homology to HutA as well as to other outer membrane haem receptors. Membrane fractionation confirmed that HutR is present in the outer membrane. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. A hutA, hutR mutant was significantly impaired, but not completely defective, in the ability to use haemin as the sole iron source. HasR is most similar to the haemophore-utilizing haem receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three haem receptors was unable to use haemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2, but haemin transport through either receptor was more efficient in strains carrying the tonB1 system genes. In contrast, haemin uptake through HasR was TonB2 dependent. Efficient utilization of haemoglobin as an iron source required HutA and TonB1. The triple haem receptor mutant exhibited no defect in its ability to compete with its Vib- parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae used haem derived from marine invertebrate haemoglobins, suggesting that haem may be available to V. cholerae growing in the marine environment.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Haemophore functions revisited

Haem is the major iron source for bacteria that develop in higher organisms. In these hosts, bacteria have to cope with nutritional immunity imposed by the host, since haem and iron are tightly bound to carrier and storage proteins. Siderophores were the first recognized fighters in the battle for iron between bacteria and host. They are non-proteinaceus organic molecules having an extremely high affinity for Fe(3+) and able to extract it from host proteins. Haemophores, that display functional analogy with siderophores, were more recently discovered. They are a class of secreted proteins with a high affinity for haem; they are able to extract haem from host haemoproteins and deliver it to specific receptors that internalize haem. In the past few years, a wealth of data has accumulated on haem acquisition systems that are dependent on surface exposed/secreted bacterial proteins. They promote haem transfer from its initial source (in most cases, a eukaryotic haem binding protein) to the transporter that carries out the membrane crossing step. Here we review recent discoveries in this field, with particular emphasis on similar and dissimilar mechanisms in haemophores and siderophores, from the initial host source to the binding protein/receptor at the cell surface.     

Mechanisms of iron and haem transport by Listeria monocytogenes

Listeria monocytogenes, the causative agent of listeriosis, is a virulent foodborne Gram-positive bacterial pathogen, with 20-30% mortality. It has a broad ability to transport iron, either in the form of ferric siderophores, or by extracting it from mammalian iron binding proteins. In this review we focus on the mechanisms of ferric siderophore and haem transport into the listerial cell. Despite the fact that it does not synthesize siderophores, L. monocytogenes transports ferric siderophores in the wild environment by the actions of cytoplasmic membrane ABC-transporter systems. The bacterium acquires haem, on the other hand, by two mechanisms. At low (nanomolar) concentrations, sortase B-dependent, peptidoglycan-anchored proteins scavenge the iron porphyrin in human or animal tissues, and transfer it to the underlying ABC-transporters in the cytoplasmic membrane for uptake. At concentrations at or above 50 nM, however, haem transport becomes sortase-independent, and occurs by direct interactions of the iron porphyrin with the same ABC-transporter complexes. The architecture of the Gram-positive cell envelope plays a fundamental role in these mechanisms, and the haem acquisition abilities of L. monocytogenes are an element of its ability to cause infectious disease.     

Entamoeba histolytica secretes two haem-binding proteins to scavenge haem

Entamoeba histolytica is a human pathogen which can grow using different sources of iron such as free iron, lactoferrin, transferrin, ferritin or haemoglobin. In the present study, we found that E. histolytica was also capable of supporting its growth in the presence of haem as the sole iron supply. In addition, when trophozoites were maintained in cultures supplemented with haemoglobin as the only iron source, the haem was released and thus it was introduced into cells. Interestingly, the Ehhmbp26 and Ehhmbp45 proteins could be related to the mechanism of iron acquisition in this protozoan, since they were secreted to the medium under iron-starvation conditions, and presented higher binding affinity for haem than for haemoglobin. In addition, both proteins were unable to bind free iron or transferrin in the presence of haem. Taken together, our results suggest that Ehhmbp26 and Ehhmbp45 could function as haemophores, secreted by this parasite to facilitate the scavenging of haem from the host environment during the infective process.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Haem transport to the liver by haemopexin. Receptor-mediated uptake with recycling of the protein

Rat [(59)Fe]haem-(125)I-labelled haemopexin complexes (700pmol/rat) associate rapidly and exclusively with the liver after intravenous injection into anaesthetized rats. The two isotopes exhibit different patterns of accumulation. Liver (125)I-labelled haemopexin is maximum 10min after injection (20+/-4.9pmol/g of liver) and then declines by 2h to the low values (about 3pmol/g of liver) seen after injection of the apoprotein. In contrast, [(59)Fe]haem accumulates in the liver for at least 2h. Haemopexin undergoes no extensive proteolysis during 2h of haem transport as shown by precipitation with acid (98%) and specific antiserum (92%) and by electrophoresis. Moreover, only 1-2% of the dose is located in extrahepatic tissues, and there is no significant urinary excretion of either (125)I or (59)Fe. Hepatic uptake at 10min is saturable, reaching 200pmol of haemopexin/g of liver and 350pmol of haem/g of liver at a dose of 9nmol/rat, whereas uptake of the apoprotein is 3-5% of the dose. This suggests that the interaction of haem-haemopexin with the liver is a specific receptor-mediated process. The complex probably interacts via the protein moiety, since the haem analogues mesohaem and deuterohaem do not affect association of the protein with the liver but the species of haemopexin does. Increasing amounts of protein are associated with the liver 5min after injection in the order: human>rabbit>rat, and haem uptake is consistently increased. For both rat and rabbit haemopexin saturation is reached at the same concentration of protein, i.e. 180-200pmol/g of liver, indicating that the different protein species bind to a common receptor. We propose that haemopexin transports haem to the liver by a specific receptor-mediated process and then returns to the circulation.     

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 frpB1 gene of Helicobacter pylori is regulated by iron and encodes a membrane protein capable of binding haem and haemoglobin

FrpB1 is a novel membrane protein of Helicobacter pylori that is capable of binding both haem and haemoglobin but consistently shows more affinity for haem. The mRNA levels of frpB1 were repressed by iron and lightly modulated by haem or haemoglobin. The overexpression of the frpB1 gene supported cellular growth when haem or haemoglobin were supplied as the only iron source. Three-dimensional modelling revealed the presence of motifs necessary to bind either haem or haemoglobin. Our overall results support the idea that FrpB1 is a membrane protein of H. pylori that allows this pathogen to survive in the human stomach.     

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.     

Structural biology of bacterial iron uptake

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.