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.     

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.     

Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules

Numerous bacteria are able to use free and haemoprotein-bound haem as iron sources because of the action of small secreted proteins called haemophores. Haemophores have very high affinity for haem, and can therefore extract haem from the haem-carrier proteins and deliver it to the cells by means of specific cell surface receptors. Haem is then taken up and the empty haemophores are recycled. Here, we report a study of the regulation of the Serratia marcescens has operon which is involved in haemophore-dependent haem acquisition. We characterized two genes encoding proteins homologous to specific ECF sigma and antisigma factors. We showed that they regulate the synthesis of the haemophore-specific outer membrane receptor, HasR, by a signal transduction mechanism similar to the siderophore surface-signalling systems. We also showed the essential role of HasR itself in this process. Using haem-loaded and haem-free haemophore, we identified the stimulus for the HasR-mediated signal transduction as being the binding of the haem-loaded haemophore to HasR. Thus, unlike siderophore-uptake systems, in which the signalling molecule is the transported substrate itself, in the haemophore-dependent haem uptake system the inducer and the transported substrate are different compounds.     

Effect on ribonucleotide reductase of novel lipophilic iron chelators: the desferri-exochelins

Desferri-exochelins are siderophores secreted by Mycobacterium tuberculosis that are both lipid- and water-soluble and have a high binding affinity for iron. Desferri-exochelin 772SM inhibits DNA replication and ribonucleotide reductase activity at 10-fold less concentration than the lipid-insoluble iron chelator deferoxamine, which is currently in clinical use. Neither chelator can extract iron directly from ribonucleotide reductase. However, because of its lipid-solubility and high binding affinity, desferri-exochelin is able to enter cells rapidly and access intracellular iron, while deferoxamine has limited capacity to cross the cell membrane.     

Iron acquisition by Streptococcus species: An updated review

Streptococcus is a genus of spherical Gram-positive bacteria responsible for many cases of meningitis, bacterial pneumonia, endocarditis, erysipelas, and necro-tizing fasciitis. To survive in the host environment with limited free iron available, Streptococcus species have developed various mechanisms to uptake iron as an essential nutrient. They can directly extract the metal ions from host iron-containing proteins such as ferritin, transferrin, lactoferrin, and hemoproteins. Other iron-uptake strategies, which are broadly distributed in the strains, include the employment of specialized secreted hemophores to acquire heme and the usage of small molecules called siderophores as high-affinity ferric chelators. This review intends to discuss the most recent discoveries of these iron acquisition systems and their relevant regulators in Streptococcus species.     

Siderophore uptake in bacteria and the battle for iron with the host; a bird’s eye view

Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe³⁺). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.     

Emerging strategies in microbial haem capture

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.     

Interaction of lactoferrin and transferrins with the outer membrane of Bordetella pertussis

Bordetella pertussis was able to grow in vitro under conditions where the only iron present was bound to the iron-binding proteins ovotransferrin, transferrin or lactoferrin. Under these conditions the bacteria produced neither hydroxamate nor phenolate-catecholate siderophores to assist in the procurement of iron. Examination of B. pertussis outer-membrane preparations by SDS-PAGE and immunoblotting showed that the iron-binding protein ovotransferrin was bound directly to the bacterial surface. Assays of the binding of radiolabelled transferrin by the bacteria showed that the association was a specific process and that there was turnover of the bound proteins. Competitive binding assays indicated that lactoferrin could be bound in the same way. It is suggested that B. pertussis obtains iron directly from host iron-binding proteins during infection.     

Iron acquisition by Streptococcus species: An updated review

Streptococcus is a genus of spherical Gram-positive bacteria responsible for many cases of meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis. To survive in the host environment with limited free iron available, Streptococcus species have developed various mechanisms to uptake iron as an essential nutrient. They can directly extract the metal ions from host iron-containing proteins such as ferritin, transferrin, lactoferrin, and hemoproteins. Other iron-uptake strategies, which are broadly distributed in the strains, include the employment of specialized secreted hemophores to acquire heme and the usage of small molecules called siderophores as high-affinity ferric chelators. This review intends to discuss the most recent discoveries of these iron acquisition systems and their relevant regulators in Streptococcus species.     

Acquisition of iron from host sources by mesophilic Aeromonas species.

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.     

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.     

Iron and virulence in Shigella

Iron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high-affinity iron-binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron-acquisition systems, such as the ability to utilize haem-iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell-surface haem-binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.     

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.     

Shr of group A streptococcus is a new type of composite NEAT protein involved in sequestering haem from methaemoglobin

A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in haem acquisition and trafficking across the cell envelope of Gram‐positive bacteria. Group A streptococcus (GAS), a β‐haemolytic human pathogen, expresses a NEAT protein, Shr, which binds several haemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane‐anchored protein, with a unique N‐terminal domain (NTD) and two NEAT domains separated by a central leucine‐rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains haem in solution and furthermore reduces the haem iron; this is the first report of haem reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding haem, although they are missing a critical tyrosine residue found in the ligand‐binding pocket of other haem‐binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methaemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methaemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester haem directly from methaemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methaemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methaemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in haem uptake found in pyogenic streptococci and Clostridium novyi.     

Mechanisms of iron and haem transport by Listeria monocytogenes

Abstract

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.     

High affinity iron-uptake systems in Vibrio damsela: role in the acquisition of iron from transferrin

In this work, the high affinity iron-acquisition systems displayed by virulent and avirulent strains of Vibrio damsela have been investigated. This species is an autochthonous member of marine ecosystems that can behave as an opportunistic pathogen for fish and mammals. All strains tested (i) were able to grow under the restricted conditions imposed by the iron chelators transferrin (Tf) and EDDHA, (ii) secreted siderophores of hydroxamic type, other than aerobactin and desferal, that were able to stimulate the growth of the auxotroph mutant Arthrobacter flavescens JG9, and (iii) expressed common iron-regulated outer membrane proteins (IROMPs). No change in LPS patterns was observed in response to iron restriction. Results from the assays with transferrin suggest that these siderophores could be utilized to sequester iron from Tf, a protein for which no surface receptor was detected in any strain. In summary, the overall data demonstrate that V. damsela expresses siderophore-mediated iron-uptake systems. These systems are probably involved in the survival of the species in the different environments that it can colonize, i.e. water and several vertebrate hosts.     

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。     

War-Fe-re: iron at the core of fungal virulence and host immunity

Iron acquisition is a bona fide virulence determinant. The successful colonization of the mammalian host requires that microorganisms overcome the Fe aridity of this milieu in which the levels of circulating Fe are maintained exceedingly low both through the compartmentalization of this nutrient within cells as well as the tight binding of Fe to host circulating proteins and ligands. Microbes notoriously employ multiple strategies for high affinity Fe acquisition from the host that rely either on the expression of receptors for host Fe-binding proteins and ligands, its reduction by cell surface reductases or the utilization of siderophores, small organic molecules with very high affinity for Fe³⁺. This review will discuss the multiple mechanisms deployed by fungal pathogens in Fe acquisition focusing on the role of siderophore utilization in virulence as well as host immune strategies of iron withholding and emerging clinical evidence that human disorders of Fe homeostasis can act as modifiers of infectious disease.