Iron, mycobacteria and tuberculosis

ARTICLE IN PRESS Tuberculosis (2004) 84, 110–130

Tuberculosis www.elsevierhealth.com/journals/tube

REVIEW

Iron, mycobacteria and tuberculosis Colin Ratledge Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK

KEYWORDS Bacterioferritin; Carboxymycobactin; Disease management; Exochelin; Inhibitors; Mycobactin; PAS; Salicylic acid; Siderophores; Tuberculosis

Summary The role of iron in the growth and metabolism of M. tuberculosis and other mycobacteria is discussed in relation to the acquisiton of iron from host sources, such as transferrin, lactoferrin and ferritin, and its subsequent assimilation and utilization by the bacteria. Key components involved in the acquisition of iron (as ferric ion) and its initial transport into the mycobacterial cell are extracellular iron binding agents (siderophores) which, in pathogenic mycobacteria, are the carboxymycobactins and, in saprophytic mycobacteria, are the exochelins. In both cases, iron may be transferred to an intra-envelope, short-term storage molecule, mycobactin. For transport across the cell membrane, a reductase is used which converts FeIIImycobactin to the FeII form. The ferrous ion, possibly complexed with salicylic acid, is then shuttled across the membrane either for direct incorporation into various porphyrins and apoproteins or, for storage of iron within the bacterial cytoplasm, bacterioferritin. The overall process of iron acquisition and its utilization is under very genetic tight control. The importance of iron in the virulence of mycobacteria is discussed in relationship to the development of tuberculosis. The management of dietary iron can therefore be influential in aiding the outcome of this disease. The role of the old anti-TB compound, p-aminosalicylate (PAS), is discussed in its action as an inhibitor of iron assimilation, together with the prospects of being able to synthesize further selective inhibitors of iron metabolism that may be useful as future chemotherapeutic agents. & 2003 Elsevier Ltd. All rights reserved.

Introduction This review seeks to describe the important role of iron in the development of tuberculosis. Iron, as we all recognize, is an important micronutrient for our own well being and conditions of anaemia, and complications arising from it, are all too well known when there has been an inadequate intake of iron in our diet. Equally, however, iron is also essential for growth of pathogenic bacteria, including Mycobacterium tuberculosis and M. leprae, the causative organisms, respectively, of tuberculosis and leprosy and which are the subject of this E-mail address: [email protected] (C. Ratledge).

monograph. Pathogenic mycobacteria, in order to grow and cause disease within a host, must therefore compete against the host for its supply of iron. Pathogenicity, however, is a multi-faceted phenomenon and, whilst it may be asserted that without the ability to gain iron from the host the pathogen cannot grow and cause disease, the converse is not necessarily true that other related organisms which may share the same mechanism for iron acquisition are equally likely to be pathogenic. There are many other defence mechanisms that have to be overcome before an invading organism is capable of becoming pathogenic. Iron acquisition is just one of many events that must occur for a bacterium to become a successful pathogen.

1472-9792/$ - see front matter & 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2003.08.012

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

With tuberculosis continuing to be the world’s worst bacterial disease in terms of the number of deaths per year, as well as the total number of sufferers, an important question to ask of any work with the mycobacteria is what relevance does this have to understanding the disease process itself, and then also to ask how can this knowledge be used to improve either the treatment of tuberculosis or even the design of novel anti-mycobacterial agents. I hope in the coverage of this topic that I will be able to show just how important iron is to the understanding of the development of this disease and how it has important implications for the manner in which patients suffering from tuberculosis should be treated with regard to their iron status. Finally, I hope to indicate that it is possible to consider the process of iron acquisition as a possible target for the design of novel chemotherapeutic agents. With the increased awareness of the continuing problem of tuberculosis on a world-wide basis and of the importance of iron in the metabolism of the mycobacteria, there has been a burgeoning of interest in this topic over the past 6 or 7 years. What was a topic of minor interest to relatively few research groups in the 1970s and 1980s has now attracted the attention of many experienced researchers and their teams. Aided by the genomic sequence of the tubercle bacillus being published in 1998, it has been possible for giant steps to be taken in solving many of the problems in mycobacterial iron metabolism that had been identified in the previous decades but which had been too complex to solve by conventional biochemical approaches. Many of the significant advances in our understanding of this subject have therefore occurred over the past five years. A number of reviews on iron metabolism in pathogenic bacteria in general1–5 and in mycobacteria in particular6–8 have appeared over the past few years, and the diligent reader is directed to these for further information and knowledge on the various issues that are discussed in the pages that follow. Other recent reviews of mycobacteria have also included details of iron metabolism9,10 which may also be usefully consulted.

Iron Iron is an essential nutrient for all living cells, except perhaps for the lactic acid bacteria. It is required for the functioning of a large number of enzymes where it may be attached as part of a heme nucleus or to various amino acids at or around

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the active site of the protein. Iron is thus involved with many enzymes as a vital co-factor and also in various oxygenases, hydroxylases and oxygen-transferring enzymes; it is also involved with the many cytochromes of the cells where it participates in crucial reactions involved with oxidative phosphorylation and energy production. In this role, iron is a key inorganic element for electron transport in that it can readily undergo oxidation and reduction reactions (i.e. redox reactions) in which iron oscillates between its oxidized form, ferric iron (FeIII, sometimes written as Fe3 þ ) and its reduced form, ferrous iron (FeII or Fe2 þ ), reactions involving the donation (oxidation) or acceptance (reduction) of an electron. In higher animals, iron is also involved in the process of oxygen transport in the form of hemoglobin and related proteins as well as in the storage of oxygen in the form of myoglobin. Without an adequate supply of iron, cells are unable to function correctly, cannot generate sufficient ATP to meet their energy requirements and will become moribund and may even die. Bacteria in their requirements for iron are no different to other cells and cell systems. Iron, however, is alone of all the nutrients required by cells in being virtually insoluble in water at pH values around neutrality. This is because iron is readily oxidized by O2 into its ferric form which then forms a highly insoluble ferric hydroxide. Older calculations usually give the solubility of the ferric ion at pH 7 as being about 1018 M. However, a more recent examination of this phenomenon has found that the major form of iron at pH 7 is not Fe(OH)3 but is instead Fe(OH)2þ 11 and which has a solubility of approx. 1.4  109 M. Although this is 109 M less than the previous value it is still too low for cells to be able to acquire iron from without some appropriate complexing molecule. However it should be noted that the solubility of FeIII increases by 103 M for every unit that the pH drops so that, for example, at pH 5 the solubility of FeIII is now 103 M making its accessibility entirely feasible without the need for any particular chelating or carrier molecules to those species of bacteria that can grow at this pH. For M. tuberculosis growing as an infectious agent within the phagocytic vacuoles of the macrophages (see below) the pH is between 6.1 and 6.512 at which values the maximum concentration of free FeIII is only between 1 and 10 ng/ml. In host tissues, this concentration will be considerably lowered, however, by its sequestration by the iron binding components therein (see next paragraph). This means that cells need a specific mechanism for acquiring iron from the environment (if the cell is a microorganism or a plant) or from its diet if it is an

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animal. For a pathogenic microorganism growing within a host animal, iron has to acquired from the animal itself and must therefore compete against the host supplies of iron to fulfil its own iron requirements. In animals, iron is taken up from various sources as part of the dietary assimilatory process. Of crucial importance in this role are two very different molecules. Transferrin, and the related molecule of lactoferrin or lactotransferrin, is a glycoprotein of some 80 kDa, that can bind with up to two atoms of FeIII and serves to transport iron around the various tissues of the body so that the many cells that require iron can remain supplied with iron. Iron is down-loaded from transferrin usually to the second important molecule, ferritin. Ferritin is composed of a shell of protein subunits that envelope a nucleus of iron which can comprise up to 5000 atoms of FeIII per molecule of ferritin. Ferritin then acts as an iron storage molecule and is found in almost every cell type in the body of higher animals. Iron is removed, probably by a reductive process, and donated to various apoproteins thereby turning them into holoproteins with the iron now giving functionality to that protein whatever it may be. In this way, hemes are synthesized to give hemoglobin, cytochromes, etc., and apoproteins become metalloproteins. Detailed reviews on the chemistry and biochemistry of both these molecules have appeared recently.13– 15 All these bodily forms of iron are capable of being used by pathogenic microorganisms when causing infection within a host (see Fig. 1). Upon infection with a pathogen, the animal body seeks to protect itself in numerous ways. With respect to iron, this is seen in the body restricting the amount of circulating iron in the body. Because invading bacteria must acquire iron from the host for their own growth, the body responds by restricting the amount of iron that is circulating in the body in the form of transferrin and also by restricting the assimilation of dietary iron.

Dietary Iron

Transferrin, Lactoferrin

Ferritin

Iron-proteins including heme proteins

Uptake mechanisms

Microbial Iron

Figure 1 Outline of the principal forms of iron within a host body that can serve as sources of iron for pathogenic microorganisms.

Although transferrin can bind two atoms of iron per molecule, the normal level is no more than 0.4 atoms/molecule and this becomes even lower upon infection. In this way, the body attempts to exert a nutritional deficiency upon the pathogen. Further manifestations of this nutritional withholding mechanism, is that the human patient may appear to be slightly anaemic but this is again because of the iron-withholding mechanism being put into operation. This aspect is discussed in more detail in the penultimate section of this review. Weinberg16,17 has extensively reviewed this important role of iron-withholding in disease control and draws specific attention to the dangers of trying to reverse apparent anaemia in patients by increasing iron in their diet with often drastic consequences. As pathogens are, by definition, successful in being able to cause disease, this means that they are able to succeed in their quest for iron and can in fact acquire it from the host tissue. However with many pathogens the withholding of iron by the host does limit the extent of the initial infection and allows the host to deal with small numbers of bacteria below their minimum infective dose entering the body. This vital role of iron in the establishment of infections can be seen from early experiments in which experimental animals were challenged with a sub-lethal dose of a pathogen and could survive this infection. However, if the pathogen was given along with iron being simultaneously administered to the animal, either in the diet or by intravenous injection, then invariably the pathogen would then overwhelm the animal and would quickly cause its death. The administration of iron to the animal was then the key to rampant bacterial proliferation, the establishment of infection and death of the host. Conversely, the withholding of iron from the bacteria prevents their multiplication and the onset of disease. There are indeed many pathogens, including fungi, protozoa, as well as bacteria, whose growth in bodily tissues and fluids in increased by the administration of iron (see 16). With respect to M. tuberculosis, Kochan18 showed that its growth in human serum was inhibited due to the iron-withholding action of transferrin. Barclay and Ratledge19 then showed that this bacteriostatic effect of serum, due to the presence of transferrin, on the growth of M. avium and M. paratuberculosis was reversed by adding either carboxymycobactin (then termed ‘exochelin’) or mycobactin, which are the bacteria’s own iron chelating compounds (see next section). Gobin and Horwitz,20 later confirmed the ability of carboxymycobactin to remove iron not only from transferrin but also, though more slowly, from

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

ferritin to promote the growth of M. tuberculosis. Douvas et al.21 showed that iron added to M. avium in macrophages enhanced bacterial growth and that serum and transferrin, but not holotransferrin (i.e. 98% replete with iron) or serum from which the transferrin had been removed, could inhibit bacterial multiplication. More recent confirmation of the role of transferrin in the prevention of bacterial multiplication in animals, and of the necessity to prevent M. tuberculosis gaining access to iron, has been provided by Lounis et al.22 These workers showed that multiplication of the tubercle bacillus in lungs and spleens of experimentally infected mice was significantly enhanced if iron was administered intraperitoneally. Without the iron, the number of bacilli remained low. Thus, the iron-withholding ability of transferrin appears of crucial importance for the prevention of bacillary multiplication and development of tuberculosis. But even with this iron withholding mechanism, pathogens do cause disease and therefore must be able to defeat the host’s attempts at nutritional deprivation. It is now known that bacteria have developed a number of ways in which they can gain iron from the host animal that they have invaded. These aspects have been reviewed numerous times with respect to pathogenic bacteria in general1,3,23,4,5 and to mycobacteria in particular.6–8 The most obvious way for iron acquisition would be for the bacteria to attack a particular source of iron and remove the iron when the compound to which it has been attached is degraded. In the simplest of terms this is what happens with hemolytic bacteria which are able to lyse red blood cells, degrade the hemoglobin therein and capture the iron for their own use. The iron may be transported into the bacterial cell still attached to the hemoglobin or may be released and then resolubilized by the bacteria secreting an iron-solubilizing molecule. The existence of specific iron-solubilizing molecules is now known to be a wide-spread phenomenon amongst many microorganisms, and not just pathogenic ones, as the acquisition of iron is still an acute problem for microorganisms living in other ecosystems.

Iron and mycobacteria The production of microbial iron solubilizing compounds has been widely reviewed by numerous authors and has been the subject of several monographs.1,3,24,23,4,5

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Although many different structures of the siderophores are known, they fall into three main categories: 1. Phenolic ring based structures in which hydroxy or dihydroxybenzoic acid is used as a chelating centre; 2. Hydroxamate structures in which the –N(OH)– CO– group is used to chelate iron often as an oN-hydroxy-N-acyl derivative of ornithine or lysine; 3. Mixed ligands of types 1 and 2. There are however some exceptions to these structures but these are rather rare. For readers requiring further information on the range and diversity of the siderophores, the reviews by Griffiths and Williams25 and by Winkelmann26,23 may be recommended. For mycobacteria, their siderophores are of the hydroxamate and mixed ligand types. Mycobacteria, together with some species of the related genera of Nocardia and Rhodocococcus, though are unique amongst microorganisms in that they synthesize both an intracellular molecule, termed mycobactin, and an extracellular siderophore which can vary in composition depending on whether the species is a pathogen or a saprophyte. These extracellular iron-chelating molecules are termed, respectively, carboxymycobactin and exochelin. The following sections discuss these molecules in further detail.

Mycobactins Mycobactins were first discovered by Alan Snow27 and his colleagues during the 1950s and 1960s. They were found as a result of a search for a growth factor for M. paratuberculosis which is the causative organism of Johne’s disease in cattle and which continues to cause serious problems today not only in cattle themselves but also in goats and possibly deer. M. paratuberculosis is a difficult-to-cultivate mycobacterium and early work had shown that the addition of heat-killed cells of M. tuberculosis to its culture medium led to its growth. The factor within M. tuberculosis which was found to be responsible for allowing M. paratuberculosis to grow was identified by Snow and co-workers as an ironbinding compound to which they gave the name ‘mycobactin’. Mycobactin is now known to be ubiquitous amongst mycobacteria though there are just a few exceptions. The molecular structures of the mycobactin from M. tuberculosis, termed mycobactin T, is shown in Fig. 2.

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* O * HO

(CH2)n

C

R

N

* OH

O * N

N H

serine

lysine

CH3

3-hydroxybutyric acid

* OH N

C O

O

salicylic acid

H N

O

* O

O

lysine

Figure 2 Structure of the mycobactin siderophores of M. tuberculosis. For mycobactin T, which occurs wholly intracellularly, n ¼ 19 (also 17) and R ¼ aCH3 ; for carboxymycobactin T, which occurs extracellularly, n ¼ 2  9 and R ¼ aCOOH: The three pairs of chelating groups responsible for the binding of Fe(III) are indicated by asterisks (*).

Mycobactins bind one atom of iron per molecule and have a very high affinity (described by its stability constant) for iron which is of the order of 1030 and it thus ideally suited to being able to hold iron from other competing molecules. It is also capable of removing iron from both transferrin and ferritin but, in practice, it does not appear to come into contact with these molecules due to its intracellular locationFsee below. The mycobactin structure (see Fig. 2) is modified with respect to the short methyl and ethyl sidechains on the core nucleus7,3,27 according to the species from which it is isolated. It is generally considered that each mycobacterial species produces its own unique variation on the basic mycobactin structure so that it would be technically possible to identify an unknown mycobacterium from its mycobactin structure. However, there are simpler techniques now available for speciation amongst mycobacteria and, because of the complexities in determining its molecular structure, mycobactin is no longer considered an appropriate molecule for such identification work. Mycobactins are virtually insoluble in water because of the long alkyl chain attached to the molecule (see Fig. 2). They must be extracted from mycobacterial cells using an appropriate organic solvent and ethanol is usually considered the most suitable for this purpose. Various methods for the purification and characterization of the molecule have been described28–33 with HPLC giving a clear, diagnostic fingerprint of the molecule that can be used for identification purposes but without involving any structural analysis.

Like all siderophores, mycobactin is produced in highest concentrations when the mycobacteria are grown with a deficiency of iron in the medium. In some cases the content of mycobactin has been recorded as being up to 10 per cent of the cell dry weight.7 However, it is not imagined that this reflects the situation for a pathogenic mycobacterium growing within an animal tissue as this would be far in excess of the requirements of the bacterium. Such high levels are considered to be produced only in the contrived, laboratory situation as the culture medium is deliberately made deficient in the quantity of iron being added to it and, once all the iron has been used, the cells then up-regulate mycobactin biosynthesis. However, being within a laboratory culture flask, the signal to stop synthesizing mycobactin, i.e. the arrival of soluble iron into the cells, is never attained and, consequently without a signal to repress mycobactin synthesis, the cells continue to synthesize mycobactin at the expense of other molecules. When mycobacteria are within a host tissue it is considered that the siderophores being produced by them are able to acquire iron from the host so that the signal to repress mycobactin synthesis (i.e. the arrival of iron) is received and thus the level of mycobactin and other components of the mycobacterial iron uptake system remain very low.7,34 It is noteworthy that attempts to isolate mycobactin from mycobacteria recovered from experimental infections have been singularly unsuccessful.35 This does not, though, then indicate that mycobactin is of no importance to the infective mycobacteria but only that the amounts of it may be small in the in vivo situation.

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

The unequivocal importance of mycobactin to the growth of M. tuberculosis has been recently established by showing that a mutant of M. tuberculosis lacking a gene from mycobactin biosynthesis had a considerably decreased ability to grow in human macrophages.36 The mutant, however, could grow in iron-replete medium, but not in iron-limiting medium in the laboratory. From this result, De Voss et al.36 were able to assert that iron acquisition from host sources of iron must be an essential pre-requisite for mycobacteria to grow and become pathogenic. Mycobactin, or its accompanying extracellular counterpart, carboxymycobactin (see below), or even both these molecules together, must therefore be crucial to enable the mycobacteria to grow in vivo. The mutant that De Voss et al.36 had used to show the necessity of iron acquisition for virulence was blocked at the earliest stage of mycobactin/carboxymycobactin biosynthesis that is at the condensation reaction between salicylate and serine (see Fig. 6), and thus the synthesis of both these siderophores would have ceased. The extent to which the bacterial cells experience a deficiency of iron when within the macrophages to trigger the synthesis of these siderophores is not known but it would appear from studies carried out on other components of the iron uptake system that the mycobacteria growing in vivo are probably on the cusp of iron deficiency and therefore are probably synthesizing small amounts of the siderophores.7,34 Up-regulation of genes involved in the regulation of iron metabolism (see below) has been shown to occur with M. tuberculosis within macrophages.37

Extracellular siderophores: exochelins and carboxymycobactins Early work on mycobacteria growing iron deficiently revealed that salicylic acid (2-hydroxybenzoic acid) was produced as an extracellular metabolite.38 Its possible role as a siderophore was initially suggested but, when the uptake of Fe(III)-salicylate was followed into M. smegmatis in the presence of phosphate, it was realized that the chelating power of salicylate was insufficient to hold the iron in solution in the presence of competing phosphate ions which readily formed insoluble ferric phosphate. No significant uptake of iron occurred from ferric salicylate under these conditions though it did do so when the phosphate was omitted from the uptake system. Although other workers have claimed that salicylic acid functions as a siderophore for the uptake of iron

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in other bacteria, notably pseudomonads where it also occurs as an extracellular metabolite in increased quantities during iron deficient growth (39,40 but also see 3), it is now clear from a theoretical study of ferric salicylate and its behaviour in the presence of phosphate ions at neutral pH values that salicylate cannot function as a siderophore.11 Arising from the realization that salicylate was not the extracellular siderophore of mycobacteria,41 an investigation was undertaken to identify what were the iron chelating molecules produced by mycobacteria.42,43 Two different types of molecule were recognized as siderophores in the mycobacteria; in the nonpathogenic bacteria the siderophore is a watersoluble entity that cannot be extracted into any organic solvent including partitioning into ethanol from saturated solutions of KCO3. In the pathogenic mycobacteria, the siderophore when converted into its ferric complex can be extracted into solvents such as chloroform.7 As the structures of these molecules were as then unknown, they were collectively called the exochelins42 even though it was appreciated that their molecular structures were likely to be entirely different. The structures of two of the exochelins, from M. smegmatis and from M. neoaurum, have now been elucidated44,45 and their structures are given in Fig. 3. They are, respectively, linear penta- and hexa-peptides in which hydroxamate groups form the major iron binding centres although, in the latter exochelin, there is a unique b-hydroxyhistidine residue which forms one of the three iron binding pairs of ligands in the molecule. In the pathogenic mycobacteria, the structures of the extracellular siderophores have now been elucidated in M. tuberculosis and M. avium46,47 and subsequently in M. bovis strains.48,49 They are variations of the mycobactin molecule (see Fig. 2) in which the long alkyl chain, which is responsible for the insolubility of mycobactin in aqueous solutions, is replaced by a shorter chain terminating in a carboxylic acid group. Because of the presence of this group, the name ‘carboxymycobactin’ has been given as an appropriate name in the first publication announcing this structure.47 This name therefore has precedence over other alternative names, such as exomycobactin50 that was rather regrettably suggested later but merely serves to confuse the nomenclature and has no validity because of its lack of priority. The name ‘carboxymycobactin’ now supersedes the older name of ‘exochelin’, which was given only as a working collective term for all mycobacterial extracellular siderophores irrespective of origin.

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Figure 3 The structure of two exochelins from non-pathogenic mycobacteria. A: exochelin MS from M. smegmatis; B: exochelin MN from M. neoaurum. (From 7 but see also 44,45.)

The name ‘exochelin’ is therefore retained only as a descriptor of the water-soluble siderophores of the saprophytic mycobacteria and serves to distinguish this group of siderophores from the ones from the pathogenic species. Whilst the exochelins are confined to the saprophytic mycobacteria, the carboxymycobactins are not confined to the pathogens but are also found in small amounts in the non-pathogenic species accompanying the exochelins themselves.51,52 As both a carboxymycobactin and an exochelin occur simultaneously in M. smegmatis52 and M. neoaurum (unpublished work) it is clearly inappropriate to retain the single name of ‘exochelins’ for both molecules and it is to be hoped that those researchers working with the carboxymycobactins now adopted this unambiguous nomenclature.

Solubilization and uptake of iron Both the exochelins and carboxymycobactins are able to remove iron from host sources of iron such as transferrin and ferritin as well as being able to solubilize it from inorganic sources such as ferric hydroxide or ferric phosphate. The mechanism by which the two siderophores are taken up into the mycobacterial cell though are different.

Uptake of the ferric exochelins into the saprophytic mycobacteria, as typified by M. smegmatis in which the majority of this work has been done, involves the transfer of the complete molecule, metal and ligand, and requires the input of metabolic energy as the process is affected by energy poisons and uncouplers of oxidative phosphorylation.53 Various proteins are now known to be involved in the uptake process,54,55,50 which have been deduced by identification of the corresponding genes, as well as a putative exochelin receptor molecule of molecular size 29 kDa56 isolated by affinity chromatography of envelope proteins from M. smegmatis.57 These proteins may be involved in the uptake process in the manner suggested by Pavelka58Fsee Fig. 4Fin which uptake of ferri-exochelin, after recognition by its specific receptor,56 is taken across the cell envelope, possibly in association with the FxuD protein, and is then transferred through the cytoplasmic membrane involving the participation of at least three proteins: FxuA, FxuB and FxuC (Fxu ¼ ferriexochelin uptake). These three proteins share amino acid sequence homology with the proteins, FepG, FepC and FepD, respectively, that are involved in the uptake of ferri-enterochelin into E. coli.54 The release of iron occurs on the cytoplasmic side of the membrane and will almost certainly involve reduction of the ferric iron to ferrous iron involving an appropriate reductase.

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

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Envelope

Extracellular

Membrane

Cytoplasm

Mycobactin iron overspill FxuC FxuB Ferri-exochelin

Rec

ATP NAD(P)H

FxuD FxuA FxuB

ADP Reductase ADP

Fe (III)

Exochelin

Fe (II)

Exit

(see also Fig 5) ATP

Figure 4 Proposed mechanism for ferri-exochelin uptake into M. smegmatis (adapted from 58 and 7). Exochelin is able to acquire iron from sources such as transferrin and ferritin (see Fig. 1). It is recognized at the cell surface by a receptor protein (Rec) and the complete ferri-exochelin is then taken across the envelope, possibly in conjunction with FxuD, and across the membrane via the FxuA, FxuB, FxuC system. The reductase for the release of iron (as FeII) may possible be associated with membrane itself (see Fig. 5). The Fe(II) then can be incorporated into a variety of apoproteins including bacterioferritin. If acceptor molecules for the Fe(II) are not available, Fe(III) will be sequestered from ferriexochelin by mycobactin in a process not involving the input of metabolic energy. Iron will be released from mycobactin as shown in Fig. 5.

[Ferri-mycobactin reductase, which may represent a non-specific NAD(P)-dependent siderophore reductase, is discussed below.] The exochelin, after releasing its iron into the cytoplasm, is transferred back into the extracellular environment of the cells using a specific exiting protein, ExiT,50 possibly operating in conjunction with other proteins and probably involving the input of energy.58 It is likely that if ferri-exochelin is not processed immediately by the cells, because of the inability of the cells to assimilate the iron intracellularly, then its proximity to mycobactin in the cell envelope will allow mycobactin to take the iron from exochelin. Thus ferri-exochelin will have a finite half-life in the envelope: it either donates the iron into the cytoplasm or the iron is removed from it by mycobactin. Mycobactin, see below, then serves as a store of iron in the cell envelope. The uptake of carboxymycobactin, the siderophore of the pathogenic mycobacteria, is taken up by a process which is not energy-linked as it was unaffected by energy poisons and uncouplers of ATP biosynthesis.59 It is thus distinct from the active transport system functioning for the uptake of ferri-exochelin. Although nothing substantive is known about the mechanism of this energy-independent process, it could involve the participation of a porin protein such as those described in M. smegmatis and M. chelonae60,61 and which also may occur in M. tuberculosis.10 The maximum diameter

of a molecule that can pass through the porin of M. chelonae is about 2.2 nm; the X-ray crystal structure of ferri-mycobactin indicates it to be ‘‘roughly spherical’’ with an effective diameter of about 1.1–1.4 nm.62 The size of carboxymycobactin would be similar to that of mycobactin and thus could be taken up by facilitated diffusion, as originally suggested,59 in a porin-mediated manner. Calder and Horwitz63 have identified two ironregulated proteins from M. tuberculosis that they have suggested as being involved in the uptake of ferri-carboxymycobactin. The two proteins, Irp10 and Mta72, by their close homology to metaltransporting P-type ATPases, could then function as a two-component metal transport system. However, this would imply that ATP is involved in the process but the prior results of Stephenson and Ratledge59 would suggest that ATP was not involved in this process. Thus, the role and function of Irp10 and Mta72 proteins may not be wholly analogous to what happens in E. coli, though clearly more work is needed to elucidate the exact mechanism of ferri-carboxymycobactin uptake. Iron could be released from the carboxymycobactin by the same reductase mechanism that is used with mycobactin (see below); however, it would also seem that iron should be able to be transferred from carboxymycobactin to mycobactin in keeping with the role of mycobactin as a intraenvelope store of iron (see Fig. 5). Gobin and

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Extracellular

Envelope

Membrane

Cytoplasm

? Irp10 & Mta72

Fe (III)

Carboxymycobactin

NAD(P)H

protoheme iron proteins bacterioferritin

salicylate? Porin Ferri-carboxymycobactin

Reductase Porin

Fe (II) salicylate? excess iron Mycobactin

NAD(P)+

protoporphyrin apoproteins bacterioferritin

Figure 5 Proposed mechanism for uptake of iron as mediated by carboxymycobactin. Uptake is via a facilitated diffusion mechanism59 possibly involving a porin10,60,61 which may funnel the ferri-carboxymycobactin across the envelope and directly to a ferri-reductase. This process, either at the membrane site of within the cytoplasm, may involve two other proteins, Irp10 and Mta72, suggested as being involved as a two-component metal transport system63 though where these fit into the scheme is uncertain. As with uptake mediated by exochelin (see Fig. 4), excess iron would be transferred to mycobactin for the intra-envelope storage of iron. Iron would be released from mycobactin by the same reductase.

Horwitz20 have indeed shown that an exchange of iron occurs from ferri-carboxymycobactin into the mycobactin within the cell envelope of M. tuberculosis even though both molecules will have exactly the same binding affinities for iron. Simple exchange of iron between the two molecules will occur and will be in favour of it going from carboxymycobactin to mycobactin as the latter will be at a greater concentration within the envelope than carboxymycobactin entering the cell (see Fig. 5). The exchange reaction need not necessarily be a rapid one and, indeed, in the experiment conducted by Gobin and Horwitz20 an incubation of 3 h was allowed to show the exchange of iron. This slow rate will be in keeping with the rate of slow growth rate of the pathogenic mycobacteria. The demand for iron by the developing cells will therefore be more than matched by the rate of exchange of iron from carboxymycobactin to mycobactin. A proposed scheme for the uptake of iron as mediated by carboxymycobactin is shown in Fig. 5.

Reduction of mycobactin (and carboxymycobactin) The mechanism of the release of iron from mycobactin is by a ferric mycobactin reductase in which the ferric iron is reduced in the presence of NAD(P)H to ferrous iron.64–66 It is likely that ferric

carboxymycobactin will be reduced similarly. Fe(II) has little affinity for mycobactin and can therefore be removed from mycobactin and inserted into appropriate receptor molecules. For the biosynthesis of hemes this is via ferrochelatase which has recently been described in M. tuberculosis.67 The exact mechanism of iron transfer is not understood although it has been shown that salicylate can function as an acceptor of Fe(II) after reduction of ferri-mycobactin65 but this requires further substantiation in view of more recent work (J. Chipperfield and C. Ratledge, unpublished work) that suggests that salicylate and Fe(II) may not form a sufficiently stable complex at pH 7 for it to act as a ‘shuttle’ compound between mycobactin and protoporphyrin IX, which is the preferred substrate for ferrochelatase.67 However, some means of transferring the iron across the cytoplasmic membrane is essential because of the spatial difference in location of mycobactin and enzymes within the cytoplasm that are responsible for the synthesis of iron-containing proteins. It should be appreciated that if mycobactin, ferri-mycobactin reductase and, say, ferrochelatase (see Fig. 5) should form an aggregate, perhaps associated in whole or in part with the cytoplasmic membrane, then transfer of Fe(II) within the aggregate would essentially be in a non-aqueous environment. Under such conditions ferro-salicylate may be sufficiently stable to function as the transfer molecule.

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

Why mycobactin is necessary as well as an extracellular siderophore The question may be asked why mycobacteria require two siderophores: mycobactin as a wholly intracellular molecule and carboxymycobactin (or the equivalent exochelin in the non-pathogen) as an extracellular iron-solubilizing agent. Surely one extracellular siderophore would be sufficient to solubilize iron and transport it into the cell as has been found in almost all other pathogens. If mycobactin were redundant, why have the mycobacteria not evolved to lose this non-essential biosynthetic activity? We may surmise, somewhat tautologically, that as the molecule occurs, it must have a functional role. The answer to this seeming duplication of siderophores possibly lies in the ability of mycobacteria to repress porphyrin biosynthesis when the supply of iron becomes limiting to growth (68 R. Barclay and C. Ratledge, unpublished work). Under such conditions, without porphyrins being available within the cell, if iron were to become suddenly available, the cells would not have the precursor molecules to synthesize heme and the various heme-containing molecules. Iron would therefore have to remain unassimilated or, if it were taken up without acceptor molecules being present, it could be highly injurious to the bacteria. As mycobacteria may be unique for their ability to repress porphyrin biosynthesis when iron is not available, this then means that they need a mechanism to store iron in a form that would subsequently allow its gradual uptake into the cell. This role is then fulfilled by mycobactin. Mycobactin is found within the cell envelope of the mycobacteria and appears to be located physically next to the cytoplasmic membrane but is not part of the membrane structure itself7,69 nor is it within the cytoplasm of the cell. When iron becomes available to the bacteria after a period of deprivation, the signal to de-repress biosynthesis of a variety of molecules, including that of porphyrins, is now given so that their synthesis begins and they gradually become available within the mycobacterial cell. Under such conditions, iron is mobilized from mycobactin in a controlled manner and balances the requirement for iron by the newly synthesized molecules. Iron is thus considered to be held by mycobactin as a temporary, but important, store of iron so that its premature and unwanted release into the cytoplasm is prevented. Again, as iron becomes generally available to the mycobacterial cell, a further molecule involved in iron storage is synthesized. This is bacterioferritin.70 Bacteriofer-

119 ritin is related to the human ferritin molecule14 but is synthesized only when iron is in excess and the bacteria then need to hold iron in a form other than mycobactin whose synthesis, of course, becomes repressed under these conditions. Bacterioferritin, unlike mycobactin, is located within the cytoplasm of the mycobacterial cell and can serve to donate iron as it is required to all manner of iron-requiring proteins as they become synthesized.

Biosynthesis of exochelin, carboxymycobactin and mycobactin Exochelin MS Genes for the synthesis of exochelin MS (see Fig. 3) have been identified in M. smegmatis54,55,50 and designated as fxbA, B and C (fxb ¼ ferriexochelin biosynthesis). The first gene was found54 to code for the N-formyltransferase that is responsible for the attachment of the terminal formyl group to the pentapeptide (see Fig. 3). Subsequently, the other two genes were identified50 as coding for proteins that were similar to other proteins known to be involved in the non-ribosomal synthesis of various small peptides. These two proteins are both large: FxbB is 257 kDa and FxbC is 497 kDa55 and thus, presumptively, as no other genes have been identified, would be responsible for the complete assemblage of the exochelin molecule by sequential attachment of ornithine to b-alanine, then to ornithine and on to the attachments of threonine and the final ornithine (see Fig. 3). Although the mechanism of this process is not known in any detail, by analogy to other nonribosomal peptide synthetases, an ATP would be needed to activate each amino acid and, indeed, six ATP-binding domains have been identified between the two proteinsFtwo on the B protein and four on the C protein.55 This, though, is somewhat puzzling as exochelin MS consists of only five amino acids (Fig. 3) and has led to the authors suggesting that the exochelin may in fact be synthesized initially as a hexapeptide; possibly the sixth (and unknown) amino acid might be displaced by the incoming formyl group or, as suggested by the authors, the hexapeptide is unstable. In addition to the ATP binding domains, Yu et al.55 showed that there were three epimerase domains within the two proteins to epimerize the L-(S)amino acids to the corresponding R stereoisomers: thus there would be separate domains for the epimerization of each of the two ornithine residues

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that need to be converted and the third domain would be for the threonine residue. No studies on the biosynthesis of exochelin MN have been done in spite of it showing several unique features for a siderophore.

They were aided in this study by there being a close similarity in the proteins involved in mycobactin synthesis to those involved in the biosynthesis of the siderophore from Yersinia pestis, yersiniabactin (72,73 and see also 3). The genes identified from M. tuberculosis were mbtA to J with two other genes coding for phosphopantetheinyl transferases (ppT and acpS).71 The proteins and their proposed activities arising from each of these 12 genes have been listed by Quadri et al.71 The projected biosynthetic sequence then followed the predicted pathway (see 27) of assembly of mycobactin starting with the synthesis of salicylic acid from isochorismic acid (see also 74) going through the addition of the various amino acids to the final addition of the long alkyl chain to the molecule. These reactions are shown in Fig. 6 and are also discussed and described in greater detail by De Voss et al.6 with accompanying structural formulae of the intermediates. Of key interest is the manner in which both mycobactin and carboxymycobactin, which share the common nucleus (see Fig. 2), will be synthesized as separate entities. The gene cluster as described applies equally to both mycobactin and

Carboxymycobactin and mycobactin The sequencing of the genome of M. tuberculosis by Stewart Cole and his colleagues in 1998 opened up a myriad of opportunities to investigate the metabolism of the mycobacteria in ways that had hitherto been unavailable to researchers. One of the first groups to take advantage of this was that of Christopher Walsh who identified the complete gene cluster in the M. tuberculosis genome that was responsible for encoding the proteins involved in the biosynthesis of mycobactin and carboxymycobactin.71 Without needing to carry out a complete array of biochemical assays of the individual enzymes, (indeed no specific biochemical assay for any single enzyme was done) it was possible to assign functional roles to the proteins that were deduced to be involved from the gene sequences.

salicylic acid

1) shikimic acid

3) salicyloyl

4)

MbtB

salicyloyl

MbtB

2) salicylic acid + ATP +

salicyloyl-seryl (SS)

+ serine + ATP

ε-RHN-lysine + ATP +

ε-RHN–lysyl

MbtE

5) SS- MbtB + ε-RHN-lysyl

MbtE

8)

9) SSLB

MbtF

ε-RHN-lysyl

+ ATP

MbtE + ε-RHN-lysyl

MbtF

MbtF

MbtE

+ MbtB

MbtC/D

SSL-O-(3-butyryl) (SSLB)

MbtC/D

AcT1 10) SSLBL

MbtE

3-hydroxybutyryl

MbtE + 3-hydroxybutyryl

-RHN-lysine +

MbtB

SS-ε-RHN-lysyl (SSL)

6) acetyl-CoA + malonyl-CoA 7) SSL

MbtB

MbtE

+ MbtC/D

MbtF

SSLB-(ε-RHN)-lysyl (SSLBL)

MbtF

+ MbtE

carboxymycobactin

[X] AcT2

mycobactin

Figure 6 Proposed scheme for the biosynthesis of mycobactin T and carboxymycobactin T (adapted from 6) as deduced from the genomic region of M. tuberculosis71. All reactions after the formation of salicylic acid (rn1) involve the attachment of the intermediates to various carrier proteins: MbtB, MbtE, MbtC/D and MbtF. The final intermediate arising from rn 9 must undergo cyclization of the terminal lysine group, together with hydroxylation of its eN atom (refer to Fig. 2), and also hydroxylation of the eN atom of the central lysine group coupled with acylation of this eN atom. In order to form both mycobactin and carboxymycobactin a common intermediate [X] may be envisaged which is acted upon by two separate acyltransferases (AcT1 and AcT2). However, the formation of carboxymycobactin may be more complex than is shown by this simple acyltransferase reaction (see text). [It should be noted that none of the reactions indicated (except for reactions 1 and 2) have been demonstrated biochemically.]

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

carboxymycobactin but there must be a common intermediate (‘pre-mycobactin’, X in Fig. 6) beyond which the two pathways will separate. As the final step in both syntheses will probably be the introduction of the long acyl chain on the central lysine residue (see Fig. 2) it may be suggested that, initially, the pre-mycobactin nucleus is synthesized with an acetyl group attached to both N6 atoms of the lysine residues (R ¼ aCOCH3 in Fig. 6). Both acetyl groups will remain until the hydroxylation of both lysyl-N6 atoms has occurred (coded for by MbtG, the lysine N-oxygenase) to give the two hydroxamate groups. The acetyl group on the terminal lysine residue will be lost upon cyclization of the hydroxylysine (possibly by the MbtJ protein described as an acetyl hydrolase) but the acetyl group on the central hydroxylysine would be expected to be replaced by a long chain fatty acyl group (possibly as its coenzyme A thioester) using an acyl transferase for the synthesis of mycobactin. Such an enzyme was not, however, identified in the gene cluster. Nevertheless this could happen as the final intermediate, pre-mycobactin (X in Fig. 6), must be transferred from the cytoplasm, where it is presumably synthesized, through the membrane for the mycobactin to be finally lodged in the cell envelope. This translocation could be accomplished by a membrane-associated acyl transferase. The fatty acyl groups being transferred to mycobactin would be synthesized in the conventional manner (and therefore will probably be membrane associated) and thus would explain why there are a variety of chain lengths to the alkyl group, varying by C2 units, in mycobactin which would then be in keeping with the products arising from normal fatty acid biosynthesis. The final step in carboxymycobactin synthesis must be different in a number of respects, firstly, as the alkyl chain now terminates in a carboxylic acid (see Fig. 2) this might suggest that a dicarboxylic acid is involved in the final acyl transferase. However, the different lengths of the acyl chains of carboxymycobactin vary by only a single carbon atom.51 This is not in keeping with them arising from a dicarboxylic fatty acid which would be produced by omega-oxidation of conventional fatty acids and thus would be expected to show a variation in the length of the chain by C2 units. It would seem possible that a fatty acid of conventional chain length is introduced on to the premycobactin nucleus which is cleaved (perhaps at a central double bond) upon movement of the molecule through the cytoplasmic membrane thereby giving rise to a series of shorter carboxylic acid chains. Nothing however is known about this

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final step and unfortunately the decoding of the genome does not indicate how it may be accomplished. As mycobactin does not though appear to be a precursor of carboxymycobactin28,19 the subtleties of this final step must await further investigation.

Regulation of iron metabolism There are many enzymes and other proteins whose activity changes according to the concentration of iron prevailing within the cell. There are also changes in the content of nucleic acids, polysaccharides and other components of the cell when iron is either growth-limiting or replete. Early work on these changes was initiated by Frank Winder and his colleagues, including the present author, in Trinity College, Dublin, who worked for many years on the consequences of trace metal deficiencies, including iron, in mycobacteria (see 75). There are changes both to the content of various ironcontaining proteins, such as the cytochromes and porphyrins68 and a number of enzymes, whose connection with iron metabolism may not be immediately obvious.76 Changes may result in increases in the level of a particular protein upon the cell being deprived of iron but equally there are decreases in the levels of other proteins. Thus the varied responses of the cell to changes in the amounts of iron available to it indicate that there is a complex array of both inductive and repressive effects at the gene level. It is not just a matter of the mycobacteria responding to iron deficient growth conditions (whether in vivo or in vitro) by producing their various siderophores but the entire metabolism of the cells must change to meet the challenge of iron insufficiency: energy metabolism is affected which in turn implies that there will be changes to glycolysis and to the tricarboxylic acid cycle activities; there will be changes in pool sizes of various amino acids and nucleotides thereby affecting protein and nucleic acid synthesis. This therefore means that the response to changes in iron concentrations within the cell must be carefully orchestrated and this can only be at the genetic level. We now know that many genes can be either induced (up-regulated) or repressed (down-regulated) according to prevailing level of iron that then enable the cell to adapt to the changing growth conditions. The coordinated response to iron deficiency is mediated by a small number of regulators chief amongst which, in bacteria, is the Fur ( ¼ ferric uptake regulation) protein. In E. coli, the Fur

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Figure 7 Diagrammatic representation of gene regulation in M. tuberculosis by the IdeR protein. (Adapted from 3) The product of the ideR gene, IdeR, combines with Fe(II) and this complex represses the expression of a number of genes (dappled rectangles) around the chromosome by physical attachment to the operator genes (black circles) at specific ‘iron boxes’ of the DNA regions. In low iron conditions, the IdeR-Fe(II) complex is not formed and the various genes are now able to be expressed. To prevent a ‘gene-lock’ situation arising by IdeR–Fe(II) preventing its own formation, regulation of the IdeR gene is probably also controlled by a catabolite activator protein. Other genes, such as those for coding bacterioferritin biosynthesis, may be regulated in the opposite (positive) manner so that they are only expressed when IdeR–FeII has bound to the promoter sequencing, i.e. under high iron growth conditions.79

protein is relatively small with a molecular size of 17 kDa and comprises 148 amino acids. Although mycobacteria contain two putative fur genes,77,78 a Fur protein has not yet been positively identified though its presence has been suggested.76 Instead of Fur, this role is undertaken by a protein known as IdeR ( ¼ iron-dependent regulator), which is closely related to diphtheria toxin repressor ( ¼ DtxR) protein of Corynebacterium diphtheriae, and which is also analogous in its functional role to Fur. DxtRlike proteins have recognized in a number of other bacteria including M. smegmatis.54 The role of IdeR in the regulating the metabolism of M. tuberculosis in response to iron deprivation has been extensively studied by the group of Issar Smith in New York University (see 9 for a review). The manner in which IdeR is considered to work is the same as that of the DxtR protein in C. diphtheriae and the Fur protein in E. coli where up to 60 genes are now known to be controlled

through its interaction with DNA (see 3). Fig. 7 shows the suggested way in which IdeR probably operates based on what is known about the operation of DxtR and Fur: for the protein to bind to DNA it must first bind with FeII to form a complex that then interacts a so-called ‘iron-box’, or IdeR box, that is located at the 10 position of each gene promoter.79 Over 40 genes have been identified in M. tuberculosis by the Smith group as having putative IdeR boxes in their promoter regions.79 The expression of the genes is prevented by the binding of the IdeR–FeII complex to the promoter. In the absence of available iron, that is under conditions of iron deprivation, the IdeR–FeII complex cannot be formed and no binding on to DNA takes place with the result that these genes are now expressed. Examples of this control of gene expression under low iron conditions were evidenced by showing that the various genes known to be associated with mycobactin synthesis (see above

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

and Fig. 6) had appropriate IdeR binding sites. However, many of the genes that are regulated by IdeR appear to have no connection with iron metabolism; genes that are so regulated include ones involved in lipid and membrane biosynthesis.79 This, as the authors suggested, raises the intriguing possibility that M. tuberculosis alters its membrane structure when deprived of iron which then would have repercussions as to how it may survive in vivo where iron deprivation would occur. These authors also showed that the gene for bacterioferritin synthesis (bfrA) was up-regulated in high-iron conditions (as is required for the bacterioferritin to act as an ironstorage protein under iron replete conditions) and this was also found to be due to IdeR. In this case, though, the bfrA promoter was found to have two IdeR boxes in tandem and it is surmised that, under high iron conditions when the IdeR–FeII complex would have been formed, this complex when bound to the promoter would cause the gene to be expressed. Thus, IdeR can control gene expression in both high and low iron conditions and that this includes genes essential for the virulence of M. tuberculosis.80 The work of Gold et al.79 has confirmed that genes for mycobactin synthesis are indeed activated when M. tuberculosis is within macrophages and that virulence of the bacterium in mice was attenuated (weakened) when a dxtR allele was mutated that then prevented the bacteria from acquiring iron from the host. Thus, there is clear evidence that M. tuberculosis is physically deprived of iron when it is within macrophages and, unless it is able to acquire iron presumably by synthesizing mycobactin and carboxymycobactin, then its virulence is greatly diminished, if not completely, removed. These conclusions are reinforced by the earlier work of Manabe et al.80 and more recently by Hobson et al.37

Iron and tuberculosis Iron is essential for the growth of all pathogenic bacteria and the ability to acquire it from the host is an absolute requirement before they can begin to grow and multiply and to cause disease. Although, as explained above, pathogenicity is a multifactorial process and the ability to acquire iron is not the sole criterion for bacteria to become pathogens, nevertheless it is one of the more important aspects for the initiation of a microbial infection. It therefore follows that the status of iron within the person or other animal who has

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become infected with a pathogen, including M. tuberculosis, is of paramount importance. One of the earliest, if not the earliest, advocates for withholding iron from patients with quiescent tuberculosis was the French physician, Trousseau, who in 1872 was alerting his medical students in Paris in a seminal lecture series81 to the dangers of giving iron to infected patients. His evidence was that patients to whom iron was administered in their diet as a form of ‘tonic’ invariably had a poorer outcome than those patients who received no additional iron and were, in fact, somewhat anaemic. Tuberculosis cases amongst the wealthy people, where there was a good chance of a nutritious diet with iron supplementation to correct any anaemia, often fared worse than poorer people who had a diet that was inadequate in its iron content. Giving ‘tonics’ of unprocessed animal blood to tuberculosis sufferers was seemingly widely practised in the 19th century and such a practice could not in fact have been worse for the outcome of the disease. More recently, Weinberg82 has discussed this earlier work of Trousseau and has cited many other examples of the enhancement of infections by viruses, other bacteria and various parasitic organisms by the mistaken view of giving iron to patients suffering from these various infections. The virulence of many pathogens is now known to be enhanced by iron. The list of such organisms is indeed a long one (see 83,16) and to which now must be added both M. tuberculosis22 and M. avium.84,85,21,86 There has been much previous, though indirect, evidence that mycobacterial infections are indeed enhanced by iron but the recent papers of Lounis et al.22 and Gomes et al.86 now confirm these findings unequivocally. These two separate research teams have clearly shown that the outcome of experimental infections of M. tuberculosis and M. avium in mice is considerably worsened by iron supplementation with the clear implication that addition of iron to the diet of human sufferers, including AIDS patients suffering from M. avium infections, must be avoided. Somewhat supportive of this, is a recent report of three cases of M. avium infection in men working in an iron foundry in Japan.87 The implication is that the high concentration of iron in the environment where these men were working may have been a major factor in the development of their infections as these three men were otherwise healthy and were not immune-compromised. To find three such cases in close proximity was therefore a very rare event and must indicate that some common factor was in operation for the

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bacteria to develop into full-blown infections. Iron appears as if it could be that common factor. With iron fulfilling a key role in the development of infectious diseases, there have even been suggestions that administration of transferrin, without it being saturated with iron, or even treating diseases with low molecular weight iron chelators, may improve the prognosis for certain diseases including cancer.17 However, there is an intrinsic danger than such compounds might possibly work in the opposite direction and aid the uptake of iron by the pathogen thereby contributing to the rapid dissemination of the disease throughout the patient. This strategy therefore must be viewed with considerable caution but the conceptual idea of preventing the infective agent gaining its iron from the host is undoubtedly sound. This aim though might be best achieved by other means (see the following section). The iron-withholding ability of the host, as manifested through the transferrin and lactoferrin proteins (see above), is vital as a defence mechanism against infection and, if this is compromised by the administration of iron, then the consequences can indeed be dire for the patient as the iron will preferentially feed the infection and not the patient. The physician who recommends an iron supplement in the diet for a patient suffering from some chronic infection, like tuberculosis, because the patient is simultaneously presenting as being anaemic, i.e. has a low red blood cell count, is in fact endangering the life of that patient. It is much better for a satisfactory outcome in the treatment of tuberculosis and other long-lasting infections, to avoid giving the patient any iron supplement or indeed foods, such as red meats, that are particularly rich in available iron. Mild anaemia is of positive benefit to the patient particularly in the early stages of the disease as this prevents the mycobacteria gaining the iron they need for growth. Of course, severe anaemia can be damaging to the tuberculosis sufferer though there are obvious dangers in trying to rectify this too quickly. A slow improvement in the iron status of the patient should therefore be aimed at so that there is no sudden influx of available iron in the diet or by injection; as most foods have some adventitious iron within them a reasonably well-balanced diet should suffice to improve the iron levels in the body to the point where the patient no longer feels impaired by the lack of iron. But, it should be clearly emphasized, iron supplements, and certainly injections of iron, should not be given to a tuberculosis sufferer until the disease has been brought under control by appropriate antibiotics and chemotherapy. Then, and only then, should the

C. Ratledge

iron status of the patient be slowly increased, preferably by improvements in the diet alone.

Drugs and the inhibition of iron metabolism One of the oldest anti-tuberculosis drugs, paminosalicylic acid (PAS),88 probably exerts its inhibitory effect by the inhibition of mycobactin biosynthesis48,89,90 rather than being an inhibitor of folic acid biosynthesis. Many current text books on the mode of action of antimicrobial agents continue to assert that PAS is a metabolic analogue of p-aminobenzoic acid and, as such, then inhibits the biosynthesis of folic acid. This assertion was originally made before the discovery of mycobactin27 and that salicylic acid, which is formed as an extracellular metabolite also under iron deficient growth conditions,38 is a direct precursor of mycobactin. However, once an initial assertion has been made it is very difficult to change this as clearly authors of textbooks rarely read the relevant literature outside their own fields of interest. Nevertheless, all the available evidence now points to PAS inhibiting the conversion of salicylic acid to mycobactin and carboxymycobactin probably in the first reactions involving its interaction with ATP or its subsequent condensation with serine (see 13 and 28, Fig. 6). Treatment of M. smegmatis and M. bovis with PAS is always more effective when the cells are growing with a sufficiency of iron than with a deficiency of iron. Under high iron conditions the genes for mycobactin and carboxymycobactin biosynthesis are repressed79 and thus the few copies of the various enzymes that remain are adequately inhibited by PAS. However when the cells are iron deficient, PAS is less effective because its intracellular concentration is, in effect, diluted by the greater abundance of the target enzyme(s). Nevertheless under these conditions the formation of mycobactin is strongly inhibited by PAS89 though that of carboxymycobactin is less effected and may even be increased,48 and the concentration of salicylic acid is increased considerably, in some cases by over 15-fold,48 by the action of PAS. These inhibitions though do not lead to the same degree of cell killing as occurs with the iron sufficiently grown mycobacteria. The conclusion is therefore that PAS exerts its primary action in the conversion of salicylate to mycobactin and thus its effectiveness as an anti-TB compound is by it preventing the bacteria from acquiring iron from the host. This then reinforces

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

the conclusion that mycobactin is absolutely essential for in vivo growth of mycobacteria.36 Salicylate, though, may carry out other functions besides being a precursor of mycobactin and carboxymycobactin as auxotrophic mutants of M. smegmatis that require salicylic acid for growth48,91 do not respond when supplied with mycobactin or carboxymycobactin or both together. This therefore implies, as both mycobactin and carboxymycobactin can be taken up by the bacteria, that salicylate must have another function besides being the precursor of these siderophores. What that function may be is uncertain though it has been suggested that salicylate may function in the transfer of FeII, following its formation by reduction of FeIII-mycobactin (see Fig. 5), and its donation into protoporphyrin for the formation of protoheme67 and also for incorporation of iron into bacterioferritin.7 Salicylate though may also be involved in the formation of the IdeR– FeII complex (see above and also Fig. 7) which is involved in the regulation of multiple gene expression as clearly some means of inserting FeII into this protein needs to be considered.7,3 However, no firm evidence for such an involvement of salicylic acid in the incorporation of iron into specific proteins is yet available and it remains a matter of speculation what might be this secondary role of salicylic acid. Whatever it may be, it could also be a target for the secondary action of PAS. Although improvements in the efficacy of PAS have been pursued with many structural variations on the basic molecule having been explored, none of these have proved to be better than PAS and hardly any its equal.92,93 Nevertheless the effectiveness of PAS as an anti-TB agent indicates that inhibition of mycobactin synthesis is a possible target for the design of novel inhibitors of mycobacteria. Most of the enzymes that are involved in the synthesis of mycobactin would not have an equivalent in animals, which do not synthesis hydroxamate compounds or other siderophores, and therefore would be safe targets for any drug as adverse inhibitory effects would be avoided. Targets could include therefore the enzymes involved in salicylic acid synthesis itself from shikimic acid, chorismic and isochorismic acids,74 the formation of N-hydroxylysine and the final acyltransferase involved in putting the long chain acyl group on to the molecule, presumably by exchanging with an acetyl group on the N-hydroxylysine (see Fig. 6). The enzymes involved in the synthesis of the 3-hydroxybutyrate moiety, and possibly the peptide synthetases involved in the creation of the peptide bonds within mycobactin

125 (see 6,71), would probably not be suitable targets as these may be too close to those involved in the synthesis of fatty acids or of peptides which do occur in animals. Of the three possible targets given above, none are particularly easy to develop for examining potential inhibitors. If one takes as a guideline the current work that is seeking to identify inhibitors of isocitrate lyase (see elsewhere in this volume) then knowledge of the structure of the enzyme is of considerable help which implies the ability to isolate it, to purify it to homogeneity and to use it in an in vitro assay to examine the effectiveness of a putative inhibitor. Even if purification of the enzyme is not feasible, at the very least one should be able to use enzyme assays to screen inhibitors, particularly if one is using combinatorial chemistry to generate a whole series of novel compounds for evaluation. Again, this implies that cell-free extracts can be prepared with the key enzyme then still active. If this cannot be done, then one would be forced to test the inhibitors in an empirical manner using whole cells and it would then be uncertain whether the target protein was indeed being affected unless one had some specific (non-enzyme) assay that could be used to detect this. Of the targets suggested for inhibiting mycobactin biosynthesis, that of inhibiting the formation of N-hydroxylysine would probably be the easiest to achieve as the synthesis of this entity by lysine-Noxygenase could probably be developed into a target more easily than inhibiting salicylate synthesis where the various intermediates are very unstable and no satisfactory enzyme assay could be easily developed.74 The final acyltransferase, although also a possible target, may be too close to animal fatty acyltransferases to be sufficiently distinctive but, to add to the difficulties, the gene that codes for this enzyme has not been identified within the mycobactin synthesis region of the M. tuberculosis genome6 and its activity would therefore pose difficulties to assay as the substrates for the enzyme are unknown and probably are unavailable commercially. However, lysine-N-oxygenase is itself likely to be a difficult enzyme to work with as many oxygenases are multi-component complexes often involving a cytochrome c and a cytochrome c reductase. Early work to identify the biosynthesis of this compound was unsuccessful94 and attempts to identify related activities of hydroxamate or hydroxylysine formation in other bacteria have not been particularly easy. One would therefore need a concerted effort to develop this enzyme as a target but it is certainly achievable given current advances in working with oxygenase enzymes.

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Although it is attractive to propose mycobactin synthesis as a likely target for novel inhibitors of mycobacteria, accomplishing this may pose some considerable difficulties if one needs to identify a potential enzyme that could be examined in detail. However, it may be possible to inhibit the actual process of iron assimilation by the physical chelation of iron with some appropriate agent. Following the observation that transferrin itself can inhibit the growth of mycobacteria,19,21,18 Douvas et al.95 examined the effect of a new iron chelating drug, known as deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one), also referred to as L1, or CAS 30652-11-0, on the growth of M. avium in macrophage culture. This agent was originally developed as a treatment for thalassaemia and other iron-overloaded patients and clearly is an effective agent for chelating iron in the body and withholding it from transferrin and the other iron binding proteins. However, instead of inhibiting bacterial growth, the drug positively stimulated proliferation of M. avium, suggesting that the drug was actually donating iron to the cells. (Possibly the drug was abstracting iron from transferrin and then the iron was removed from the drug by carboxymycobactin.) A second drug that was tested along with L1, though, did show promise of inhibiting mycobacterial growth. This was the chelating agent 2-pyridinecarboxyaldehyde-2quinolylhydrazone (PCQH) which was effective in inhibiting mycobacterial growth in the macrophage culture test at 0.1–1 mg/ml. Therefore although the use of iron chelating agents to inhibit growth of mycobacteria produces variable responses, this work95 illustrates that it may be feasible to design an iron chelating drug as an anti-TB agent but that it is also advisable to evaluate these agents in macrophage cultures of mycobacteria rather than relying on simple tests in laboratory media. Other attempts to inhibit iron assimilation in mycobacteria have included the use of ‘xenosiderophores’, i.e. siderophores originating from organisms other than mycobacteria, that could then withhold iron from the mycobacteria.96–99 Although some of these siderophores clearly may exert an inhibitory action against mycobacteria in laboratory growth media, they and other related molecules all suffer from the same defect: siderophores will be readily degraded by many enzymes of the host as they invariably contain simple peptide or ester linkages. They will therefore not be able to pass intact through the stomach unless they can be successfully enterically coated and, moreover, shown to be able to resist further degradation either in the blood or within the tissues or organs of the body. Even if the siderophores can be taken up

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from the intestine before their hydrolysis, they would still have to enter the blood stream, reach the site of infection and then be able to penetrate into the macrophages where the mycobacteria will be residing. The large molecule size of the siderophores may preclude any one of these events from occurring, particularly the final step of entry into the macrophage. Such considerations would apply equally to the various mycobactin analogues that have been reported as being inhibitory against M. tuberculosis:100,101 delivery to the target will be a major problem. It is also unlikely that the strategy of substituting a metal ion other than iron into mycobactin (or carboxymycobactin) would be a successful strategy to adopt for an inhibitor,102 as there would be far too great a chance of the metal ion becoming displaced by iron. This would turn the inhibitor into a growth promoter with disastrous consequences. Gallium protoporphyrin has, though, been suggested as an anti-mycobacterial agent and appeared to exert its in vitro effect by being taken up via the heme transport system in the bacteria;103 whether or not such a material would be able to be taken up from the intestine without degradation or substitution of the gallium by iron remains uncertain. Of some potential has been the suggestion by the group of Marvin Miller in the USA (see 104) to use the siderophores of mycobacteria, and indeed of other bacteria, as a means of coupling them to a known bacterial inhibitor, such as an antibiotic, and for this then to be taken up by the pathogen. This principle was established by earlier work showing that various novel antibiotics were taken up by pathogens via their siderophore uptake system.105–107 This is the concept of the Trojan horse: to smuggle into the bacteria an inhibitor that ordinarily would be excluded, but, once inside the bacteria the siderophore and the agent would be hydrolysed, and the agent would then be able to effectively kill the cell. However this Trojan horse strategy has the inherent danger of allowing the bacteria to become resistant to the agent more easily than they do to a drug not requiring in situ unmasking.108 Applying this concept, to say, the attachment of penicillin or cephalosporin, which should be inhibitory towards mycobacteria but are not, presumably because of their exclusion from the cells,10,109 to a molecule like carboxymycobactin may then be attractive as a means of taking the antibiotic into the cell. However, the problem of hydrolysis of such conjugates by host enzymes, and of hydrolysis of the carboxymycobactin, still remains as does the ensuing difficulty of targeting

ARTICLE IN PRESS Iron, mycobacteria and tuberculosis

such materials to the macrophages containing the mycobacteria. Unless such problems can be adequately solved, then the major hopes for antimycobacterial chemotherapy must reside in the design, rational or otherwise, of novel chemicals that are not degraded by hydrolytic enzymes of the host, that will be adequately and actively taken up from the jejunum or small intestine, will reach the macrophages without degradation in the blood or rapid voidance through the liver, will be taken up by the macrophages, and then finally enter the mycobacterial cell itself. There seems little chance of molecules that contain groups which are vulnerable to enzymatic hydrolysis overcoming such difficulties and being effective agents. We would also be lucky if naturally occurring antibiotics could be found that would be specifically effective against mycobactin, its synthesis or function. Thus the hope for using iron metabolism as a target for chemotherapy of tuberculosis must rely upon the skills of the chemist to synthesize appropriate molecules and of the mycobacteriologist to identify the key target of this process whose inhibition would then lead to the death of the tubercle bacillus.

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haem/Hb uptake systems of pathogenic bacteria. Mol Microbiol 1999;31:429–42. 104. Miller MJ, Darwish I, Ghosh A, Ghosh M, Hansel G, Hu J, Niu C, Ritter A, Scheidt K, Suling C, Sun S, Zhang D, Budde A, De Clercq E, Leong S, Malouin F, Moellmann U. Design, synthesis and studies of new antibacterial, antifungal and antiviral agents. In: Bentley PH, O’Hanlon PJ, editors. Antiinfectives. Recent advances in chemistry and structureactivity relationships. Cambridge, UK: Royal Society of Chemistry; 1997. p. 116–38. 105. Basker MJ, Edmondson RA, Knott SJ, Ponford RJ, Slocombe B, White SJ. In vitro antibacterial properties of BRL 36650, a novel 6a-substituted penicillin. Antimicrob Agents Chemother 1984;26:734–40.

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106. Basker MJ, Frydrych CH, Harrington FP, Milner PH. Antibacterial activity of catecholic piperacillin analogues. J Antibiot 1989;42:1328–30. 107. Critchley IA, Basker MJ, Edmondson RA, Knott SJ. Uptake of a catecholic cephalosporin by the iron transport system of Escherichia coli. J Antimicrob Chemother 1991;28:377–88. 108. Barry III CE, Slayden RA, Sampson AE, Lee RE. Use of genomics and combinational chemistry in the development of new antimycobacterial drugs. Biochem Pharmacol 2000;59:221–31. 109. Webb V, Davies J. Antibiotics and antibiotic resistance in mycobacteria. In: Ratledge C, Dale JW, editors. Mycobacteria virulence and molecular biology. Oxford, UK: Blackwell Science; 1999. p. 287–306.