Iron acquisition strategies in mycobacteria.

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YTUBE1259_proof ■ 18 January 2015 ■ 1/8

Tuberculosis xxx (2015) 1e8

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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube

REVIEW

Iron acquisition strategies in mycobacteria Q2

Zhuo Fang a, *, Samantha Leigh Sampson a, Robin Mark Warren a, Nicolaas Claudius Gey van Pittius a, Mae Newton-Foot b a

DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, US/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Stellenbosch, Francie van Zijl Drive, Tygerberg, 7505, South Africa b Division of Medical Microbiology, Department of Pathology, Faculty of Medicine and Health Sciences, University of Stellenbosch, Francie van Zijl Drive, Tygerberg, 7505, South Africa

a r t i c l e i n f o

s u m m a r y

Article history: Received 22 August 2014 Received in revised form 1 January 2015 Accepted 7 January 2015

Iron is an essential element to most life forms including mycobacterial species. However, in the oxidative atmosphere iron exists as insoluble salts. Free and soluble iron ions are scarce in both the extracellular and intracellular environment which makes iron assimilation very challenging to mycobacteria. Tuberculosis, caused by the pathogen, Mycobacterium tuberculosis, is one of the most infectious and deadly diseases in the world. Extensive studies regarding iron acquisition strategies have been documented in mycobacteria, including work on the mycobacterial iron chelators (siderophores), the iron-responsive regulon, and iron transport and utilization pathways. Under low iron conditions, expressions of the genes encoding iron importers, exporters and siderophore biosynthetic enzymes are up-regulated significantly increasing the ability of the bacteria to acquire limited host iron. Disabling these proteins impairs the growth of mycobacteria under low iron conditions both in vitro and in vivo, and that of pathogenic mycobacteria in animal models. Drugs targeting siderophore-mediated iron transport could offer promising therapeutic options. However, the discovery and characterization of an alternative iron acquisition mechanism, the heme transport and utilization pathway, questions the effectiveness of the siderophore-centered therapeutic strategy. Links have been found between these two distinct iron acquisition mechanisms, thus, targeting a few candidate proteins or mechanisms may influence both pathways, leading to effective elimination of the bacteria in the host. © 2015 Published by Elsevier Ltd.

Keywords: Iron Heme ESX Mycobacteria

1. Introduction Mycobacteria, like most other living organisms, require iron for many vital functions. Iron forms the essential catalytic centre of the active site of various enzymes enabling enzymatic reactions. The ability of iron to receive and donate an electron thereby oscillating between the ferrous (Fe2þ) and ferric (Fe3þ) oxidative states enables the enzyme to catalyse redox reactions. For this reason, iron is often associated with cytochromes responsible for oxidative phosphorylation and energy production [1]. Ironesulfur clusters are essential cofactors of many enzymes involved in amino acid and pyrimidine biogenesis, the tricarboxylic acid cycle as well as * Corresponding author. Tel.: þ27 21 938 9073. E-mail addresses: [email protected] (Z. Fang), [email protected] (S.L. Sampson), [email protected] (R.M. Warren), [email protected] (N.C. Gey van Pittius), maen@sun. ac.za (M. Newton-Foot).

electron transport [2]. Iron is one of the most abundant elements on earth, however, under the earth's oxidizing environment at physiological pH, iron exists predominantly as insoluble ferric salts such as iron oxide, iron hydroxide and iron phosphate which cannot be assimilated by bacteria. Free iron ions are therefore scarce. Iron acquisition is even more challenging for pathogenic bacteria because iron ions are bound to host iron-binding proteins, such as transferrin and lactoferrin which serve as host iron transporters, the iron storage protein ferritin and iron-protoporphyrins in hemoproteins [3]. During infection, the host restricts the amount of circulating transferrin-bound iron in the body and reduces the uptake of dietary iron utilising iron-deprivation as a host antimicrobial defense mechanism [4e6]. Pathogenic mycobacteria are, however, able to cause disease despite the severely iron limited host environment. To overcome iron-deprivation mycobacterial pathogens have evolved iron acquiring pathways which are more efficient than those of their vertebrate hosts. In this review, the

http://dx.doi.org/10.1016/j.tube.2015.01.004 1472-9792/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Fang Z, et al., Iron acquisition strategies in mycobacteria, Tuberculosis (2015), http://dx.doi.org/10.1016/ j.tube.2015.01.004

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Z. Fang et al. / Tuberculosis xxx (2015) 1e8

strategies mycobacteria use to access iron and the iron-dependent gene regulation responsible for the maintenance of iron homeostasis in mycobacteria are discussed. Additionally, a brief opinion is given regarding the selection of iron acquisition machineries as drug targets. 2. Siderophore-mediated iron acquisition A major mechanism employed by mycobacteria to compete for the limited available iron is the use of high affinity iron chelators, siderophores, which are predominantly produced during iron deprivation [7]. There are three types of siderophores, mycobactin, carboxymycobactin and exochelin, where mycobactin and carboxymycobactin share a core structure which is distinct from exochelin. Mycobactin is cell envelope-associated and facilitates the transport of iron through the cell envelope into the cytoplasm while carboxymycobactin and exochelin are secreted ironchelators which acquire iron in the extracellular milieu and transport iron into the cytoplasm of the bacteria [7]. 2.1. The siderophores: mycobactin, carboxymycobactin and exochelin Mycobactins can be isolated from almost all mycobacterial species except Mycobacterium microti [8], Mycobacterium paratuberculosis, and Mycobacterium vaccae [9]. They have a core structure comprising 5 amino acids with a characteristic phenyloxazolidine ring derived from salicylate with saturated or unsaturated alkyl side chains of various lengths on the hydroxylated lysine residue at the centre of the molecule [10] (Figure 1 and Figure 2a). The long alkyl chain confers the lipophilic property of mycobactin and thereby locates it within the cell envelope [7]. Early studies created confusion by using the term “exochelin” to describe all extracellular siderophores isolated from both saprophytic and pathogenic mycobacteria. This became inappropriate when “exochelin” from pathogenic mycobacteria was found to have a different core structure from that of saprophytic mycobacteria. The former has a core structure similar to that of mycobactin, but with shorter alkyl side chains that terminate with either a carboxyl group or a methyl ester (Figure 2b) [12,13]. This feature makes these “exochelin” molecules more hydrophilic than the lipophilic mycobactins. These molecules were renamed carboxymycobactin [14], although less frequently “exomycobactin” is used.

Figure 1. The general structure of mycobactin from M. tuberculosis. The structure consists of 5 amino acids including one salicylic acid, one serine, two lysines, and one 3-hydroxybutyric acid. A long alkyl chain extends from the side chain of the middle lysine residue where the length of the R-group may vary amongst the mycobacteria species. The asterisk (*) indicates the chelating group for the ferric ion binding [6]. Copyright (2004) Elsevier, license number for the permission of reuse: 3451920576018.

The extracellular siderophores from saprophytic mycobacteria retained the name exochelin. This water-soluble molecule has a different core structure to mycobactin/carboxymycobactin. consisting of a formylated pentapeptide: N-(d-N-formayl, d-N-hydroxy-R-ornithyl)-balaninyl-d-N-hydroxy-R-ornithinyl-R-allothreoninyl-d-N-hydroxy-S-ornithine (Figure 2c) [15]. The two peptide bonds within the molecule are atypical making them resistant to peptidase hydrolysis. The structure of exochelin varies in some mycobacterial species, e.g. in Mycobacterium neoaurum exochelin is a hexapeptide with an unusual b-hydroxyhistidine residue [16]. Carboxymycobactin was thought to be exclusive to pathogenic mycobacteria until it was found to comprise about 5% of the total siderophore content in Mycobacterium smegmatis [14]. The coexistence of carboxymycobactin and exochelin in M. smegmatis further assisted in resolving the historical confusion between the two siderophores. The difference in core structure of mycobactin/ carboxymycobactin and exochelin necessitates distinctive biosynthetic pathways and transport mechanisms for the two unrelated siderophores. The identification of the two biosynthetic pathways further clarified the distinction between the two types of siderophores. The machinery for mycobactin biosynthesis is encoded in two gene clusters, mbtA-J [17] and mbtK-N [18] in both Mycobacterium tuberculosis and M. smegmatis. The enzymes responsible for exochelin biosynthesis are encoded by fxbA, fxbB and fxbC genes in M. smegmatis [19e21]. The biosynthetic pathways of the mycobactin [22,23] and exochelin [21] are described in detail elsewhere.

Figure 2. Examples of mycobacterial siderophores. M. tuberculosis mycobactin (a) and carboxymycobactin (exomycobactin) structures contain a typical phenyloxazolidine ring (b); The difference between the two molecules is that the N-acyl chain on the hydroxylated lysine is a 19-carbon alkyl group (a) while that of carboxymycobactin is a 6-carbon alkyl group coupled with a carboxylic acid (b) [11]. M. smegmatis exochelin structure containing 5 amino acids connected with 2 unusual peptide bonds (c) [7]. Copyright (2000) National Academy of Science, U.S.A.


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2.2. Siderophore transport The import and export of the secreted siderophores, carboxymycobactin and exochelin utilises unique mechanisms and machineries. Carboxymycobactin transport is dominant in pathogenic mycobacteria while exochelin transport is more important in saprophytic species. 2.2.1. Carboxymycobactin transport Hydrophilic deferri-carboxymycobactin is secreted into the extracellular milieu to acquire iron from the environment, and presumably the host transferrin [12] and at a slower rate, ferritin [24]. The ferri-carboxymycobactin is transported back through the mycobacterial outer membrane via the Msp family of porins [25]. The Msp porins are abundant membrane proteins which allow a variety of nutrients to enter the cell by non-specific diffusion [25,26]. In the mycobacterial periplasmic space, ferricarboxymycobactin either transfers its iron to the cell-wall associated mycobactin or delivers it to the plasma membrane-bound protein complex, IrtAB (iron-regulated transporter A and B, Rv1348 and Rv1349) [27]. IrtA and IrtB are ATP-binding cassette

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(ABC) transporters, which are homologues of the YbtPQ system involved in iron transport in Yersinia pestis [28]. Inactivation of this gene pair impairs the utilization of ferri-carboxymycobactin and affects the optimal growth of M. tuberculosis under low iron conditions but does not abolish the production and secretion of the siderophores [27]. It has been proposed that IrtA possesses the dual function of iron import and ferric iron reduction. It is thought to release the bound iron in the ferrous form, possibly due to the reductase role of its FAD-binding domain [29]. However, this activity was not obtained in vitro, possibly because other components are also required to assist in this role [29]. A possible candidate is ViuB (Rv2895c), whose homologue in Vibrio cholera encodes a cytosolic protein required for the utilization of ferric-vibriobactin [30]. ViuB forms a complex with IrtB [31], but is not essential for iron acquisition [32]. Whether ViuB is involved in siderophore import remains unclear. After releasing the bound iron, deferri-carboxymycobactin may either be degraded or secreted again to acquire more iron. Siderophore catabolism has been relatively neglected in the literature whereas siderophore secretion has recently been described. The carboxymycobactin export system was shown to be the membrane complexes of MmpL5/MmpS5 and MmpL4/MmpS4, where the

Figure 3. Exochelin import and export in M. smegmatis (adapted from Ratledge, 2004). The ferri-exochelin gets recognized by an unknown protein (indicated with a “?” in the figure) on the outer membrane and delivered to FxuA-B-C complex on the plasma membrane while coupled with FxuD. While in the cell envelope, ferri-exochelin may transfer the iron to mycobactin on the membrane. Once ferri-exochelin are imported into the cytoplasm, the ferric iron gets reduced by unidentified reductase and released for utilization. The deferri-exochelin is transported out of the cell via ExiT [19e21].


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MmpLs are large integral membrane proteins whereas the MmpSs are small soluble proteins tethered to the membrane and are thought to be transport accessory proteins [33,34]. The MmpS4/S5 double mutant showed an iron starvation phenotype under high iron conditions in which the uptake of carboxymycobactin was not affected but the siderophore biosynthesis and secretion were strongly impaired indicating that MmpS4/S5 is directly involved in mycobactin/carboxymycobactin export [34]. The siderophores, that are taken up and produced de novo, are able to exert a toxic effect on the MmpS4/S5 double mutant because of a blockage of siderophore recycling [33]. 2.2.2. Exochelin transport Exochelin transport has not been well characterized in mycobacteria (Figure 3). The deferri-exochelin may be exported by an ABC transporter protein, ExiT, encoded in the same operon as the exochelin synthetase genes fxbB and fxbC [20,21]. ExiT appears to be involved in both the biosynthesis and export of exochelin, directly coupling these processes [21]. After binding extracellular iron, ferriexochelin is recognized by some unknown receptors on the outer membrane and imported into the periplasmic space [25]. There, ferri-exochelin may associate with FxuD, the ferri-exochelin carrier, and either transfer the iron to mycobactin or to the ferric-exochelin

importer membrane complex formed by FxuA, FxuB, and FxuC. FxuA, FxuB and FuxC are homologues of FepG, FepC and FepD which form the membrane-bound permease which is responsible for the uptake of the siderophore ferrienterobactin in Escherichia coli [19]. In the cytoplasm, exochelin-bound ferric iron is reduced to ferrous iron by an unidentified reductase and released. 3. Heme transport and its metabolism in mycobacteria Wells and colleagues used heme as an alternative iron source to successfully rescue their M. tuberculosis mutants in which the mycobactin biosynthetic pathway was disabled under low iron conditions [34]. Heme is an iron-containing prosthetic group found in many hemoproteins, including hemoglobin which are abundant in erythrocytes. In fact, two thirds of iron in veterbrates are incorporated into the heme. Heme is therefore a reservoir of iron for many pathogenic bacteria and their heme acquisition systems have been well described [35e37]. M. tuberculosis may encounter heme or heme-containing protein both intracellularly and extracellularly during infection. Erythrocytes are degraded in the phagosomes of macrophages resulting in heme release [38,39], exposing M. tuberculosis residing in the phagosome directly to the heme. Extracellular M. tuberculosis may access hemoglobin in the

Figure 4. Relationships between iron acquisition, heme acquisition, siderophore export and the ESX-3 secretion system. It is currently thought that mycobacteria possess transport systems that may acquire iron from two types of iron source, one from transferrin and the other one from heme. Carboxymycobactin acquires iron from the host transferrin and gets recognized by Msp family porins on the outer membrane and imported to the periplasmic space where they either deliver the iron to the cell envelope associated mycobactin or to the importer protein complex IrtA and IrtB. IrtA contains an intracellularly-located FAD domain which might perform reductase function releasing ferrous iron from carboxymycobactin. The deferri-carboxymycobactin gets exported through MmpL5/S5 and MmpL4/S4 siderophore export complex and acquires more iron if necessary. Mycobacteria release heme-binding protein Rv0203 which acquires heme from hemoproteins in the host and delivers the heme to the heme importers, MmpL11 and MmpL3. Once inside the bacteria, heme either gets degraded to release iron by MhuD or possibly incorporated into bacterial hemoproteins such as cytochromes, hemoglobin, KatG, DosT and DosS, etc. Heme can also be synthesized by mycobacteria via the ferrochelatase HemZ, which catalyzes the addition of iron to protophorphyrin IX to form protoheme. The iron and heme acquisition processes are enabled by the ESX-3 components or their substrates. The detailed roles played by ESX-3 on these processes are to be investigated.


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bloodstream where hemolysins produced by M. tuberculosis facilitate the release of hemoglobin from the red blood cells [40,41]. Many studies have demonstrated the ability of M. tuberculosis to utilize heme as an iron source in low iron culture media [42e44]. The gene cluster Rv0201c-Rv0207c was shown to be responsible for heme acquisition in M. tuberculosis. Rv0203 is a secreted hemophore which has a similar heme binding affinity to its counterparts in some gram-negative bacteria [44,45]. Rv0202c (MmpL11) and Rv0206c (MmpL3) are transmembrane heme importers (Figure 4). Rv0203 is secreted via an unidentified mechanism and acquires heme from hemoglobin, transports it across the outer membrane and delivers to the extracellular domain (E1) of either MmpL11 and MmpL3 [45]. Inside M. tuberculosis, heme can either be degraded to release iron or possibly utilized for the synthesis of heme-containing proteins, which is yet to be discovered. Heme degradation is catalysed by heme oxygenase (HO) in a well characterized process in which heme is broken down to biliverdin, releasing carbon monoxide (CO) and iron ions [46]. The HO counterpart in M. tuberculosis, MhuD (Mycobacterial heme utilization Degrader, Rv3592) which shares no sequence homology with HO suggesting a distinct catalytic mechanism, however, degrades heme to mycobilin without generating CO. As CO induces M. tuberculosis to enter dormant state [47], this particular heme degradation mechanism does not interfere with the CO sensing of the bacteria in the host environment [48]. As MhuD is able to bind to two heme molecules in its inactive state, it may serve as an additional heme storage molecule [49]. Although M. tuberculosis favours heme as an iron source, it expresses a heme biosynthetic enzyme, ferrochelatase, encoded by hemZ (Rv1485). This protein contains a [2Fee2S] cluster and catalyzes the last step of heme biosynthesis in which ferrous iron is inserted into protoporthyrin IX to form protoheme [50]. This gene is essential in vitro implying that heme is not only used as an iron source by M. tuberculosis but is also an essential cofactor for some metabolic enzymes [51]. For example, the catalase-peroxidase (KatG) and the DosS/DosT redox sensing two component system proteins contain heme in their active sites [52]. It is interesting that addition of heme to the low iron media could not rescue the MmpS4/S5 double mutant, in which the mycobactin export was destroyed, unless the siderophore biosynthesis pathway was also disabled [34]. This implies that the intracellular accumulation of carboxymycobactin/mycobactin exerts a toxic effect on the bacteria perhaps by removing irons from the bacteria's own iron-containing proteins or from imported heme. 4. Additional iron acquisition pathway It is worth mentioning that an additional siderophoreindependent iron acquisition mechanism in M. tuberculosis has recently been discovered. A cell surface-localized human holotransferrin-binding enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), was found to be able to facilitate the internalization of human transferrin across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages. Overexpression of this protein may increase transferrin binding to M. tuberculosis and iron uptake [53]. 5. The role of ESX-3 in siderophore and heme transport M. tuberculosis contains five copies of the Type VII protein secretion system, namely ESX-1- to -5 [54,55]. ESX-1 and ESX-5 are involved in virulence in pathogenic mycobacteria [56e60]. ESX-3 has shown to be involved in the mycobactin-mediated iron acquisition pathway, but not the exochelin pathway in M. smegmatis [61]. In M. tuberculosis, ESX-3 is essential for in vitro growth [62,63]. The

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growth defect of the M. tuberculosis conditional ESX-3 knock-out strain in 7H9 was only rescued by the addition of extremely high concentrations of iron and zinc [64]. This implies that alternative iron acquisition systems exist in M. tuberculosis and sustain the bacteria with a basal iron supply. The transcriptional profile of the M. tuberculosis ESX-3 conditional mutant resembles that of iron starvation even under these high iron conditions [65]. Repression of ESX-3 expression has a negative effect on iron import, but ESX-3 is not directly responsible for iron uptake. This suggests that ESX-3 may participate in iron uptake by allowing the modification and secretion of unidentified factors which assist in iron acquisition, as suggested by the survival of the ESX-3 conditional mutant in the culture supernatant of wildtype M. tuberculosis [64]. Alternatively ESX-3 may modify the M. tuberculosis cell membrane surface composition, thereby facilitating the import of ferricarboxymycobactin [64] (Figure 4). ESX-3 also appears to influence the heme uptake pathway in M. tuberculosis. Heme supplementation could not rescue the M. tuberculosis ESX-3 conditional mutant under low iron conditions in the presence of tetracycline which suppressed the expression of esx-3 [65]. As the heme uptake pathway has been well characterized [44], ESX-3 is not directly involved in heme uptake, but may play a similar supportive role as observed for mycobactin-mediated iron acquisition where ESX-3 may modify and secrete unknown factors which are essential for iron and heme acquisition in M. tuberculosis. Further characterisation of the role of ESX-3 in both iron and heme acquisition is of great significance due to the essential nature of these processes. 6. Iron-dependent gene regulation Mycobacteria utilise elaborate molecular mechanisms to maintain iron homeostasis. Iron acquisition mechanisms are activated in response to low intracellular iron levels to acquire iron from the environment. When there is sufficient intracellular iron, excess iron is stored to prevent it from participating in the Fenton reaction, which would generate harmful hydroxyl radicals [66]. Mycobacteria have developed complex gene regulation mechanisms which respond to different iron levels to maintain iron homeostasis. The iron dependent regulator, IdeR, of M. tuberculosis was identified based on homology to the Diphtheria Toxin Repressor (DtxR), an iron-dependent gene expression regulator in Corynebacterium diphtheria. IdeR is essential in M. tuberculosis in vitro [67,68]. Abolishing the iron-responsive regulator leads to attenuation of M. tuberculosis in macrophages and the mouse model [67]. IdeR binding sites have been identified throughout the M. tuberculosis H37Rv genome [69,70]. The expression of 153 M. tuberculosis genes is regulated in response to iron concentration and 44 of these genes are regulated directly by IdeR (See examples in Table S1 and Table S2). Of the 40 genes that are repressed by IdeR in high iron conditions, 32 encode proteins involved in three iron acquisition related processes: 1. Iron acquisition including the ESX-3 secretion system and the siderophore import transmembrane protein pair IrtA/IrtB; 2. Siderophore biosynthesis; and 3. Siderophore export, MmpL4/S4 [68] (Table S1). The expression of four genes is induced by IdeR under high iron levels, two of which encode the iron storage bacterioferritin proteins (bfrA and bfrB). The same transcriptional response was observed in M. tuberculosis infected macrophages implying that the macrophage presents an iron-deprived condition [69]. Similarly in M. smegmatis, under high iron conditions, IdeR negatively regulates the expression of fxbA, which encodes a formyltransferase which is essential in the exochelin biosynthetic pathway [71]. Mycobacteria therefore respond to high iron levels by inhibiting the expression of the iron acquisition and transport


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machineries while increasing the production of iron storage proteins. These activities show that mycobacteria maintain iron homeostasis by preventing excessive iron uptake which could cause oxidative damage and storing excess intracellular iron for future use. Many M. tuberculosis genes were found to be regulated by iron independently of IdeR. Expression of 26 genes was repressed, and 23 induced, by a high iron concentration. These genes are involved in a variety of cellular functions, including carbohydrate metabolism, lipid metabolism, energy metabolism, cofactor biosynthesis, aerobic growth, transcriptional regulation, iron transport, and toxin-antitoxin systems (Table S2). Of the genes involved in iron acquisition related processes, the expression of mmpL5 and mmpS5 is repressed under high iron conditions, consistent with the other components of the siderophore export system, MmpL4 and MmpS4. Interestingly these four genes are not within the same IdeR regulon which may indicate that M. tuberculosis also utilises alternative iron regulators. Additionally, there is a second iron-dependent regulator in M. tuberculosis, namely FurA (ferric uptake regulator A, Rv1909c). FurA, whose gene is upstream to katG in the same operon, is able to negatively regulate the expression of KatG [72,73]. The FurA mutant showed increased resistance to H2O2 and susceptibility to isoniazid (INH) because of the upregulation of KatG [72,73]. One substrate of ESX-1, EspD, is also repressed under high iron condition. EspD might play a role in facilitating the secretion of other ESX-1 substrates thereby enhancing the virulence of M. tuberculosis [74], so the iron-deprived environment in macrophages possibly enables the functionality of ESX-1. The gene, ethA, encoding a monooxygenase which activates the anti-TB drug ethionamide, was also induced under low-iron condition. The effectiveness of this drug may correlate with different iron concentrations. Sufficient iron in the culture media allows the bacteria to synthesize ironesulfur cluster-containing metabolic enzymes including NADH dehydrogenase [75], so the transcription of this gene cluster is induced (nuoA-D, nuoH-I, and nuoK-N). 7. Iron and heme acquisition-centred drug target discovery Mycobacteria cannot survive without iron; a malfunctioning iron acquisition pathway causes a growth defect in vitro and in macrophages, and attenuates M. tuberculosis in mouse model [27,34,61,65]. Intervention strategies targeting siderophoremediated iron acquisition could be effective because this pathway is not only essential in M. tuberculosis but also is absent from the human host. Acyl sulfamoyl adenosine (acyl-AMS), an inhibitor to one mycobactin biosynthesis enzyme, MbtA, has been synthesized and proven to be effective in abolishing the pathway under low iron conditions in vitro [76]. However, as mycobacteria are also able to acquire iron from heme, an abundant iron source in the host, this may compensate the loss of the siderophore-mediated iron acquisition pathway. This approach may therefore not be successful in vivo unless heme acquisition is targeted at the same time. Drugs which target or disable both pathways would be potentially therapeutic. MmpS5 and MmpS4 are promising candidates, as disrupting them would not only abolish the siderophore-mediated iron acquisition and recycling of the siderophores, but would also cause intracellular siderophore accumulation, disrupting heme utilization as previously described where heme could not rescue the MmpS5/S4 mutant [33,34]. MmpL3 is another candidate which is essential for cell viability in vitro [77]. Abolishing MmpL3 may impair trehalose monomycolate export preventing proper assembly of the mycobacterial cell wall [78] and certainly heme uptake [44]. Two anti-TB drugs, SQ109 [78] and BM212 [79], have shown an inhibitory effect on MmpL3. Drug target exploration and

characterization regarding the heme uptake pathway is well described elsewhere [80]. Moreover, ESX-3 also influences both the iron and heme acquisition pathways [65]. The substrates of ESX-3 may play a variety of roles in fulfilling the metabolic needs of mycobacteria, although these roles remain to be determined. Drugs targeting ESX-3 may have potent therapeutic effects. IdeR, although not directly involved in iron or heme uptake, regulates the global iron-responsive metabolism. The M. tuberculosis IdeR mutant has a growth defect in vitro, in vivo and in a mouse model [67] making it a promising drug target. The other iron-dependent regulator, FurA, could also be of interest as abolishing it may increase intracellular KatG levels possibly enhancing INH potency [81]. 8. Conclusion M. tuberculosis utilises elaborate iron-responsive gene regulation and various iron acquisition strategies to maintain intracellular iron homeostasis and overcome the challenges of accessing scarce iron in their natural and host environments. Numerous studies have demonstrated the essentiality of these mechanisms for the survival and virulence of mycobacteria. Therapeutic strategies targeting those pathways offer promising medical solutions for relieving the global burden of tuberculosis. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tube.2015.01.004 Funding:

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Q1

None declared. Not required

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Iron Acquisition Strategies of Vibrio anguillarum.

Extracellular iron acquisition by mycobacteria: role of the exochelins and evidence against the participation of mycobactin.

Recent developments in understanding the iron acquisition strategies of gram positive pathogens.

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Shigella Iron Acquisition Systems and their Regulation.

Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity.

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Polyacrylic acid-coated iron oxide nanoparticles for targeting drug resistance in mycobacteria.

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