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Interdependence of mycobacterial iron regulation, oxidative-stress response and isoniazid resistance
REVIEWS
Interdependence of mycobacterial iron regulation, oxidative-stress response and isoniazid resistance Olivier Dussurget and Issar Smith
I
ron is required by almost Iron is an essential cofactor for vital is a major bacterial antigen all living organisms as an functions in microorganisms. Bacterial in cattle infected with Mycoessential cofactor of propathogens have developed efficient ironbacterium paratuberculosis6. teins with important cellular acquisition systems to counteract the Mycobacterium tuberculosis functions, such as those redefensive sequestration of iron by their exochelins can also remove iron quired for DNA and amino hosts. In mycobacteria, the recently from human transferrin and acid synthesis, detoxification described protein, IdeR, negatively lactoferrin and transfer iron to and respiration. Thus, iron controls iron-uptake systems. This protein desferri-mycobactins in the cell acquisition is a vital function also has a role in the oxidative-stress wall7 (Fig. 1). The process of for survival1. The concenresponse, as well as in resistance to the iron assimilation in vivo is not tration of free iron in the envi- frontline antimycobacterial drug isoniazid. known and might vary, deronment and in human serum pending on the environment (~10218 M) is far below the O. Dussurget and I. Smith* are in the Public Health of mycobacteria (extracellular Research Institute, 455 1st Avenue, New York, nutritional requirements of vs. intracellular). A definitive NY 10016, USA; O. Dussurget is also in the UFR microorganisms. In response answer to the question of iron de Biochimie, Université Paris 7, 75251 Paris to low iron availability in the acquisition and mycobacterial Cedex 05, France; *tel: +1 212 578 0868, environment and the acutedisease awaits the isolation of fax: +1 212 578 0804, phase response hypoferremia M. tuberculosis mutants with e-mail: [email protected] that they face during infection, defects in the iron-uptake sysmicroorganisms have develtem for virulence assessment. oped several mechanisms for iron uptake2. In Gram-negative bacteria and some Gram-positive Many microorganisms secrete siderophores: low- bacteria, the expression of the genes involved in the molecular-weight, high-affinity iron chelators that uptake of iron is controlled by Fur, a repressor prosolubilize iron complexed in the environment or tein that employs ferrous iron as a co-repressor1. bound to compounds from their hosts, such as trans- DtxR, another transcriptional repressor, has been ferrin and lactoferrin. In addition, microorganisms characterized in Corynebacterium diphtheriae8. DtxR can acquire iron by various mechanisms, including controls the expression of genes for the iron-uptake secretion of soluble reductants that reduce ferric iron machinery, as well as the gene encoding the diphto ferrous iron, synthesis of specific receptors for theria toxin. In mycobacteria, IdeR, a homolog of transferrin and/or lactoferrin, production of hemo- DtxR, has been described9 and shown to regulate total lysins and toxins, utilization of heme compounds, siderophore biosynthesis10 (Fig. 1), suggesting it is a and uptake by low-affinity systems1,2. major regulator of the mycobacterial iron-acquisition However, an excess of iron can be deleterious, as apparatus. However, IdeR does not account for all this element can catalyze the formation of reactive adaptive responses to iron starvation10, and additionoxygen species (ROS) in aerobically growing cells3; al control mechanisms probably play a role in mycoconsequently, all living organisms have developed bacterial iron regulation. In this regard, two open systems to carefully regulate iron levels. reading frames with high similarity to Fur (FurA and FurB) have been observed in mycobacteria11,12 and Mycobacterial iron uptake regulation their role in iron regulation is being investigated. The In response to iron starvation, mycobacteria produce first gene to be characterized that is involved in iron several siderophores (Table 1), iron-storage proteins acquisition in mycobacteria is fxbA, which encodes a and putative receptors4. Currently, little is known putative formyltransferase necessary for exochelin about the role of mycobacterial siderophores and biosynthesis in Mycobacterium smegmatis13. The iron regulation in the infected host, and the impor- fxbA gene possesses an IdeR-regulated and irontance of these compounds in mycobacterial patho- regulated promoter, to which IdeR binds (Table 2; genesis is not clear4,5. However, it is likely that iron O. Dussurget et al., unpublished). A recent search in acquisition and metabolism are important in this the M. tuberculosis genome database has isolated process. The iron storage protein bacterioferritin several genes with potential IdeR-binding sequences Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS
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(iron boxes) in their promoter regions. Two of these genes, irg1 and irg2, encode HisE, which is involved in histidine biosynthesis, and a putative membrane protein of unknown function, respectively. The expression of these genes is negatively regulated by iron in M. tuberculosis, and their promoter regions bind IdeR at the predicted iron box sequence (Table 2; M. Rodriguez et al., unpublished). Mycobacterial response to oxidative stress Ferri-transferrin delivered to the phagosome14 and oxidative metabolism of alveolar macrophages15 are potential sources of iron and ROS, respectively, for mycobacteria multiplying intracellularly. Iron levels and oxidative stress are closely linked in aerobic organisms. Oxidative stress occurs when abnormally high levels of ROS are generated, resulting in DNA, protein and lipid damage. Iron deficiency can lead to oxidative stress, presumably by decreasing the activity of heme-containing enzymes that are involved in protection against ROS (Ref. 16), such as the M. tuberculosis catalase/peroxidase KatG. Iron overload as a result of deregulation of iron metabolism, for example in Escherichia coli fur mutants, leads to oxidative stress and DNA, protein and lipid damage via the Fenton reaction17, in which ferrous iron catalyzes the synthesis of highly reactive hydroxyl radicals from hydrogen peroxide. Consequently, aerobic organisms have evolved a set of protection systems to counteract the potentially lethal iron–ROS partnership. These include superoxide dismutases (SODs), which convert superoxides to hydrogen peroxide, and the related catalases and peroxidases, which degrade simple and organic peroxides. In enteric bacteria, the transcriptional regulators OxyR, SoxR/SoxS and RpoS, which activate the expression of several genes encoding anti-oxidant enzymes, are well characterized18. By contrast, little is known about the control of the oxidative-stress response in mycobacteria. Several protective enzymes have been described, including a catalase/peroxidase (KatG), a superoxide dismutase (SodA), and an alkyl hydroperoxide reductase (AhpC) (Table 3; Fig. 1). At least one of these, KatG, plays a role in mycobacterial pathogenesis19. Low levels of hydrogen peroxide can induce a protective response to oxidative stress in M. smegmatis: an OxyR-like response. However, an OxyR homolog has not been described in M. smegmatis. In the pathogens Mycobacterium avium, Mycobacterium bovis, bacillus Calmette– Guérin (BCG)20 and M. tuberculosis21, hydrogen peroxide induces the synthesis of KatG, but this response is not protective. Interestingly, M. tuberculosis complex strains do have an oxyR homolog, but this open reading frame contains deletions, frameshifts and nonsense mutations that result in its inactivation20,21. This could be responsible for the failure to elaborate a protective response in these microorganisms11. The existence of other OxyR-like regulatory proteins or alternative detoxification pathways controlled by other inducible regulons cannot be ruled out. Pathogenic mycobacteria, unlike enterobacteria or sapro-
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Table 1. Siderophores in mycobacteria Class Characteristics Mycobactin
Exochelin
Family Solubility
Hydroxamate Water-soluble or a chloroform-extractable Secreted 1025–1030
Hydroxamate Lipophilic
Location Cell-wall associated Affinity constant 1035 for iron a
Also called carboxymycobactins.
phytic mycobacteria, respond to hydrogen peroxideinduced stress by producing a very limited repertoire of proteins20. This suggests the presence of a constitutive mechanism for some of the protection against oxidative stress. It has been hypothesized that the mycobacterial cell wall could be part of this constitutive defense against ROS (Ref. 20), and cell-wallassociated phenolic glycolipid I derivatives from M. leprae have been shown to be efficient ROS scavengers22. In addition, cyclopropanation of mycolic acids of pathogenic mycobacteria decreases the sensitivity to lipid peroxidation and might, therefore, be another constitutive line of defense against ROS (Ref. 23). As SoxR/SoxS-like proteins have not yet been described in the newly sequenced M. tuberculosis genome, the M. tuberculosis response to superoxide appears to be different from the E. coli and Salmonella typhimurium models. Exposure of M. tuberculosis to menadione, a superoxide-radical generator, has been shown to significantly induce heat-shock proteins but not the SOD (Ref. 24). Taken together, these data indicate that enteric bacteria and mycobacterial species have major differences in their responses to oxidative stress but that the latter possess equally complex regulatory networks. IdeR, the repressor of the mycobacterial iron regulon discussed above, is necessary for a robust oxidative-stress response in M. smegmatis10. Recent results indicate that IdeR positively regulates the levels of some antioxidant enzymes (Fig. 1), as M. smegmatis ideR mutants have lower levels of katG and sodA mRNA and protein than the wild-type strain (O. Dussurget et al., unpublished). The mechanism by which IdeR positively regulates these genes is currently unknown. Isoniazid resistance Isoniazid (INH) is a very effective, inexpensive drug that is central to current antituberculosis treatment. There has been much information relating mycobacterial INH resistance and oxidative-stress response. The mycobacterial KatG is involved in the peroxidatic conversion of the inactive form of INH to its active form, which inhibits one or possibly two enzymatic steps in mycolic acid biosynthesis25,26 (Fig. 1). As discussed above, these lipids are a major component of mycobacterial cell envelopes and are believed to be
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O2
Fe-transferrin
Mycobacterial cell wall Exochelin
ROH
Fe-exochelin
IdeR AhpC
O2
Mycobactin Fe(III)
OxyR
ROOH Receptor
Fe(II) RH .
O2−
INHi
Iron storage proteins
FeS clusters SodA
IdeR
H2O2
Fe(II)
KatG
OxyR
H2O
ROS Mycolic acids
Damage
INHa
Nucleic acids Proteins Lipids Carbohydrates
Other targets
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Fig. 1. (facing page) Model showing possible connections between iron, oxidants and isoniazid (INH) in mycobacteria. Hypothetical pathways are shown by dashed lines, green arrows indicate positive regulation, and red lines indicate negative regulation. Yellow, orange and blue backgrounds indicate INH activation and mode of action, oxygen metabolism and iron metabolism, respectively. Mycobacteria secrete exochelins, which solubilize and bind iron from ferri-transferrin (Fe-transferrin) or ferri-lactoferrin. The ferri-exochelin (Fe-exochelin) complex is taken up by an energy-dependent mechanism via a specific receptor, and iron is transferred to mycobactins, which presumably act as an iron reservoir. The mechanism of iron removal from siderophores might involve reduction to ferrous iron [Fe(II)]. Once in the cytoplasm, iron is incorporated into heme and non-heme iron molecules, such as iron–sulfur (FeS) clusters. Superoxides, which are generated by reduction of molecular oxygen and autoxidation of redox enzymes, are able to inactivate some FeS clusters by excision of iron and release of Fe(II). Hydrogen peroxide (H2O2), which is the product of superoxide dismutation, and Fe(II) might react to generate hydroxyl radicals via the Fenton reaction. Mycobacteria have specific enzymatic defense systems against reactive oxygen species (ROS): the superoxide dismutase (SodA) for superoxides (O22?), the catalase/peroxidase (KatG) for H2O2 and the alkyl hydroperoxide reductase (AhpC) for organic peroxides (ROOH). In addition to its protective role against ROS, KatG is involved in the conversion of the inactive form of INH (INHi) to its active form (INHa), which inhibits mycolic acid biosynthesis and presumably other targets. The production of ROS during activation of INH could lead to oxidative damage. Antioxidant enzymes and their putative positive regulators (e.g. AhpC, SodA, IdeR and OxyR) might protect against the toxicity of INH byproducts and/or intermediates. IdeR might have a central role in this network: as a regulator of siderophore biosynthesis and some antioxidant enzymes, and in INH resistance.
Table 2. DtxR- and IdeR-binding sites from iron-regulated genes a
b
Bacterium
Gene
Iron box
Corynebacterium diphtheriae
Consensus
T
A T
A
G G
T
T
A G
G C T C
A
A C
C
T
T A
A
Mycobacterium tuberculosis Mycobacterium smegmatis
tox irg1/irg2 fxbA
T A A
T T A
A A A
G G G G G G
A T T
T T A
A G C T T A G G C T A G G C T
T A T
A C C C A C
C C C
T A T A A A
A G T
The consensus sequence for DtxR binding has been identified by in vitro affinity selection8. tox codes for the diphtheria toxin, irg1 encodes HisE, which is involved in the histidine biosynthetic pathway, and irg2 encodes a protein of unknown function. irg1 and irg2 are divergently transcribed and have the same iron box. fxbA codes for a putative formyltransferase necessary for exochelin biosynthesis. b The bases that are underlined share identity with the consensus sequence. a
a
Table 3. Mycobacterial defenses against oxidative stress b
Enzyme
Gene
Notes
Catalase/peroxidase
katG
Catalase Mn-SOD Fe-SOD Cambialistic SOD
katE sodA sodA sodA
Thioredoxin Alkyl hydroperoxide reductase
trxA/trxB ahpC
Found in most mycobacteria; inactive in Mycobacterium leprae; found in Mycobacterium tuberculosis; associated with resistance to INH and virulence in guinea pigs Found in a limited number of mycobacteria Most mycobacteria have an Mn-SOD c M. tuberculosis has an Fe-SOD Mycobacterium smegmatis SOD is active with either iron or manganese as a metal ion ligand Found in many mycobacteria, including M. leprae and M. tuberculosis Found in many mycobacteria, including M. leprae, M. tuberculosis, M. smegmatis and Mycobacterium avium
a
Abbreviations: INH, isoniazid; SOD, superoxide dismutase. In Escherichia coli, KatG and AhpC are positively regulated by OxyR, the Mn-SOD is positively regulated by SoxR/SoxS, and KatE is positively regulated by KatF (RpoS). c An open reading frame coding for a putative Cu/Zn-SOD is found in the M. tuberculosis genome. b
essential for survival. There might be other targets for INH in mycobacteria because enteric bacteria lacking mycolic acids can be made susceptible to INH (Ref. 27). It has been postulated that ROS generated as byproducts of the INH-activation process can also lead to INH toxicity28–30. In addition, M. smegmatis treated with superoxide-generating drugs is more susceptible to INH, an effect that can be reversed by overexpressing SOD (Ref. 31). Overproduction of ROS, or defective systems of protection against ROS,
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results in increased levels of ROS, which can lead to cellular toxicity3. This could partly explain why M. tuberculosis, which possesses an inactive OxyR, is so susceptible to INH (Refs 11,32). Interestingly, some DKatG–INH-resistant M. tuberculosis strains have been shown to overproduce AhpC (Refs 32,33), the small subunit of the alkyl hydroperoxide reductase that has been implicated in the oxidative-stress response. The loss of KatG induces protection of M. tuberculosis against INH, but it also reduces
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Questions for future research • What is the concentration and availability of iron in a phagosome infected by Mycobacterium tuberculosis? • Are siderophores required for multiplication and virulence of M. tuberculosis? • Can siderophore biosynthetic genes and iron-uptake genes be targets for new antimycobacterial agents? • Are there any proteins with OxyR- and SoxR/SoxS-like function that can regulate oxidative-stress response in M. tuberculosis? • What other genes are controlled by IdeR in addition to those involved in iron uptake and oxidative-stress response? • What are the other mechanisms mediating isoniazid resistance in M. tuberculosis?
mycobacterial defenses that are important for survival during infection. Therefore, the overexpression of ahpC might compensate for the loss of KatG. A relationship between AhpC and INH resistance is also observed in M. smegmatis, because AhpC mutants are more susceptible to INH (Ref. 34). It is possible that AhpC may detoxify ROS formed during INH activation or that a defect in AhpC indirectly induces overexpression of KatG via a regulatory mechanism. Further evidence linking oxidative-stress enzymes and INH resistance has been obtained recently: M. smegmatis ideR mutants, showing decreased levels of KatG and SodA activity and wild-type levels of AhpC, are more susceptible to INH (O. Dussurget et al., unpublished). This result is puzzling, because reduced KatG levels are usually associated with resistance to INH. This effect does not seem to be caused by reduced levels of SodA, because M. smegmatis ideR mutants are more sensitive to INH than M. smegmatis sodA mutants (O. Dussurget et al., unpublished). As a controller of iron levels and oxidativestress response that is also necessary for INH resistance, IdeR might have a central role in this interrelated network. It can be hypothesized that, in addition to AhpC and SodA, which are putative detoxifiers of INH byproducts, there are other enzymes and putative regulators protecting against ROS (e.g. FurA and/or FurB). These might be expected to play a role, directly or indirectly, in the detoxification of INH and/or its byproducts, thereby leading to reduced susceptibility of mycobacteria to the drug. Greater knowledge of mycobacterial physiology and of INH activation and mode of action can be used to develop new drugs to treat tuberculosis, which is still the biggest killer among infectious diseases. Acknowledgements We thank B.R. Byers for sharing unpublished data. This work was supported by a fellowship from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (to O.D.) and NIH grant GM32651 (to I.S.). References 1 Earhardt, C.F. (1996) in Escherichia coli and Salmonella (Neidhardt, F.C., ed.), pp. 1075–1090, ASM Press 2 Litwin, C.M. and Calderwood, S.B. (1993) Clin. Microbiol. Rev. 6, 137–149
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3 Lynch, A.S. and Lin, E.C.C. (1996) in Escherichia coli and Salmonella (Neidhardt, F.C., ed.), pp. 1526–1538, ASM Press 4 Wheeler, P.R. and Ratledge, C. (1994) in Tuberculosis: pathogenesis, protection and control (Bloom, B.R., ed.), pp. 353–385, ASM Press 5 Lambrecht, R.S. and Collins, M.T. (1993) Microb. Pathog. 14, 229–238 6 Brooks, B.W. et al. (1991) J. Clin. Invest. 29, 1652–1658 7 Gobin, J. and Horwitz, M.A. (1996) J. Exp. Med. 183, 1527–1532 8 Tao, X. et al. (1994) Mol. Microbiol. 14, 191–197 9 Schmitt, M.P. et al. (1995) Infect. Immun. 63, 4284–4289 10 Dussurget, O., Rodriguez, M. and Smith, I. (1996) Mol. Microbiol. 22, 535–544 11 Deretic, V., Song, J. and Pagan-Ramos, E. (1997) Trends Microbiol. 5, 367–372 12 Smith, I. et al. (1997) 32nd U.S.–Japan Cooperative Medical Science Program Tuberculosis-Leprosy Research Conference, Cleveland, OH, pp. 32–36 13 Fiss, E.H., Yu, S., and Jacobs, W.R., Jr. (1994) Mol. Microbiol. 14, 557–569 14 Clemens, L.C. and Horwitz, M.A. (1996) J. Exp. Med. 184, 1349–1355 15 Kuo, H.P. et al. (1996) Tuber. Lung Dis. 77, 468–475 16 Lundrigan, M.D. et al. (1997) Biometals 10, 215–225 17 Touati, D. et al. (1995) J. Bacteriol. 177, 2305–2314 18 Rosner, J.L. and Storz, G. (1997) Curr. Top. Cell Regul. 35, 163–177 19 Collins, D.M. (1996) Trends Microbiol. 4, 426–430 20 Sherman, D.R. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6625–6629 21 Deretic, V. et al. (1995) Mol. Microbiol. 17, 889–900 22 Chan, J. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2453–2457 23 Yuan, Y. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6630–6634 24 Garbe, T.R., Hibler, N.R. and Deretic, V. (1996) Mol. Med. 2, 134–142 25 Banerjee, A. et al. (1994) Science 263, 227–230 26 Mdluli, K. et al. (1996) J. Infect. Dis. 174, 1085–1090 27 Rosner, J.L. (1993) Antimicrob. Agents Chemother. 37, 2251–2253 28 Shoeb, H.A. et al. (1985) Antimicrob. Agents Chemother. 27, 408–412 29 Shoeb, H.A. et al. (1985) Antimicrob. Agents Chemother. 27, 404–407 30 Ito, K., Yamamoto, K. and Kawanishi, S. (1992) Biochemistry 31, 11606–11613 31 Wang, J.Y., Burger, R.M. and Drlica, K. (1998) Antimicrob. Agents Chemother. 42, 709–711 32 Dhandayuthapani, S. et al. (1996) J. Bacteriol. 178, 3641–3649 33 Sherman, D.R. et al. (1996) Science 272, 1641–1643 34 Zhang, Y., Dhandayuthapani, S. and Deretic, V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13212–13216
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VOL. 6 NO. 9 SEPTEMBER 1998
Interdependence of mycobacterial iron regulation, oxidative-stress response and isoniazid resistance Olivier Dussurget and Issar Smith
I
ron is required by almost Iron is an essential cofactor for vital is a major bacterial antigen all living organisms as an functions in microorganisms. Bacterial in cattle infected with Mycoessential cofactor of propathogens have developed efficient ironbacterium paratuberculosis6. teins with important cellular acquisition systems to counteract the Mycobacterium tuberculosis functions, such as those redefensive sequestration of iron by their exochelins can also remove iron quired for DNA and amino hosts. In mycobacteria, the recently from human transferrin and acid synthesis, detoxification described protein, IdeR, negatively lactoferrin and transfer iron to and respiration. Thus, iron controls iron-uptake systems. This protein desferri-mycobactins in the cell acquisition is a vital function also has a role in the oxidative-stress wall7 (Fig. 1). The process of for survival1. The concenresponse, as well as in resistance to the iron assimilation in vivo is not tration of free iron in the envi- frontline antimycobacterial drug isoniazid. known and might vary, deronment and in human serum pending on the environment (~10218 M) is far below the O. Dussurget and I. Smith* are in the Public Health of mycobacteria (extracellular Research Institute, 455 1st Avenue, New York, nutritional requirements of vs. intracellular). A definitive NY 10016, USA; O. Dussurget is also in the UFR microorganisms. In response answer to the question of iron de Biochimie, Université Paris 7, 75251 Paris to low iron availability in the acquisition and mycobacterial Cedex 05, France; *tel: +1 212 578 0868, environment and the acutedisease awaits the isolation of fax: +1 212 578 0804, phase response hypoferremia M. tuberculosis mutants with e-mail: [email protected] that they face during infection, defects in the iron-uptake sysmicroorganisms have develtem for virulence assessment. oped several mechanisms for iron uptake2. In Gram-negative bacteria and some Gram-positive Many microorganisms secrete siderophores: low- bacteria, the expression of the genes involved in the molecular-weight, high-affinity iron chelators that uptake of iron is controlled by Fur, a repressor prosolubilize iron complexed in the environment or tein that employs ferrous iron as a co-repressor1. bound to compounds from their hosts, such as trans- DtxR, another transcriptional repressor, has been ferrin and lactoferrin. In addition, microorganisms characterized in Corynebacterium diphtheriae8. DtxR can acquire iron by various mechanisms, including controls the expression of genes for the iron-uptake secretion of soluble reductants that reduce ferric iron machinery, as well as the gene encoding the diphto ferrous iron, synthesis of specific receptors for theria toxin. In mycobacteria, IdeR, a homolog of transferrin and/or lactoferrin, production of hemo- DtxR, has been described9 and shown to regulate total lysins and toxins, utilization of heme compounds, siderophore biosynthesis10 (Fig. 1), suggesting it is a and uptake by low-affinity systems1,2. major regulator of the mycobacterial iron-acquisition However, an excess of iron can be deleterious, as apparatus. However, IdeR does not account for all this element can catalyze the formation of reactive adaptive responses to iron starvation10, and additionoxygen species (ROS) in aerobically growing cells3; al control mechanisms probably play a role in mycoconsequently, all living organisms have developed bacterial iron regulation. In this regard, two open systems to carefully regulate iron levels. reading frames with high similarity to Fur (FurA and FurB) have been observed in mycobacteria11,12 and Mycobacterial iron uptake regulation their role in iron regulation is being investigated. The In response to iron starvation, mycobacteria produce first gene to be characterized that is involved in iron several siderophores (Table 1), iron-storage proteins acquisition in mycobacteria is fxbA, which encodes a and putative receptors4. Currently, little is known putative formyltransferase necessary for exochelin about the role of mycobacterial siderophores and biosynthesis in Mycobacterium smegmatis13. The iron regulation in the infected host, and the impor- fxbA gene possesses an IdeR-regulated and irontance of these compounds in mycobacterial patho- regulated promoter, to which IdeR binds (Table 2; genesis is not clear4,5. However, it is likely that iron O. Dussurget et al., unpublished). A recent search in acquisition and metabolism are important in this the M. tuberculosis genome database has isolated process. The iron storage protein bacterioferritin several genes with potential IdeR-binding sequences Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS
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PII: S0966-842X(98)01307-9
VOL. 6 NO. 9 SEPTEMBER 1998
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(iron boxes) in their promoter regions. Two of these genes, irg1 and irg2, encode HisE, which is involved in histidine biosynthesis, and a putative membrane protein of unknown function, respectively. The expression of these genes is negatively regulated by iron in M. tuberculosis, and their promoter regions bind IdeR at the predicted iron box sequence (Table 2; M. Rodriguez et al., unpublished). Mycobacterial response to oxidative stress Ferri-transferrin delivered to the phagosome14 and oxidative metabolism of alveolar macrophages15 are potential sources of iron and ROS, respectively, for mycobacteria multiplying intracellularly. Iron levels and oxidative stress are closely linked in aerobic organisms. Oxidative stress occurs when abnormally high levels of ROS are generated, resulting in DNA, protein and lipid damage. Iron deficiency can lead to oxidative stress, presumably by decreasing the activity of heme-containing enzymes that are involved in protection against ROS (Ref. 16), such as the M. tuberculosis catalase/peroxidase KatG. Iron overload as a result of deregulation of iron metabolism, for example in Escherichia coli fur mutants, leads to oxidative stress and DNA, protein and lipid damage via the Fenton reaction17, in which ferrous iron catalyzes the synthesis of highly reactive hydroxyl radicals from hydrogen peroxide. Consequently, aerobic organisms have evolved a set of protection systems to counteract the potentially lethal iron–ROS partnership. These include superoxide dismutases (SODs), which convert superoxides to hydrogen peroxide, and the related catalases and peroxidases, which degrade simple and organic peroxides. In enteric bacteria, the transcriptional regulators OxyR, SoxR/SoxS and RpoS, which activate the expression of several genes encoding anti-oxidant enzymes, are well characterized18. By contrast, little is known about the control of the oxidative-stress response in mycobacteria. Several protective enzymes have been described, including a catalase/peroxidase (KatG), a superoxide dismutase (SodA), and an alkyl hydroperoxide reductase (AhpC) (Table 3; Fig. 1). At least one of these, KatG, plays a role in mycobacterial pathogenesis19. Low levels of hydrogen peroxide can induce a protective response to oxidative stress in M. smegmatis: an OxyR-like response. However, an OxyR homolog has not been described in M. smegmatis. In the pathogens Mycobacterium avium, Mycobacterium bovis, bacillus Calmette– Guérin (BCG)20 and M. tuberculosis21, hydrogen peroxide induces the synthesis of KatG, but this response is not protective. Interestingly, M. tuberculosis complex strains do have an oxyR homolog, but this open reading frame contains deletions, frameshifts and nonsense mutations that result in its inactivation20,21. This could be responsible for the failure to elaborate a protective response in these microorganisms11. The existence of other OxyR-like regulatory proteins or alternative detoxification pathways controlled by other inducible regulons cannot be ruled out. Pathogenic mycobacteria, unlike enterobacteria or sapro-
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Table 1. Siderophores in mycobacteria Class Characteristics Mycobactin
Exochelin
Family Solubility
Hydroxamate Water-soluble or a chloroform-extractable Secreted 1025–1030
Hydroxamate Lipophilic
Location Cell-wall associated Affinity constant 1035 for iron a
Also called carboxymycobactins.
phytic mycobacteria, respond to hydrogen peroxideinduced stress by producing a very limited repertoire of proteins20. This suggests the presence of a constitutive mechanism for some of the protection against oxidative stress. It has been hypothesized that the mycobacterial cell wall could be part of this constitutive defense against ROS (Ref. 20), and cell-wallassociated phenolic glycolipid I derivatives from M. leprae have been shown to be efficient ROS scavengers22. In addition, cyclopropanation of mycolic acids of pathogenic mycobacteria decreases the sensitivity to lipid peroxidation and might, therefore, be another constitutive line of defense against ROS (Ref. 23). As SoxR/SoxS-like proteins have not yet been described in the newly sequenced M. tuberculosis genome, the M. tuberculosis response to superoxide appears to be different from the E. coli and Salmonella typhimurium models. Exposure of M. tuberculosis to menadione, a superoxide-radical generator, has been shown to significantly induce heat-shock proteins but not the SOD (Ref. 24). Taken together, these data indicate that enteric bacteria and mycobacterial species have major differences in their responses to oxidative stress but that the latter possess equally complex regulatory networks. IdeR, the repressor of the mycobacterial iron regulon discussed above, is necessary for a robust oxidative-stress response in M. smegmatis10. Recent results indicate that IdeR positively regulates the levels of some antioxidant enzymes (Fig. 1), as M. smegmatis ideR mutants have lower levels of katG and sodA mRNA and protein than the wild-type strain (O. Dussurget et al., unpublished). The mechanism by which IdeR positively regulates these genes is currently unknown. Isoniazid resistance Isoniazid (INH) is a very effective, inexpensive drug that is central to current antituberculosis treatment. There has been much information relating mycobacterial INH resistance and oxidative-stress response. The mycobacterial KatG is involved in the peroxidatic conversion of the inactive form of INH to its active form, which inhibits one or possibly two enzymatic steps in mycolic acid biosynthesis25,26 (Fig. 1). As discussed above, these lipids are a major component of mycobacterial cell envelopes and are believed to be
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O2
Fe-transferrin
Mycobacterial cell wall Exochelin
ROH
Fe-exochelin
IdeR AhpC
O2
Mycobactin Fe(III)
OxyR
ROOH Receptor
Fe(II) RH .
O2−
INHi
Iron storage proteins
FeS clusters SodA
IdeR
H2O2
Fe(II)
KatG
OxyR
H2O
ROS Mycolic acids
Damage
INHa
Nucleic acids Proteins Lipids Carbohydrates
Other targets
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Fig. 1. (facing page) Model showing possible connections between iron, oxidants and isoniazid (INH) in mycobacteria. Hypothetical pathways are shown by dashed lines, green arrows indicate positive regulation, and red lines indicate negative regulation. Yellow, orange and blue backgrounds indicate INH activation and mode of action, oxygen metabolism and iron metabolism, respectively. Mycobacteria secrete exochelins, which solubilize and bind iron from ferri-transferrin (Fe-transferrin) or ferri-lactoferrin. The ferri-exochelin (Fe-exochelin) complex is taken up by an energy-dependent mechanism via a specific receptor, and iron is transferred to mycobactins, which presumably act as an iron reservoir. The mechanism of iron removal from siderophores might involve reduction to ferrous iron [Fe(II)]. Once in the cytoplasm, iron is incorporated into heme and non-heme iron molecules, such as iron–sulfur (FeS) clusters. Superoxides, which are generated by reduction of molecular oxygen and autoxidation of redox enzymes, are able to inactivate some FeS clusters by excision of iron and release of Fe(II). Hydrogen peroxide (H2O2), which is the product of superoxide dismutation, and Fe(II) might react to generate hydroxyl radicals via the Fenton reaction. Mycobacteria have specific enzymatic defense systems against reactive oxygen species (ROS): the superoxide dismutase (SodA) for superoxides (O22?), the catalase/peroxidase (KatG) for H2O2 and the alkyl hydroperoxide reductase (AhpC) for organic peroxides (ROOH). In addition to its protective role against ROS, KatG is involved in the conversion of the inactive form of INH (INHi) to its active form (INHa), which inhibits mycolic acid biosynthesis and presumably other targets. The production of ROS during activation of INH could lead to oxidative damage. Antioxidant enzymes and their putative positive regulators (e.g. AhpC, SodA, IdeR and OxyR) might protect against the toxicity of INH byproducts and/or intermediates. IdeR might have a central role in this network: as a regulator of siderophore biosynthesis and some antioxidant enzymes, and in INH resistance.
Table 2. DtxR- and IdeR-binding sites from iron-regulated genes a
b
Bacterium
Gene
Iron box
Corynebacterium diphtheriae
Consensus
T
A T
A
G G
T
T
A G
G C T C
A
A C
C
T
T A
A
Mycobacterium tuberculosis Mycobacterium smegmatis
tox irg1/irg2 fxbA
T A A
T T A
A A A
G G G G G G
A T T
T T A
A G C T T A G G C T A G G C T
T A T
A C C C A C
C C C
T A T A A A
A G T
The consensus sequence for DtxR binding has been identified by in vitro affinity selection8. tox codes for the diphtheria toxin, irg1 encodes HisE, which is involved in the histidine biosynthetic pathway, and irg2 encodes a protein of unknown function. irg1 and irg2 are divergently transcribed and have the same iron box. fxbA codes for a putative formyltransferase necessary for exochelin biosynthesis. b The bases that are underlined share identity with the consensus sequence. a
a
Table 3. Mycobacterial defenses against oxidative stress b
Enzyme
Gene
Notes
Catalase/peroxidase
katG
Catalase Mn-SOD Fe-SOD Cambialistic SOD
katE sodA sodA sodA
Thioredoxin Alkyl hydroperoxide reductase
trxA/trxB ahpC
Found in most mycobacteria; inactive in Mycobacterium leprae; found in Mycobacterium tuberculosis; associated with resistance to INH and virulence in guinea pigs Found in a limited number of mycobacteria Most mycobacteria have an Mn-SOD c M. tuberculosis has an Fe-SOD Mycobacterium smegmatis SOD is active with either iron or manganese as a metal ion ligand Found in many mycobacteria, including M. leprae and M. tuberculosis Found in many mycobacteria, including M. leprae, M. tuberculosis, M. smegmatis and Mycobacterium avium
a
Abbreviations: INH, isoniazid; SOD, superoxide dismutase. In Escherichia coli, KatG and AhpC are positively regulated by OxyR, the Mn-SOD is positively regulated by SoxR/SoxS, and KatE is positively regulated by KatF (RpoS). c An open reading frame coding for a putative Cu/Zn-SOD is found in the M. tuberculosis genome. b
essential for survival. There might be other targets for INH in mycobacteria because enteric bacteria lacking mycolic acids can be made susceptible to INH (Ref. 27). It has been postulated that ROS generated as byproducts of the INH-activation process can also lead to INH toxicity28–30. In addition, M. smegmatis treated with superoxide-generating drugs is more susceptible to INH, an effect that can be reversed by overexpressing SOD (Ref. 31). Overproduction of ROS, or defective systems of protection against ROS,
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results in increased levels of ROS, which can lead to cellular toxicity3. This could partly explain why M. tuberculosis, which possesses an inactive OxyR, is so susceptible to INH (Refs 11,32). Interestingly, some DKatG–INH-resistant M. tuberculosis strains have been shown to overproduce AhpC (Refs 32,33), the small subunit of the alkyl hydroperoxide reductase that has been implicated in the oxidative-stress response. The loss of KatG induces protection of M. tuberculosis against INH, but it also reduces
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Questions for future research • What is the concentration and availability of iron in a phagosome infected by Mycobacterium tuberculosis? • Are siderophores required for multiplication and virulence of M. tuberculosis? • Can siderophore biosynthetic genes and iron-uptake genes be targets for new antimycobacterial agents? • Are there any proteins with OxyR- and SoxR/SoxS-like function that can regulate oxidative-stress response in M. tuberculosis? • What other genes are controlled by IdeR in addition to those involved in iron uptake and oxidative-stress response? • What are the other mechanisms mediating isoniazid resistance in M. tuberculosis?
mycobacterial defenses that are important for survival during infection. Therefore, the overexpression of ahpC might compensate for the loss of KatG. A relationship between AhpC and INH resistance is also observed in M. smegmatis, because AhpC mutants are more susceptible to INH (Ref. 34). It is possible that AhpC may detoxify ROS formed during INH activation or that a defect in AhpC indirectly induces overexpression of KatG via a regulatory mechanism. Further evidence linking oxidative-stress enzymes and INH resistance has been obtained recently: M. smegmatis ideR mutants, showing decreased levels of KatG and SodA activity and wild-type levels of AhpC, are more susceptible to INH (O. Dussurget et al., unpublished). This result is puzzling, because reduced KatG levels are usually associated with resistance to INH. This effect does not seem to be caused by reduced levels of SodA, because M. smegmatis ideR mutants are more sensitive to INH than M. smegmatis sodA mutants (O. Dussurget et al., unpublished). As a controller of iron levels and oxidativestress response that is also necessary for INH resistance, IdeR might have a central role in this interrelated network. It can be hypothesized that, in addition to AhpC and SodA, which are putative detoxifiers of INH byproducts, there are other enzymes and putative regulators protecting against ROS (e.g. FurA and/or FurB). These might be expected to play a role, directly or indirectly, in the detoxification of INH and/or its byproducts, thereby leading to reduced susceptibility of mycobacteria to the drug. Greater knowledge of mycobacterial physiology and of INH activation and mode of action can be used to develop new drugs to treat tuberculosis, which is still the biggest killer among infectious diseases. Acknowledgements We thank B.R. Byers for sharing unpublished data. This work was supported by a fellowship from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (to O.D.) and NIH grant GM32651 (to I.S.). References 1 Earhardt, C.F. (1996) in Escherichia coli and Salmonella (Neidhardt, F.C., ed.), pp. 1075–1090, ASM Press 2 Litwin, C.M. and Calderwood, S.B. (1993) Clin. Microbiol. Rev. 6, 137–149
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3 Lynch, A.S. and Lin, E.C.C. (1996) in Escherichia coli and Salmonella (Neidhardt, F.C., ed.), pp. 1526–1538, ASM Press 4 Wheeler, P.R. and Ratledge, C. (1994) in Tuberculosis: pathogenesis, protection and control (Bloom, B.R., ed.), pp. 353–385, ASM Press 5 Lambrecht, R.S. and Collins, M.T. (1993) Microb. Pathog. 14, 229–238 6 Brooks, B.W. et al. (1991) J. Clin. Invest. 29, 1652–1658 7 Gobin, J. and Horwitz, M.A. (1996) J. Exp. Med. 183, 1527–1532 8 Tao, X. et al. (1994) Mol. Microbiol. 14, 191–197 9 Schmitt, M.P. et al. (1995) Infect. Immun. 63, 4284–4289 10 Dussurget, O., Rodriguez, M. and Smith, I. (1996) Mol. Microbiol. 22, 535–544 11 Deretic, V., Song, J. and Pagan-Ramos, E. (1997) Trends Microbiol. 5, 367–372 12 Smith, I. et al. (1997) 32nd U.S.–Japan Cooperative Medical Science Program Tuberculosis-Leprosy Research Conference, Cleveland, OH, pp. 32–36 13 Fiss, E.H., Yu, S., and Jacobs, W.R., Jr. (1994) Mol. Microbiol. 14, 557–569 14 Clemens, L.C. and Horwitz, M.A. (1996) J. Exp. Med. 184, 1349–1355 15 Kuo, H.P. et al. (1996) Tuber. Lung Dis. 77, 468–475 16 Lundrigan, M.D. et al. (1997) Biometals 10, 215–225 17 Touati, D. et al. (1995) J. Bacteriol. 177, 2305–2314 18 Rosner, J.L. and Storz, G. (1997) Curr. Top. Cell Regul. 35, 163–177 19 Collins, D.M. (1996) Trends Microbiol. 4, 426–430 20 Sherman, D.R. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6625–6629 21 Deretic, V. et al. (1995) Mol. Microbiol. 17, 889–900 22 Chan, J. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2453–2457 23 Yuan, Y. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6630–6634 24 Garbe, T.R., Hibler, N.R. and Deretic, V. (1996) Mol. Med. 2, 134–142 25 Banerjee, A. et al. (1994) Science 263, 227–230 26 Mdluli, K. et al. (1996) J. Infect. Dis. 174, 1085–1090 27 Rosner, J.L. (1993) Antimicrob. Agents Chemother. 37, 2251–2253 28 Shoeb, H.A. et al. (1985) Antimicrob. Agents Chemother. 27, 408–412 29 Shoeb, H.A. et al. (1985) Antimicrob. Agents Chemother. 27, 404–407 30 Ito, K., Yamamoto, K. and Kawanishi, S. (1992) Biochemistry 31, 11606–11613 31 Wang, J.Y., Burger, R.M. and Drlica, K. (1998) Antimicrob. Agents Chemother. 42, 709–711 32 Dhandayuthapani, S. et al. (1996) J. Bacteriol. 178, 3641–3649 33 Sherman, D.R. et al. (1996) Science 272, 1641–1643 34 Zhang, Y., Dhandayuthapani, S. and Deretic, V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13212–13216
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