A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptibleto Killing by Antibiotics and Is Unable To Establish a ChronicInfection in Mice

Ruchi Pandey and G. Marcela Rodriguez

Public Health Research Institute and Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NewJersey, USA

Iron is an essential, elusive, and potentially toxic nutrient for most pathogens, including Mycobacterium tuberculosis. Due to thepoor solubility of ferric iron under aerobic conditions, free iron is not found in the host. M. tuberculosis requires specializediron acquisition systems to replicate and cause disease. It also depends on a strict control of iron metabolism and intracellulariron levels to prevent iron-mediated toxicity. Under conditions of iron sufficiency, M. tuberculosis represses iron acquisition andinduces iron storage, suggesting an important role for iron storage proteins in iron homeostasis. M. tuberculosis synthesizes twoiron storage proteins, a ferritin (BfrB) and a bacterioferritin (BfrA). The individual contributions of these proteins to the adap-tive response of M. tuberculosis to changes in iron availability are not clear. By generating individual knockout strains of bfrAand bfrB, the contribution of each one of these proteins to the maintenance of iron homeostasis was determined. The effect ofaltered iron homeostasis, resulting from impaired iron storage, on the resistance of M. tuberculosis to in vitro and in vivostresses was examined. The results show that ferritin is required to maintain iron homeostasis, whereas bacterioferritin seems tobe dispensable for this function. M. tuberculosis lacking ferritin suffers from iron-mediated toxicity, is unable to persist in mice,and, most importantly, is highly susceptible to killing by antibiotics, showing that endogenous oxidative stress can enhance theantibiotic killing of this important pathogen. These results are relevant for the design of new therapeutic strategies against M.tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, con-tinues to be a major cause of morbidity and mortality world-

wide. The capacity to control the current TB pandemic is chal-lenged by the association of TB and HIV infection and the increasein numbers of multidrug-resistant M. tuberculosis strains. An un-derstanding of the molecular mechanisms enabling M. tuberculo-sis to adapt to different nutritional environments and adverse con-ditions encountered in the host is important for the design of newtools against this pathogen. Like most living organisms, M. tuber-culosis requires iron as a cofactor of enzymes involved in vitalcellular functions. Due to the poor aqueous solubility of ferric ion(Fe3�) in the presence of oxygen and at a neutral pH, free iron isnot found in the mammalian host but is sequestered in complexeswith iron binding proteins such as transferrin, ferritin, and lacto-ferrin or bound to protoporphyrins in heme and hemoproteins(40).

The capacity to acquire iron in the host is indispensable for M.tuberculosis to proliferate and cause disease (27). To obtain iron,M. tuberculosis synthesizes and secretes high-affinity ferric ironchelators (mycobactins) (33), and in vitro, it also utilizes heme asan iron source (18, 34). Although essential, iron can also be toxic,due to the ability of Fe2� to catalyze the generation of deleteriousoxygen radicals from normal products of aerobic metabolismthorough the Fenton reaction (12, 17). To prevent iron-mediatedtoxicity, aerobic bacteria tightly control intracellular iron levels byregulating the transcription of genes involved in iron acquisition,transport, and storage. In M. tuberculosis, this is accomplishedthrough the action of IdeR, an essential transcriptional regulatorthat binds iron and represses genes involved in siderophore syn-thesis (mbt) and transport (irtAB), while it upregulates iron stor-age genes (bfrA and bfrB) under conditions of iron sufficiency (15,

26). The synthesis of iron storage proteins is central to iron ho-meostasis in most aerobic organisms. Eubacteria, archaebacteria,and eukaryotes store iron in ferritin (2). Some bacteria and fungisynthesize ferritin-like proteins containing heme b, which areknown as bacterioferritins. Ferritins have been shown to be essen-tial for the survival of several pathogenic organisms in infectedhosts (37). Some bacteria have only one ferritin or bacterioferri-tin, others have two ferritins or two bacterioferritins, and a fewbacteria, including M. tuberculosis, Vibrio cholerae, Clostridiumacetobutylicum, Erwinia chrysanthemi, and Escherichia coli, con-tain one ferritin and one bacterioferritin (2). The basic role offerritins and bacterioferritins is conserved; however, there is sig-nificant variation in the function and regulation of these proteinsin different bacterial species. For example, E. coli and Campylobac-ter jejuni depend on ferritin for growth in low levels of iron (1, 36).However, C. jejuni but not E. coli ferritin confers protectionagainst iron-mediated oxidative stress. Bacterioferritin can serveas an iron source during an iron deficiency in Neisseria gonor-rhoeae (6) but is dispensable for this role in E. coli (1). In fact, nophenotype has been identified for E. coli bacterioferritin mutants,

Received 6 March 2012 Returned for modification 28 March 2012Accepted 28 June 2012

Published ahead of print 16 July 2012

Editor: J. L. Flynn

Address correspondence to G. Marcela Rodriguez, rodrigg2@umdnj.edu.

Supplemental material for this article may be found at http://iai.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00229-12

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and its role in E. coli remains unknown. Ferritin subunits form aspherical shell, where up to 4,500 atoms of Fe3� can be seques-tered. The uptake of iron by ferritins involves an initial step ofoxidation of Fe2� by molecular oxygen at the ferroxidase center,and iron is stored as Fe3�. Ferritins can release stored iron in timesof need, while they exert a protective antioxidant function bykeeping iron inside the cavity in a nonreactive state under condi-tions of iron sufficiency (5).

M. tuberculosis possesses one gene (Rv1876) encoding a bacte-rioferritin (BfrA) and one ferritin (BfrB)-encoding gene (Rv3841)(8). The crystal structures of M. tuberculosis bacterioferritin (16)and ferritin (19) show the highly conserved architecture of theferritin superfamily of a cage-like hollow shell formed by 24monomers with the characteristic fold of a four-helical bundlecontaining the ferroxidase catalytic center and, in bacterioferritin,a heme group in each subunit pair interface. Consistent with theirpostulated function, bfrA and bfrB are induced in M. tuberculosiscells cultured under conditions of iron sufficiency (15, 28). How-ever, bfrB is also upregulated in M. tuberculosis cells subjected tolow levels of oxygen (32) or NO (14, 24) and during the stationaryphase (35), reflecting possible functional differences between BfrAand BfrB.

Previously, an M. tuberculosis bfrA bfrB double mutant wasreported to be deficient for growth under low-iron conditions, inmacrophages, and in guinea pigs (25). However, the individualcontributions of BfrA and BfrB to iron storage, iron homeostasis,and virulence remain unclear. Here, we report that BfrB is impor-tant whereas BfrA is dispensable for adaptation to iron limitation,resistance to oxidative stress, and the maintenance of iron homeo-stasis. M. tuberculosis lacking BfrB was found to be unable to per-sist in mice and exhibited enhanced susceptibility to antibiotickilling. Our results have implications for the development of moreeffective therapeutic strategies against M. tuberculosis and possiblyother pathogens.

MATERIALS AND METHODSBacterial strains, media, and chemicals. Escherichia coli strains JM109and XL-10 (Stratagene) were used for cloning and were grown in Luria-Bertani (LB) broth. M. tuberculosis strains were maintained in 7H10 agar(Difco) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10%ADN supplement (0.5% albumin, 0.2% dextrose, 0.085% NaCl). M. tu-berculosis cells were grown in liquid 7H9 medium (Difco) or in low-irondefined medium (LIMM) prepared as previously described (27). Briefly,LIMM contains 0.5% (wt/vol) L-asparagine, 0.5% (wt/vol) KH2PO4, 2%glycerol, 0.05% Tween 80, and 10% ADN. The pH was adjusted to 6.8. To

lower the level of trace metal contamination, the medium was treated withChelex-100 (Bio-Rad Laboratories) according to the manufacturer’s in-structions. Chelex was removed by filtration, and before use, the mediumwas supplemented with 0.5 mg ZnCl2/liter, 0.1 mg/liter MnSO4, and 40mg/liter MgSO4. This medium contained less than 2 �M residual iron, asdetermined by atomic absorption spectroscopy.

Transformants were selected in medium supplemented with 100�g/ml of hygromycin (Hyg), 20 �g/ml of streptomycin (Strp), and 75�g/ml of spectinomycin (Spec), as indicated. For antibiotic sensitivityassays, antibiotics were used at the specified concentrations.

DNA manipulation and analysis. Standard procedures for cloning,PCR, and restriction digestions were performed as described previously(30). Plasmid DNA from E. coli and PCR products were isolated by usingQiagen kits. All modifying and restriction enzymes were obtained fromNew England BioLabs (NEB), unless otherwise indicated, and were usedas specified. All constructs were verified by DNA sequencing. Analysis ofDNA sequences was performed by using Vector NTI (Invitrogen).

Construction of bfrA and bfrB deletion mutants. bfrA and bfrB dele-tion mutants were created via specialized transduction and allelic ex-change by using a method developed previously (3). Briefly, 500 bp ofDNA upstream and downstream of bfrA and bfrB was amplified by PCR(for the primers used, see Table S1 in the supplemental material) includ-ing appropriate restrictions sites and cloned into the cosmid vectorpjsc284, flanking a Hyg resistance cassette, generating plasmids pSM822and pSM821, respectively (Table 1). pSM821 and pSM822 were digestedwith PacI and ligated into DNA from the mycobacteriophage shuttlephasmid phAE87. The ligation mixture was packed by using Gigapack KL(Stratagene), and phage particles were used to transduce E. coli HB101.Transductants were selected on agar plates containing Hyg. PhasmidDNA was isolated from Hygr E. coli transformants and electroporated intoM. smegmatis mc21551 cells. Transducing phages were plaque purified,titrated, and used to transduce M. tuberculosis. Transductants were se-lected in 7H10 agar containing 100 �g/ml Hyg. Chromosomal DNA wasisolated from Hygr colonies for PCR amplification. PCR amplification wasdone by using the Failsafe PCR system (Epicenter). PCRs were performedfor each candidate mutant by using primers in the Hyg cassette and thesequence flanking the predicted insertion site, as indicated in Fig. S1. PCRfragments were purified and sequenced to verify correct allelic exchangeand the deletion of bfrA or bfrB in M. tuberculosis. The deletion of bfrA wasalso confirmed by Southern blotting (see Fig. S1 in the supplementalmaterial).

Complementation of the bfrB mutant. bfrB with its own promoterwas PCR amplified from chromosomal DNA by using primersBfrBUPFw200 and BfrBDRev32 (see Table S1 in the supplemental mate-rial). The PCR product was cloned at the StuI site of pSM316 (22), anintegrative plasmid containing Strp, Spec, and kanamycin (Kan) resis-tance cassettes. The resulting complementing plasmid, pSM852, was elec-troporated into bfrB mutant strain ST214, and transformants were se-

TABLE 1 Plasmids and strains

Plasmid or strain Characteristic(s) Reference or source

PlasmidspJSC284 Vector to generate substrate for homologous recombination; Hygr 3pSM316 Integrative vector (Strp Spec Kan) 22pSM821 pJSC284 containing allelic exchange substrate for bfrB deletion This workpSM822 pJCS284 containing allelic exchange substrate for bfrA deletion This workpSM852 bfrB and its promoter into pSM316 This work

M. tuberculosis strainsH37Rv Wild-type strain ATCCST214 �bfrB This workST215 �bfrA This workST216 �bfrB::bfrB This work

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lected on 7H10 agar containing Strp and Spec. The complemented strainwas named ST216.

Oxidative stress sensitivity assays. Sensitivity to H2O2 and the super-oxide generator menadione was tested by zone inhibition assays, as pre-viously described (28). Briefly, M. tuberculosis strains were grown to thelogarithmic phase (optical density at 595 nm [OD595] of 0.5) in 7H9medium, and approximately 3 � 107 bacteria were plated onto 7H10 agaror 7H10 agar plus 5 �M the iron chelator 2=,2-dypyridyl (DPI) and spreadevenly. A 6.5-mm paper disc saturated with 10 �l of a solution containing500 mM hydrogen peroxide or 40 mM menadione was placed onto thecenter of the plate. After incubation for 10 days, the diameter of the halo ofgrowth inhibition generated by each compound was measured.

Streptonigrin sensitivity assay. The microplate version of the alamar-Blue assay (MABA) (13) was used to assess the susceptibilities of myco-bacterial strains to streptonigrin. Briefly, 2 � 105 bacteria in microtiterplates were incubated at 37°C in 7H9 medium containing increasing con-centrations of streptonigrin. Twenty microliters of alamarBlue was added,and 24 h later, the fluorescence emitted by cell-reduced alamarBlue wasmeasured with a GloMax-Multi detection system (Promega). The MIC isdefined as the minimal concentration that inhibits 99% of growth.

Antibiotic susceptibility assays. For MIC determinations, 2-fold an-tibiotic serial dilutions were inoculated with a suspension of M. tubercu-losis strains, and the optical density of the cultures incubated at 37°C withagitation was monitored for 5 days. The MIC was defined as the minimalconcentration of antibiotic that inhibited the increase in the optical den-sity by at least 90% in comparison to a culture with no antibiotic. Forkilling assays, M. tuberculosis strains were treated with the different anti-biotics at the specified concentrations for 1 or 2 days in 7H9 medium withagitation at 37°C. Dilutions of the antibiotic-treated cultures as well asnontreated controls were plated onto 7H10 agar plates to enumerate CFU.Where specified, 100 �M DPI and 1 mM thiourea were added simultane-ously with each antibiotic.

Mouse infection and histopathology. For each strain tested, a 10-mlbacterial suspension of 1 � 106 bacilli/ml in saline containing 0.04%Tween 80 was used. Aerosols were generated with a Lovelace nebulizer(In-tox Products, Albuquerque, NM), and female C57BL/6 mice wereexposed to the aerosol for 30 min. Under these conditions, the number ofmicroorganisms detected in the lungs at time zero (4 h postinfection) wasapproximately 100. At the indicated time points after infection, 3 mice foreach strain were sacrificed, and lungs, spleen, and liver were removed andhomogenized in phosphate-buffered saline (PBS)–Tween 80. Dilutions ofthe homogenates were plated onto 7H10 agar to determine the numberof CFU.

For histopathology evaluations, lung tissue was removed, fixed in 10%buffered formalin (Sigma-Aldrich), and paraffin embedded. Sectionswere stained with hematoxylin-eosin (HE) or Ziehl-Neelsen (ZN) acid-fast stain for evaluation of pathology.

RESULTSRole of BfrA and BfrB in adaptation to iron limitation. bfrA andbfrB were separately deleted and replaced by a hygromycin resis-tance cassette in the chromosome of M. tuberculosis H73Rv, usingphage transduction and allelic exchange by homologous recom-bination. The creation of the �bfrB and �bfrA deletion mutantswas confirmed by PCR amplification, sequencing, and Southernblotting (see Fig. S1 in the supplemental material). To examine therole of BfrA and BfrB as iron storage proteins, the �bfrB and �bfrAmutants were grown under conditions of iron sufficiency on 7H10agar and then inoculated into LIMM (�2 �M Fe3�); the �bfrAmutant grew similarly to the wild-type strain, whereas the �bfrBmutant showed a defect in replication under conditions of irondeficiency, which was fully complemented by the introduction ofwild-type bfrB (Fig. 1). These results suggest that under normalconditions, BfrB is the main provider of stored iron to sustain M.tuberculosis replication under conditions of iron limitation.

Deletion of bfrB causes sensitivity to iron. The �bfrB mutantdid not grow as well as the wild type, the bfrB complementedstrain, or the �bfrA mutant when cultured in 7H9 medium(Fig. 2). Since 7H9 medium is an iron-rich medium (80 to 100 �MFe3�), this suggested that the �bfrB mutant could be sensitive tohigh iron levels. To test that possibility, the �bfrB mutant wasgrown in LIMM supplemented with increasing concentrations ofiron. As shown in Fig. 3A, the growth of the �bfrB mutant wasstimulated by the addition of up to 40 to 50 �M FeCl3 to LIMM,but higher concentrations, such as those in 7H9 medium, inhib-ited growth compared to supplementation with 40 �M FeCl3.Moreover, the addition of 2=,2-dypiridyl (DPI), a permeable fer-rous iron chelator, to 7H9 medium improved the growth of the�bfrB mutant, indicating that the reduced growth in 7H9 mediumwas iron dependent (Fig. 3B). DPI treatment had no effect on thegrowth of the wild-type strain in 7H9 medium (data not shown).These results suggested that in the absence of bfrB, during growthin iron-rich medium, inappropriately stored iron has a deleteriouseffect. To further examine this possibility, we compared the sen-sitivities of the wild type, the �bfrB mutant, and the bfrB comple-mented strain to streptonigrin. Streptonigrin is an antibiotic thatrequires iron for its bactericidal activity, and sensitivity to thisantibiotic is related directly to intracellular iron levels. Killing bystreptonigrin correlates with increased iron uptake, and mutantsdeficient in iron uptake exhibit increased resistance to streptoni-

FIG 1 Growth of M. tuberculosis strains under iron-deficient conditions.Wild-type strain H37Rv (}), the �bfrA mutant (Œ), the �bfrB mutant (�),and the �bfrB mutant complemented with wild-type bfrB (�) were grown inLIMM with agitation at 37°C. Growth was monitored by measuring the in-crease in the optical density (O.D) at 540 nm. Shown are mean values (� stan-dard deviations) from three experiments.

FIG 2 Growth of M. tuberculosis strains in 7H9 medium. Wild-type strainH37Rv (}), the �bfrA mutant (Œ), the �bfrB mutant (�), and the comple-mented �bfrB mutant (�) were grown in 7H9 medium with agitation at 37°C.The growth of each strain was monitored by measuring the increase in theoptical density (O.D) at 540 nm. Shown are mean values (� standard devia-tions) from three experiments.

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grin compared to wild-type strains (29, 41). The streptonigrinMIC for the �bfrB strain in 7H9 medium was 1.2 �g/ml, whereasthe MIC for the wild-type and complemented strains was 10 �g/ml, showing that the level of sensitivity to streptonigrin was highlyelevated in the �bfrB strain (Fig. 4). This result supports the ideathat the lack of bfrB increases cellular levels of reactive iron, whichinhibits the normal replication of M. tuberculosis under iron-suf-ficient conditions.

BfrB and sensitivity to oxidative stress. Fe2� enhances oxida-tive stress by reacting with H2O2 and generating the highly toxichydroxyl radical (OH·). Therefore, the increased concentration ofreactive iron as a result of deficient iron storage could compromiseresistance to oxidative stress. In addition, ferritins can have a di-rect role in protection against oxidative damage by detoxifyingdioxygen and its radical products by the ferroxidase reaction. Totest the effect of bfrB and bfrA deletions on the resistance of M.tuberculosis to oxidative stress, we examined the sensitivities of thewild-type strain and the �bfrA and �bfrB mutants to H2O2 and theredox-cycling agent menadione. This compound reduces molec-ular oxygen and generates superoxide, which reduces Fe3�

through the Haber-Weiss reaction and provides more Fe2� for theFenton reaction. Although the sensitivity of the �bfrA mutant tooxidative stress was similar to that of the wild type, the �bfrBmutant was more sensitive to oxidative stress caused by H2O2

(Fig. 5A) and menadione (Fig. 5B). The sensitivity of the wild typeto these compounds was fully restored in the complemented

strain, confirming that this was a phenotype attributable to thelack of bfrB (Fig. 5A and B). Iron chelation with DPI decreased thesensitivity of the bfrB mutant to hydrogen peroxide, indicatingthat iron contributes to oxidative stress in this strain (Fig. 5C).Together, these results indicate a major role for BfrB in protectingM. tuberculosis against oxidative damage and support an impor-tant connection between iron metabolism and resistance to oxi-dative stress.

BfrB is essential for persistence of M. tuberculosis duringchronic infection of mice. Taken together, the results indicatethat BfrB plays a major role in maintaining cellular iron homeo-stasis in M. tuberculosis. To evaluate how a deficiency in iron ho-meostasis affects the pathogenesis of M. tuberculosis, mice wereaerosol infected with the �bfrB, wild-type, and complementedstrains. The enumeration of CFU in lungs at various time pointsduring infection showed that the �bfrB mutant replicated duringthe first 4 weeks of infection albeit at a lower rate than those of thewild-type and complemented strains, but by 8 weeks, the numberof CFU decreased, and by 12 weeks, there were about 3 logs fewerbacteria in the lungs of �bfrB mutant-infected mice than in thelungs of mice infected with the wild-type and complementedstrains (Fig. 6A). The mutant strain was also attenuated for growthin the spleen (Fig. 6B) and was unable to colonize the liver, as nobacteria were detected at any time point in the liver of mice in-fected with the bfrB mutant strain (Fig. 6C). Histopathology anal-ysis of infected mouse lungs (Fig. 7) showed small to mediumperivascular and peribronchial collections of lymphocytes at 4weeks in all infected mice but more frequently in the mice infectedwith the wild-type and complemented strains than in those in-fected with the �bfrB mutant. Those small lesions progressed tolarge regions of cellular infiltrates consisting of multiple foci oflymphocytes embedded in fields of foamy macrophages in themice infected with the wild-type and complemented strains at 8weeks and persisted at 12 weeks postinfection. In the �bfrB mu-tant-infected mice, the lymphocytic lesions increased in size andnumber at 8 weeks but did not exhibit significant macrophageinfiltrates at 8 weeks or even 12 weeks.

These results indicate that impeded iron storage negatively im-pacts the ability of M. tuberculosis to resist the challenges imposedby the adaptive immune response and to establish a chronic infec-tion in mice.

Deletion of bfrB enhances susceptibility to antibiotics. Anal-yses of the mechanism of antibiotic killing in E. coli have indicated

FIG 3 Iron-dependent growth of the �bfrB mutant. (A) Growth of the �bfrB mutant in LIMM supplemented with 0 �M (�), 10 �M (�), 20 �M (}), 40 �M(Œ), 80 �M (�), or 100 �M (Œ) FeCl3. (B) Growth of the �bfrB mutant in 7H9 medium (�) and 7H9 medium with 75 �M DPI (�). Shown are mean values(� standard deviations) from three experiments.

FIG 4 Sensitivity to streptonigrin. Wild-type strain H37Rv (Œ), the �bfrBmutant (�), and the �bfrB complemented strain (}) were incubated withincreasing concentrations of streptonigrin. The fluorescence resulting fromthe reduction of alamarBlue by each strain is shown. Shown are mean values(� standard deviations) from three experiments.

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that, regardless of their primary targets, many bactericidal antibi-otics induce a common stress response involving alterations incentral metabolism and iron homeostasis leading to the produc-tion of hydroxyl radicals, which ultimately contribute to killing by

these drugs (20). Hydroxyl radical production upon treatmentwith lethal concentrations of bactericidal antibiotics was shownpreviously to be dependent on intracellular iron and its participa-tion in the Fenton reaction (20). We reasoned that the breakdown

FIG 5 Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H2O2 (A) or40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns) or 7H10 agar plus DPI (dark columns). The valuesrepresent means plus standard deviations from three experiments. �, P � 0.01.

FIG 6 Replication of M. tuberculosis strains in mice. Mice were infected with each M. tuberculosis strain as described in Materials and Methods. At the indicatedtime points, mice were sacrificed, and bacteria in the lungs (A), spleen (B), and liver (C) were plated for determinations of CFU. The data are reported as theaverages and standard deviations of the CFU per organ of three infected mice. No CFU were detected in the liver of mice infected with the �bfrB mutant, but avalue of 1 CFU was plotted on the graph. The experiment was repeated twice. �, H37Rv; o, �bfrB mutant; Œ, �bfrB complemented strain.

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of iron homeostasis resulting from the bfrB deletion could poten-tiate the action of bactericidal antibiotics if, as in E. coli, the iron-dependent generation of hydroxyl radicals contributes to antibi-otic killing in M. tuberculosis. To test this hypothesis, we comparedthe sensitivities of the wild type and the bfrB mutant to killing bykanamycin, gentamicin, spectinomycin, ciprofloxacin, rifampin,and isoniazid (INH) as representatives of different classes of anti-biotics targeting different cell functions. The aminocyclitols, ka-namycin, gentamicin, and spectinomycin inhibit protein synthe-sis; the quinolones ciprofloxacin and moxifloxacin interfere withDNA supercoiling and block DNA replication; rifampin inhibitsRNA synthesis; and isoniazid inhibits cell wall synthesis. First, wedetermined the MICs of these antibiotics for the wild-type, �bfrB,and complemented strains. As shown in Table 2, the growth of thebfrB mutant was inhibited more efficiently than the growth ofthe wild-type and complemented strains by all antibiotics, withthe exception of rifampin. The �bfrB strain was also killed moreefficiently than the wild-type strain by all antibiotics tested exceptfor rifampin (Fig. 8). Complementation with bfrB reversed theantibiotic hypersensitivity of the �bfrB strain (Tables 2 and 3).

To determine whether the hypersensitivity of the bfrB mutantto antibiotics was dependent on iron and endogenous oxidativestress, we examined the effects of iron chelation and the addition

of the hydroxyl radical quencher thiourea on the survival of thebfrB mutant to antibiotic treatment. The chelation of intracellulariron with DPI or treatment with thiourea protected the �bfrBstrain against killing by ciprofloxacin, gentamicin, kanamycin,and isoniazid (Fig. 9). The same treatments did not affect thesurvival of the wild-type strain at bactericidal antibiotic concen-trations (Table 4). These results support our hypothesis that in theabsence of bfrB, M. tuberculosis suffers from iron-mediated oxida-tive stress, which increases susceptibility to antibiotic killing. Ironchelation and radical quenching did not protect the bfrB mutantfrom killing by moxifloxacin and spectinomycin.

DISCUSSION

The work presented here analyzed the role of ferritin and bacte-rioferritin in the iron homeostasis and virulence of M. tuberculosis.Our results indicate that the functions of bacterioferritin and fer-ritin are not redundant in M. tuberculosis. BfrB is required to over-come iron limitation and for protection against oxidative stress,whereas BfrA was dispensable for successful adaptation to thosestresses. The lack of bfrB renders M. tuberculosis sensitive to highlevels of iron and hypersensitive to the iron-activated antibioticstreptonigrin and to oxidative stress, indicating that bfrB is essen-tial to prevent iron toxicity and maintain iron homeostasis in thispathogen. Although we did not detect a phenotype in a bfrA mu-tant, BfrA may function under very specific conditions not testedin this study. In a previous study (25), a marginal increase in thesensitivity of a bfrA mutant to oxidative stress was reported, whichwe did not observe for our mutant. However, the complementa-tion of that phenotype was not demonstrated; therefore, it is notpossible to conclude that it was due to a lack of bfrA. Thus, thefunction of BfrA remains unknown. Our current studies aim toidentify a phenotype in vitro or in vivo for the bfrA mutant thatreveals the function of this protein in M. tuberculosis.

Our results show that bfrB is essential for the persistence of M.tuberculosis in mice. This mutant grows more slowly than thewild-type and complemented strains during the first 4 weekspostinfection, and, coinciding with the onset of the adaptive im-mune response, it was then progressively eliminated from the

FIG 7 Histopathology of M. tuberculosis-infected lungs. Shown are lung sections stained with HE at 4 and 12 weeks after infection of mice with the wild-type,�bfrB, and complemented �bfrB strains. Data are from individual sections and are representative of three infected mice and two independent experiments.

TABLE 2 Susceptibility of M. tuberculosis strains to antibioticsa

Antibiotic

MIC (�g/ml)

H37Rv �bfrB �bfrB::bfrB

Gentamicin 20 8 20Kanamycin 20 1 NDCiprofloxacin 10 2 10Moxifloxacin 10 0.4 10Spectinomycin 25 1 NDRifampin 0.6 0.6 0.6Isoniazid 0.2 0.04 0.2a The data show MICs (�g/ml) assayed in triplicate with a range of 2-fold dilutions ofantibiotics. ND, not determined, as the complementing plasmid confers resistance tothose antibiotics.

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lungs of infected mice. This finding indicates that BfrB is necessaryto survive the stress imposed by the adaptive immune responseand to establish a chronic infection in mice. Based on the pheno-types of the �bfrB strain in vitro, one may speculate that this mu-tant may die from iron limitation and/or oxidative stress in vivo.Before the onset of the adaptive immune response, �bfrB cellsinfecting resident macrophages can use siderophores and obtainiron from the ferric transferrin present in early endosomes thatfuse with the maturation-arrested M. tuberculosis phagosome (7).

However, upon macrophage activation, phagosome-lysosome fu-sion eliminates the interaction of the phagosome with early endo-somes, restricting iron acquisition, and this combined with thelack of BfrB-stored iron may limit replication and favor the elim-ination of the bacteria by the immune defense. In addition, the

FIG 8 Sensitivity of the wild type and the �bfrB mutant to antibiotics. The wild-type (Œ) and �bfrB (�) strains were cultured for 2 days in 7H9 medium withno drug added or with increasing concentrations of antibiotics. Dilutions of the cultures were plated, and CFU were enumerated. The percent survival wascalculated for each strain treated with different concentrations of antibiotics, taking the number of CFU in the culture not exposed to antibiotics as 100%. Shownare mean values (� standard deviations) from three experiments.

TABLE 3 Complementation of the antibiotic sensitivity phenotype ofthe bfrB mutanta

Antibiotic

Mean % survival � SD

H37Rv �bfrB �bfrB::bfrB

Kanamycinb 97 � 4.2 0.1 � 0.01 NDGentamicinb 84 � 4 0.93 � 0.09 71 � 6Spectinomycinb 97 � 2 0.94 � 0.2 NDCiprofloxacinb 97 � 3.7 0.09 � 0.03 93 � 3Moxifloxacinc 65 � 6.9 0.8 � 0.08 75 � 2Isoniazidd 97 � 3 0.05 � 0.001 90 � 3a Shown are mean values (� standard deviations) from three experiments to determinepercent survival in the presence of antibiotics for the wild-type, �bfrB, and bfrBcomplemented strains. ND, not determined, since the selection cassette in thecomplementing plasmid confers resistance to that antibiotic.b Two-day treatment with 0.5 �g/ml of antibiotic.c Two-day treatment with 0.1 �g/ml of antibiotic.d One-day treatment with 0.1 �g/ml of antibiotic.

FIG 9 Effects of DPI and thiourea on survival of the �bfrB mutant to antibi-otics. Cells of the �bfrB strain were treated with antibiotic (white columns),antibiotic plus DPI (light gray columns), antibiotic plus thiourea (dark graycolumns), or antibiotic plus DPI and thiourea (black columns). The data showthe means plus standard deviations from three experiments. The percent sur-vival for each treatment was calculated by taking the number of CFU in anontreated culture as 100%. Antibiotics were added at the following concen-trations: 0.5 �g/ml ciprofloxacin (Cipro), 0.5 �g/ml gentamicin (Gent), 0.5�g/ml moxifloxacin (Moxi), 0.5 �g/ml spectinomycin (Spec), and 0.5 �g/mlkanamycin (Kan) for 2 days, and 0.1 �g/ml isoniazid (INH) for 1 day.

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bfrB mutant is likely to be more sensitive to the combined toxicityof NO and reactive oxygen species generated in activated macro-phages. Remarkably, no viable bacilli were recovered from thelivers of mice infected with the bfrB mutant at any time pointexamined, even though viable bacilli were recovered from thespleen, suggesting that the strain was able to disseminate from thelungs but was unable to proliferate in the liver. It has been notedthat in animal models and in cases of human extrapulmonarytuberculosis, the liver is the least infected organ. Studies usingmice have suggested that this is due to the microanatomical struc-ture of the liver, where the narrow sinusoids restrict the access ofM. tuberculosis to parenchymal cells, limiting the infection to pro-fessional phagocytes, which are better equipped than endothelialcells to kill M. tuberculosis cells (31). Hence, the increased sensi-tivity to the bactericidal mechanisms of liver macrophages mayunderline the inefficient colonization of this organ by the bfrBmutant.

In contrast to our results, single bfrA and bfrB mutants werereported previously to not be attenuated in guinea pigs (25). Thisdiscrepancy may reflect differences in the immune pressureagainst M. tuberculosis generated in mice and guinea pigs.

A common mechanism of cell death induced by quinolones,aminoglycosides, and �-lactams was proposed for E. coli based onthe fact that the primary drug-target interactions increased tricar-boxylic acid (TCA) cycle and respiratory activity, stimulating theoxidation of NADH in the electron transport chain and therebyincreasing superoxide formation. Superoxide damages Fe-S clus-ters, making ferrous iron available for oxidation by the Fentonreaction and the formation of hydroxyl radicals that damage pro-teins, lipids, and DNA and contribute to cell death (20). Since therole of intracellular iron in the generation of hydroxyl radicals wasat the core of this antibiotic killing mechanism, we hypothesizedthat the alteration of iron homeostasis in the �bfrB strain wouldincrease its susceptibility to antibiotics. Our results show that anM. tuberculosis strain lacking BfrB was killed more efficiently bygentamicin, kanamycin, spectinomycin, ciprofloxacin, moxi-floxacin, and isoniazid (INH), suggesting that the deletion of bfrBsensitized M. tuberculosis to a killing mechanism common to thesediverse antibiotics. The addition of a permeable ferrous iron ch-elator and thiourea, which quenches hydroxyl radicals, protectedthe bfrB mutant, but not the wild-type strain, from killing by gen-tamicin, kanamycin, ciprofloxacin, and isoniazid, indicating thatiron-dependent endogenous oxidative stress contributes to thehypersensitivity of the �bfrB strain to these antibiotics. The fact

that iron chelation and radical quenching protected the bfrB mu-tant from killing by ciprofloxacin but not by moxifloxacin is con-sistent with previously observed differences in the lethal pathwaysinduced by these two quinolones. It was shown previously thatciprofloxacin’s lethal action against M. smegmatis is sensitive tothe inhibition of protein synthesis, whereas moxifloxacin lethalityis not (21), and in E. coli, hydroxyl radical damage contributes tocell death via protein synthesis inhibitor-sensitive quinolones butnot via moxifloxacin (39).

Of special relevance is the increased killing of the bfrB mutantby the first-line antituberculosis drug INH. Although the maintarget of isoniazid is the mycolic acid biosynthesis enzyme InhA, ageneral toxicity of INH dependent on oxidative stress has beensupported by several related observations reviewed previously (9):(i) the activation of isoniazid by catalase-peroxidase (KatG) invitro generates reactive oxygen species; (ii) M. tuberculosis is hy-persensitive to INH and is a natural mutant of OxyR, the mainactivator of the oxidative stress defense response in other bacteria;(iii) M. tuberculosis transformed with OxyR from Mycobacteriumleprae (an organism that is not sensitive to isoniazid) is less sensi-tive to INH; (iv) Mycobacterium smegmatis cells treated with su-peroxide-generating drugs are more susceptible to INH, an effectwhich can be reversed by the overexpression of superoxide dismu-tase (38); and (v) an M. smegmatis ideR mutant has deregulatediron acquisition systems and is more sensitive to both oxidativestress and INH (10, 11). Our results showing that the bfrB mutantis more sensitive to INH and that this increased sensitivity isdependent on iron and reactive oxygen intermediates supportthe idea that oxidative stress potentiates the killing of M. tuber-culosis by INH.

Interestingly, our results show that the bfrB mutant was notmore sensitive to rifampin than the wild-type strain, suggestingthat endogenous oxidative stress does not contribute to the killingof M. tuberculosis by this antibiotic. Since rifampin is a very effi-cient inhibitor of transcription, cells treated with this antibioticmay not be able to mount a metabolic response and activate theoxidative damage cell death pathway that is enhanced in the �bfrBstrain. This is in agreement with the global downregulation ofgene expression observed for M. tuberculosis in response to treat-ment with rifamycins (4).

It is worth noting that according to our results, M. tuberculosisdiffers from E. coli in that iron chelation and hydroxyl radicalquenching did not protect wild-type M. tuberculosis cells fromkilling by bactericidal antibiotics (Table 4) (20). This finding sug-

TABLE 4 Effects of DPI and thiourea on survival of H37Rv against antibiotics

Antibiotic

Mean % survival of H37Rv � SDa

Ab Ab-DPI Ab-T Ab-DPI-T

Ciprofoxacinb 0.96 � 0.02 0.94 � 0.04 0.094 � 0.04 0.92 � 0.1Gentamicinc 0.82 � 0.06 0.83 � 0.01 0.81 � 0.02 0.83 � 0.02Kanamycinb 0.86 � 0.07 0.90 � 0.05 0.96 � 0.05 0.90 � 0.03Moxifloxacinb 0.05 � 0.009 0.05 � 0.005 0.045 � 0.01 0.04 � 0.01Spectinomycinb 0.92 � 0.08 1 � 0.01 0.90 � 0.06 0.99 � 0.1Isoniazidd 0.001 � 0.0003 0.001 � 0.0002 0.001 � 0.0002 0.001 � 0.0002a Shown are mean values (� standard deviations) from three experiments. Ab, antibiotic; Ab-DPI, antibiotic plus DPI; Ab-T, antibiotic plus thiourea; Ab-DPI-T, antibiotic plusDPI and thiourea.b Two-day treatment with 1 �g/ml of antibiotic.c Two-day treatment with 2 �g/ml of antibiotic.d One-day treatment with 0.4 �g/ml of antibiotic.

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gests differences in the contributions of oxidative stress to antibi-otic killing between wild-type M. tuberculosis and E. coli, whichmay originate from distinct responses to antibiotics and/or oxida-tive stress in these two bacteria.

At least 4 months of continued treatment with multiple anti-biotics, which often have undesirable secondary effects, is neces-sary to cure TB (23). The failure of antibiotic treatment to com-pletely eliminate the bacilli results in relapse, continuedtransmission, and the potential development of drug resistance.Strategies that enhance the killing activity of currently availableantibiotics could have a great impact on reducing persistence, in-creasing the efficacy of preventive therapy, and precluding thedevelopment of drug-resistant bacteria. If the bfrB mutant is alsomore sensitive to antibiotic killing in vivo, it might be possible toenvision antibiotic therapy combined with the inhibition of BfrBas a good strategy to potentiate the antibiotic killing of M. tuber-culosis. An encouraging observation is that, unlike most other fer-ritins, BfrB has an extended C terminus involved in ferroxidaseactivity (19), which may represent a target for inhibition.

In conclusion, we have shown that the ferritin BfrB plays anessential role in iron homeostasis in M. tuberculosis and is indis-pensable for the resistance of this pathogen to immune mecha-nisms of defense. We have also demonstrated that the antibiotickilling of M. tuberculosis can be enhanced by the deletion of bfrB.To the best of our knowledge, this is the first report showing thatdirect interference with cellular iron homeostasis results in in-creased susceptibility to antibiotics. This has implications for thedevelopment of new strategies to alter iron homeostasis in orderto potentiate the effect of antibiotics not only against M. tubercu-losis but also against other pathogens.

ACKNOWLEDGMENTS

We thank Issar Smith, Jeanie Dubnau, and Barun Mathema for valuablediscussions and critical reading of the manuscript and Irina Kolesnikovafor technical assistance with the mouse infections.

This work was supported NIH grant AI044856 (G.M.R.).

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