INFECTION AND IMMUNITY, Dec. 1975, p. 1242-1251 Copy:ight © 1975 American Society for Microbiology

Vol. 12, No. 6 Printed in U.SA.

Extracellular Iron Acquisition by Mycobacteria: Role of the Exochelins and Evidence Against the Participation of Mycobactin LIONEL P. MACHAM,' COLIN RATLEDGE,* AND JENNIFER C. NOCTON Department of Biochemistry, The University of Hull, Hull, HU6 7RX, England

Received for publication 8 August 1975

Mycobacterium bovis var. BCG was grown under iron-deficient conditions in the presence and absence of 1% Tween 80. Mycobactin, the iron ionophore of mycobacteria, was found solely within the bacteria grown in the absence of Tween, but low concentrations (0.75 ,ug/ml) of it appeared in the medium in the presence of the surfactant. Both types of medium contain agents, named exochelins, which could solubilize iron. 55Fe added to spent culture media was recovered only chelated to these compounds. Two exochelins were detected, isolated, and purified. Neither were precursors or breakdown products of mycobactin. In the desferri-form, exochelin MB-2, the major component, reversed the inhibitory effect of serum on the growth of BCG, and in their ferri-forms exochelins MB-1, MB-2, and MS (from Mycobacterium smegmatis) stimulated the growth of their producing organism in the presence of serum. Exochelin MB-2 could physically remove iron from ferritin, and BCG used ferritin as a source of iron during growth even when ferritin was separated from the bacteria by a dialysis membrane. As solutions of the exochelins were freely dialyzable, whereas solutions of mycobactin, even in Tween, were not, only exochelin could have been active in this experiment. The exochelins are proposed as the functional extracellular iron-binding agents of BCG and other mycobacteria, the role of mycobactin being confined to that of a cell wall iron transporter. The acquisition of iron is essential for the growth of a microorganism whether in vitro or in vivo. As this element is usually present either in vitro in an insoluble form [e.g., Fe(OH)j] or in vivo complexed to an organic molecule of the host (e.g., transferrin), microorganisms are obliged to sequester iron for themselves under most growth conditions. The situation is further complicated as the bacteria may not be able to come into direct contact with the source of iron and therefore need to release iron-chelating molecules into their environment to achieve iron solubilization. A wide range of such molecules has been described (11, 13) and they have been given the collective name "siderochromes" (7). Characteristically, the production of siderochromes is much greater in media where there is a shortage of iron (11, 13). Mycobacteria differ from other bacteria in that they elaborate more than one type of ironbinding compound. So far three compounds of distinctly different properties have been described: mycobactin (21), salicylic acid (20), and exochelins (12). ' Present address: Glasgow College of Technology, Glasgow, Scotland.

Mycobactin is a lipophilic molecule and is located in the boundary layers of the organism where it transports iron across the thick lipoidal layers of the mycobacterial cell (18). Iron is released from this carrier by conversion of the ferric ion to a ferrous ion by a reduced nicotinamide adenine dinucleotide phosphate-linked reductase (3), and the ferrous ion is then available for intracellular purposes. Salicylic acid, on the other hand, is found predominantly in the extracellular medium (20). Initially salicyclic acid was thought to fulfil the role of the extracellular iron-sequestering agent, but this particular function has been discounted as it fails to hold iron in solution at pH 7 in the presence of competing anions such as phosphate (17). The failure to demonstrate the effectiveness of salicylate, however, has led to the search for, and discovery of, a new series of extracellular, water-soluble, iron-binding compounds which do hold iron in solution under physiological conditions and to which we have given the name exochelins (12). We have already briefly described the nature and properties of exochelin MS, i.e., that from Mycobacterium smegmatis, and have reported the occurrence of a similar compound in the

1242

VOL. 12, 1975

IRON ACQUISITION BY MYCOBACTERIA

1243

culture media of M. bovis var. BCG, i.e., exo- tion fluid, and the radioactivity then was counted chelins MB. In this paper we wish to elaborate (see below). Further quantities were prepared using non-radioupon the nature of this latter compound and to FeCl3 in a similar manner. Large-scale chrodescribe experiments which lead us to ascribe active accomplished by using a column matography its function as that of the extracellular iron- (60 by 18 mm)was of neutral alumina (Brockmann grade sequestering agent. While we were carrying 1, BDH Chemicals Ltd, Poole, U.K.), the crude exoout this work, we were aware that Kochan and chelins being applied in a concentrated chloroform his associates (6, 8-10) carried out other experi- solution. A minor component termed MB-1 did not ments towards similar objectives but that they adsorb to the column and emerged immediately. concluded that the sequestering compound was MB-2, the major component, was eluted with water mycobactin. We believe this view to be mis- after washing the column with methanol. The aqueous eluate was re-extracted as above with chlotaken. roform and further purified by TLC using solvent MATERIALS AND METHODS Organisms and their growth. M. bovis var. BCG (from Glaxo Laboratories Ltd, Slough, England) and Mycobacterium smegmatis NCIB 8548 were grown with shaking in 100 ml of iron-deficient medium as previously described (16). Whenever Tween 80, at 1%, was included in the medium this is stated explicitly in the text. Unless stated otherwise, all media were iron deficient (Fe = 0.05 ,ug/ml). Harvesting of cultures and the estimation of cell dry weights were as described previously (18). Purification, identification, and estimation of mycobactin. Mycobactin was extracted from freshly harvested bacteria and purified as previously described (18, 23). For the attempted estimation of mycobactin in non-Tween-containing medium, an excess of FeCl3 was added to the filtered medium which was then extracted twice with an equal volume of chloroform. The chloroform was held over anhydrous MgSO4 and evaporated to dryness. The residue was dissolved in a minimum volume of methanol, and the whole of this was then chromatographed on thin layers of Silica Gel G using solvent system A as described below. For the estimation of mycobactin in Tween-containing medium, the above procedure was followed except that the chloroformextracted material was dissolved in toluene and applied to a column (90 by 18 mm) of neutral alumina suspended in toluene. After washing with 50 ml of toluene, the mycobactin was eluted in chloroformtoluene (3:1, vol/vol). This procedure had to be adopted to remove Tween 80 extracted into the chloroform. The sample was then subjected to thin-layer chromatography (TLC) as above. Detection, extraction, and purification of exochelin MB. (Exochelins, probably having different structures from different species, are denoted as MB, MS, etc., to avoid confusion; see above.) Initially, the presence of iron-binding compounds in culture filtrates was determined by adding a solution of -"FeCl3 in 0.1 M HCI (14.7 ng; 90 nCi) to the filtered medium taken after supporting growth of BCG for 5 days. The medium was then extracted twice with an equal volume of chloroform. The chloroform extracts were combined, held over anhydrous MgSO4, and evaporated to dryness on a rotary evaporator. The residue was dissolved in 0.5 ml of methanol and 0.05 ml then was subjected to TLC using solvent system A (see below). Bands of 5 mm were scraped off across the length of each individual chromatogram and placed directly into 10 ml of scintilla-

system B. MB-1 and MB-2 were both red materials with similar visible and ultraviolet spectra and had El%.m values of approximately 23 at 450 nm (L. P. Macham, unpublished work). Samples of purified ferriexochelin fractions (about 1 mg) were converted to their desferri-forms by dissolving in about 1 ml of 50% aqueous methanol containing about 5 mg of ethylenediaminetetraacetate. After 24 h at 20 C, the mixture was extracted three times with chloroform. The chloroform layers were combined, washed with double-distilled water, held over anhydrous MgSO4, and evaporated to dryness under vacuum. Preparation of carboxy-[14C]salicyloyl exochelin MB. An analysis of the products from acid hydrolysis of exochelin MB showed salicylic acid to be present (L. P. Macham, unpublished work). This afforded a method for preparing labeled exochelin MB. Carboxy-['4C]salicylic acid (0.5 MCi) (Radiochemical Centre, Amersham, U.K.) was sterilized by membrane filtration and added to growth medium, to give 1 zg/ml, immediately prior to inoculation. After 5 days of growth of BCG, the exochelins were recovered from the medium and then purified as described above. About 25 nCi of radioactivity was recovered in the main exochelin (MB-2) and about 3 nCi in the minor component. Possible metabolism of carboxy-['4C]salicyloyl exochelin MB-2 was checked by adding 17.5 nCi, after sterilization by membrane filtration, to a culture of BCG 3 days after inoculation, this coinciding with the onset of mycobactin formation. After a further 4 days of growth, mycobactin was subsequently extracted from the cells and purified. The possible incorporation of radioactivity was monitored by counting in the scintillant as given below. Purification of exochelin MS. Exochelins MS (from M. smegmatis) were isolated and purified by ion-exchange chromatography as previously described (12). The major component was further purified by chromatography through a column (400 by 25 mm) of Sephadex G10 (Pharmacia, Uppsala, Sweden) in distilled water. (It too is a red compound when in the ferri-form and has an E `tm of approximately 23; it does not contain salicylic acid [12; L. P. Macham, unpublished work].) TLC. For TLC, glass plates precoated with 250gm layers of Silica Gel G (Anachem Ltd, Luton, U.K.) were used either with solvent system A (petroleum ether-n-butanol-ethyl acetate, 2:3:3, vol/vol/ vol) or system B (methanol-ethyl acetate, 4:1, vol/ vol).

1244

MACHAM, RATLEDGE, AND NOCTON

INFECT. IMMUN.

Radioactive counting. Samples were counted by RESULTS liquid scintillation using as scintillant naphthalene An important difference between the results (100 g); 2,5-diphenyloxazole (10 g); and 1,4-di-(2-[5phenyloxazolyll)benzene (0.25 g) in 1 liter of toluene. of Ratledge and Marshall (18), who were unable Internal standard corrections for quenching were to detect mycobactin in the medium of M. smegmade where appropriate (see references 16, 18). matis, and Kochan et al. (10), who reported the Agar well diffusion experiments and growth in occurrence of mycobactin in BCG media, was serum. For agar well experiments, the method of the incorporation of Tween 80 into the medium Kochan et al. (9) was followed, with the following of the latter workers. Estimations of mycobacmodifications: 90-mm diameter petri dishes were tin in BCG cultures therefore were made as filled with 20 ml of Dubos serum agar medium using calf serum (Flow Laboratories, Irvine, Scotland) at carefully as possible both in cultures that con25%. The agar was inoculated with 0.3 ml of bacte- tained or lacked 1-4 Tween 80. Culture filtrates rial suspension having an extinction of 0.01 at 600 were taken at daily intervals throughout nm. The plates were incubated at 37 C for 2 h, any growth and mycobactin was extracted, as given excess medium was then drained off, and the plates above. The sensitivity of the assay for mycobacdried for a further hour. Wells were cut in the agar tin is such that 20 ng of mycobactin/ml of mewith a 1-cm-diameter glass tube and then filled with dium would have been detected. No mycobactin 120 ,ul of solutions of ferriexochelins (180 jg/ml) in double-distilled water. Zones of growth were as- was detected at any time in cultures growing without Tween 80, though the amount of mycosessed after 3 days of incubation at 37 C. For the testing of growth of BCG and M. smegma- bactin within the cells was high: after 5, 7, and tis in serum, a 30-ml tissue culture flask (A/S Nunc, 9 days, cultures contained 3, 16, and 27 mg of Denmark) containing 5 ml of undiluted calf serum mycobactin/100 ml of culture (i.e., equivalent to was inoculated with 0.5 ml of a bacterial suspension about 6%7 of the cell dry weight at 9 days). Thus, with an extinction at 600 nm of 0.1. Exochelins were the partition of mycobactin is at least 1,500:1 in added from stock solutions to give 45 ,g/ml. The favor of the cells and is probably considerably bottles were incubated without shaking at 37 C. higher than this since Ratledge and Marshall They were examined daily for 10 successive days for failed to detect mycobactin in the culture filgrowth in comparison with unsupplemented con- trates of M. smegmatis using an isotopic labeltrols. Removal of iron from ferritin using isolated ing procedure (18). They concluded that mycodesferriexochelin. Desferriexochelin MB was bactin could not be present in the medium at added to 10 ml of 50 mM phosphate buffer, pH 7.0, more than 20 pg/ml. When cultures containing 17c Tween 80 were held in a 15-ml vial to give 150 jig/ml. A dialysis bag (30 by 6 mm) containing 0.1 ml of 110 mg of ferritin examined, 230 ,g of mycobactin was recovered solution (B Grade, Calbiochem, Inc., San Diego, from 300 ml of medium taken after supporting Calif.) per ml was placed within the vial and the growth for 4 days. Thus, the addition of a surfaccontents of the vial were stirred at 15 C for 9 days. tant to cultures can, in fact, result in the apCetyl trimethylammonium bromide (0.5c%) was included in exochelin solution to prevent microbial pearance of low concentrations of mycobactin in growth. Controls, without either ferritin or exo- the medium, but even under these conditions chelin, were also run. Solutions were sampled at the partition of mycobactin is still greatly in intervals, and their extinctions at 450 nm were re- favor of the cells as the amount of mycobactin in Tween-grown cells is typically 5 to 6'7 of the corded to measure the formation of ferriexochelin. Growth of M. bovis var. BCG in the presence of cell dry weight. ferritin-containing diffusion capsules. Diffusion In the examination of the culture filtrates capsules (LH. Engineering Ltd, Stoke Poges, from both Tween-containing and non-Tween Bucks., U.K.) have been described by Pirt (15) for medium for mycobactin, the presence of ironuse in growth experiments. They are nylon cylin- solubilizing compounds was seen upon adding ders measuring 50 by 25 mm, with an orifice of 4-mm diameter at one end. Into this is sealed a membrane FeCl3 to each type of medium. Although these of Visking dialysis tubing. These capsules and the have been noted previously (12), they were dialysis membrane were made as free of iron as found to be distinct from those compounds isopossible by boiling before assembly for 15 min in 100 lated from M. smegmatis in that they were mM ethylenediaminetetraacetate, followed by thor- extractable into chloroform or, after adjusting ough rinsing in all-glass double-distilled water. As- the media to pH 3.5, into ethyl acetate. They sembled capsules, containing 1 ml of water, were were also shown to be distinct from mycobactin autoclaved at 7,030 kg/M2 for 10 min. After cooling, by TLC (Fig. 1). Solvent system A distinthe water was replaced under aseptic conditions by guished the materials from mycobactin and sol0.75 ml of ferritin solution (10 mg/ml) and the reassembled capsules were added aseptically to growth vent system B confirmed that there were two flasks containing 150 ml of medium. Suitable con- components present in the isolated material trols were run simultaneously in each experimental neither of which corresponded to exochelin MS. These components are referred to as exochelin series (see below).

IRON ACQUISITION BY MYCOBACTERIA

VOL. 12, 1975 -Solvent front A

-

Solvent frontB

0

MB-2 (see Fig. 2). No radioactivity was located in the position of mycobactin or indeed in any other compounds than the ones which are described as the exochelins. The production of these chloroform-extractable iron-binding compounds during the growth of the organism is illustrated in Fig. 3. They accumulated rapidly during active growth, reaching a maximum usually of between 15 to 20 ,ug/ml, but at the end of growth their concen2000

O

,#

It

60C

1

1245

2

1

2

-

3 28

2

FIG. 1. TLC of chloroform extracts of M. bovis var. BCG culture media. The plates were developed in solvent systems A and B (see text). Spot 1, Chloroform extract of 9-day culture medium; Spot 2, purified mycobactin from BCG; Spot 3, exochelin from M

smegmatis. Hatched lines indicate faint zones.

c

3

2

_3

44

,

,

v'

b

9

^

FIG. 2. Iron-binding compounds in M. bovis var. BCG culture media located using radio-TLC. Culture medium, labeled with 55Fe, was extracted with chloroform and the extracts were chromatographed in solvent system A (see text). Strips of the plate were removed and the 55Fe was counted. Arrowed line indicates location of ferrimycobactin which was cochromatographed.

MB-1 and exochelin MB-2, the latter having the lowest Rf value in solvent B and being the major component. To establish that either or both of these compounds was indeed capable of binding iron and 600 ^ holding it in solution in the presence of competing anions such as phosphate, the spent medium from a 5-day culture of BCG was labeled with a very small quantity of 55FeCl3 (14.7 ng; 400 _E 90 nCi). (The addition of ferric ions to uninoculated medium results in its precipitation [12].) 0~~~~ 0 By adding very small quantities of radioactive 3 _ 200, iron, only those compounds with the greatest affinity for ferric ions would become labeled. The compound or compounds with the highest affinity would presumably be functionally the O IU 4 7 < 2 3 5 6 most significant. 8 9 10 co DAYS) ME The medium was extracted twice with an equal volume of chloroform which removed 86(4 FIG. 3. Growth of M. bovis var. BCG and producof the added radioactivity. After adjusting the tion of chloroform-extractable, iron-binding compH of the remaining aqueous phase to 3.5 and pounds. Cell dry weight (-) and E450 of total chloroextracting with ethyl acetate, a further 7(4 of form-extractable material in ferri-form (0) were dethe radioactivity was removed from the termined by extracting 100 ml of medium saturated with FeCl, into chloroform. The chloroform extract aqueous phase. This fraction, however, was not was evaporated and the residue was dissolved in 5 ml examined further. TLC, using solvent system of methanol and read at 450 nm. The maximum A, of the concentrated chloroform extracts re- concentration of exochelin, which is the sole ironvealed that the 55Fe was located in two major binding compound produced in the medium under zones near the origin (Rf values 0.3 and 0.1) these conditions, corresponds, at 7 days, to 20.2 corresponding to exochelin MB-1 and exochelin pg/ml assuming ElN-m = 23. 0 0

T

1246

MACHAM, RATLEDGE, AND NOCTON

INFECT. IMMUN.

tration fell, presumably either by reabsorption instability in the medium. TLC (as in Fig. 1) of each sample taken at daily intervals (3 to 8 days) and also at day 10 showed that both exochelin MB-1 and exochelin MB-2 were present simultaneously throughout growth. The latter component was consistently the major material always comprising about 75 to 809% of the total. Mycobactin was not seen in any of these samples. Exochelins as possible precursors of mycobactin. An unlikely but possible explanation for the presence of exochelins in culture media was that, although they possessed high affinities for iron, they had no functional significance, serving only as mycobactin precursors. The mycobactin molecule contains a long alkyl chain, and a mycobactin precursor without this chain would be expected to have an increased solubility in water that might lead to its accumulation in the medium. Some weight was added to this view when salicylic acid was identified as a component of exochelin after acid hydrolysis (L. P. Macham, unpublished work) since salicylic acid also forms part of the mycobactin molecule (21). To investigate a relationship between exochelin and mycobactin, carboxy-['4C]salicyloyl exochelin MB-2 was prepared (see above) and added at 17.5 nCi (about 2 ,ug/ml) to BCG. After a further 6 days of incubation, the culture was harvested and the mycobactin within the cells was extracted, purified, and examined for radioactivity. No trace of 14C was found within it. As we would have detected an incorporation of 14C of about 20 dpm without difficulty,we calculate that any incorporation of exochelin MB-1 into mycobactin was less than 0.05Y/( and therefore could be taken as insignificant. Ability of exochelins to acquire iron from serum and supply it to mycobacteria. Any assessment of the significance of iron-binding compounds either in vitro or in vivo, must rest on a demonstration of the effectiveness of the agent in appropriate model systems. Mycobactin, for example, has been shown to be capable of overcoming the tuberculostatic effects of serum (10). Desferriexochelin MB-2 at 45 ,Lg/ml was tested for its effects on the growth of BCG in undiluted calf serum. The amount added was higher than that occurring in growth medium, but since the unsupplemented control would produce exochelin of its own volition, an excess had to be added to ensure that any stimulation of growth would be discernible. A slight pellicle of cells appeared in the presence of desferriexochelin MB-2 within 3 days, whereas without the exochelin, pellicle formation was not seen until day 4. Growth continued to be greater in

the bottles supplemented with exochelins throughout the rest of the incubation period (10 days) than in the unsupplemented control. Des-

or

ferriexochelin MS had a similar effect on M. smegmatis when tested in the same way. (Cross-checks of exochelin MS with BCG and vice versa were not performed.) When the serum was supplemented with 0.2 Aog of iron/ml as well as exochelin, better growth still occurred in the cultures containing exochelin as compared to those supplemented with iron alone. Exochelins, therefore, sequester iron and supply it to bacteria even in the presence of the competing iron-binding compounds in serum. This result was confirmed and extended by showing that ferriexochelins act as iron donors in the presence of serum. Using an agar plate diffusion method previously used with mycobactin (9), serum agar plates, their surfaces seeded with mycobacterial inocula, had wells cut in them which were filled with solutions containing exochelin MB-1, exochelin MB-2, or exochelin MS (each in the ferric form). The ability of the contents of the well to overcome the serum's inhibition of growth was registered as a zone of stimulated growth around the well. The results (Table 1) showed that both ferriexochelin MB fractions were able to supply iron for both test organisms. In contrast, ferriexochelin MS caused a large zone only, with M. smegmatis having no discernible effect on the growth of BCG. Ferrimycobactin (10 ,g), when tested against M. smegmatis in the same system, produced comparably large zones of growth in the same time (not given in Table 1). Ability of exochelins to remove iron from ferritin. The results of the previous section show that exochelins could acquire and donate their iron to mycobacteria even in environments containing competing molecules. Their ability to acquire and use iron from the iron-containing protein ferritin was also investigated. Ferritin, besides occurring in low concenTABLE 1. Activity of ferriexochelins on growth of mycobacteria on serum agar diffusion plates

Solution added"

Growth of organism (width [mm] of zones after 3-day incubation) M. bovis BCG

smegmatis

0 52 38 0

70 48 23 0

var.

Ferriexochelin MS Ferriexochelin MB 1 Ferriexochelin MB 2 Control (water) "

22 ,ug of each was added to the well.

M.

IRON ACQUISITION BY MYCOBACTERIA

VOL. 12, 1975

trations in serum (2), has been identified in macrophages (1) where it may therefore constitute the major source of iron for ingested mycobacteria. Because of the high proportion of iron in ferritin (it contains 20% iron whereas the amount of iron in transferrin is about 0.15%), it was possible to design two series of experiments that directly followed the release of iron. In the first series of experiments the ability of exochelin MB-2 to remove iron from ferritin in a model system was tested by separating desferriexochelin MB-2 from ferritin by containing the latter within a dialysis bag. Desferriexochelin was found to be freely dialyzable and passed through the membrane, whereas ferritin was retained within the bag. The acquisition of iron by exochelin MB-2 therefore was easily followed by measuring the increase in extinction at 450 nm of the external exochelin solution (Fig. 4). Neither of the controls, containing only exochelin or only ferritin, had any increase in E450 throughout the experiment (Fig. 4). The slow but steady increase in E480 of

the test experiments showed exochelin could acquire iron from ferritin. The rate of removal of iron from ferritin by exochelin, however, is comparable to the rates achieved with high affinity iron chelators such as ethylenediaminetetraacetate or desferrioxamine (5). In the second series of experiments the ability of BCG itself to remove iron from ferritin during growth was examined. The production of extracellular iron-binding compounds during iron-deficient growth is strongly suggestive of an effective role for these compounds, but normal experimental growth conditions do not ex0 08

0 0 06

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0

0

0 0

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0

0-04

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7;l" 0 02

bo 0 bo o

0

0

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0

o

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0

0

o

_

o

o-o

0

6

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o

0

0

9

( DAYS)

FIG. 4. Acquisition of iron from ferritin by exochelin MB-2. Ferritin, within a dialysis bag, was surrounded by a solution of desferriexochelin MB-2; E,,O = formation of ferriexochelin MB-2 (O). Controls omitting either desferriexochelin MB-2 (O) or ferritin (0) from the system also ran.

1247

clude the possibility that iron is acquired by simple contact with the bacteria, thereby making the production of specialized compounds superfluous. To show that iron solubilization and transport via exochelins could take place, we separated the source of iron from direct contact with the bacteria by enclosing the iron, as ferritin, within diffusion capsules (15). Here a dialysis membrane barred iron escaping from the capsule into the medium. Complete retention of iron within the capsule was confirmed in a control experiment in which diffusion capsules were filled with ferritin solution and shaken in uninoculated medium either supplemented with 1% Tween 80 or not for 7 days at 37 C. The amount of iron released, if any, was monitored by a bioassay whereby M. smegmatis was inoculated into this medium, with the diffusion capsules removed, for a further 6 days. There was no increase in dry weight of the organisms compared to growth on the same batch of irondeficient medium which had been in contact with empty diffusion capsules. Therefore, in flasks where iron was restricted within diffusion capsules, growth additional to that observed in the controls of bacteria depended on the ability of the organisms to sequester iron in a diffusible form from this source. Growth flasks were set up each containing a diffusion capsule and with the medium in half the flasks supplemented with 1% Tween 80. Capsules without ferritin were used as controls. Other controls had ferritin added directly into the medium. The results (Fig. 5A and B) were essentially similar irrespective of the presence of Tween in the medium. In the absence of any ferritin added to a flask, some growth occurred upon the traces of residual iron in the flask. When ferritin was added directly into the medium, the growth increased showing that the bacteria were acquiring the iron from ferritin. When ferritin was within the diffusion capsules, there was still a significant increase in growth.. Iron from ferritin therefore was made available for growth despite its restriction within a diffusion capsule. In the absence of Tween 80 (Fig. 5B), the growth yield and growth rate of the test flask were comparable with those of the flask containing ferritin within the medium; the only difference between these cultures was a longer lag period in the test flask. This may have been anticipated as synthesis of exochelins and subsequent mobilization of iron from within the capsule cannot occur until growth has been initiated. These experiments therefore demonstrated the ability of mycobacteria to solubilize iron in a location remote to the bacteria and implicated the freely dialyzable exochelins for this role.

1248

MACHAM, RATLEDGE, AND NOCTON

INFECT. IMMUN.

above experiments could not have been due to any mycobactin which may have been present in the medium.

Q

E

_120

E

DISCUSSION Kochan et al. have previously ascribed to mycobactin the role of extracellular iron scavenging agent (10), although we have consistently failed to detect this compound in culture filtrates (18). This contradiction is now resolved by the results presented in the first part of this paper. Mycobactin is not released from cells of M. smegmatis or BCG in the absence of a surfactant such as Tween 80. Instead, a hitherto unsuspected compound(s) is found which we have termed exochelin. The properties of exochelin from BCG are such that it could have been mistaken, in a superficial examination, for mycobactin. A comparison of the properties of exochelin from BCG and mycobactin is given in Table 2. In the presence of Tween 80 some mycobactin is found in culture filtrates, though this is almost certainly a direct result of the chemical modification of the medium and not an orga-

0

, 80

E -

0

40

L

-1

co

Time

(days)

TABLE 2. Comparison of properties of exochelin MB and mycobactin as extracellular iron-binding compounds

In

L-

E

u

Determinants

0

0-

O

Location Medium of Tweencontaining cultures Medium of nonTween-containing cultures

_1

3L O n

o

O

4

Time

6 (days)

8

Exochelin MB

Mycobactin T

Present (about 15 .g/ml)

Present (about 0.75 /.g/ml)

Present (about

Undetectable (less than 0.02 gg/ml)

15 Ztg/ml)

10

FIG. 5. Growth of M. bovis var. BCG on iron derived from ferritin. Cultures with 1 % Tween 80 (A) or without (B) grew in flasks containing diffusion capsules. Ferritin (7.5 mg) added as sole source of iron, either into the medium (a) or into a diffusion capsule (A); flasks without ferritin (0).

Kochan et al. (10), however, have proposed that mycobactin is capable of this role, but when our experimental conditions were applied to mycobactin we found that ferrimycobactin was not a readily diffusible form of iron. Ferrimycobactin, 80 ,tg/ml, in 1% Tween 80 solution was dialyzed against 100 ml of a 1% Tween 80 solution for 7 days at 4 C. There was no decrease in the extinction of the ferrimycobactin solution at 450 nm, indicating that it probably exists as aggregates or micelles too large to be freely dialyzable. Therefore, the acquisition of iron from within the diffusion capsules used in the

Biosynthesis Repression in ironsufflicient cultures

Yes

Yes

Solubility in aque-

Soluble

Insoluble

ous environments Solubility in lipid solvents (e.g.,

Soluble

Soluble

chloroform) in Diffusibility aqueous environments

Dialyzable

Positive

Nondialyzable, even in 1% Tween solutions Positive"

Positive

Negative

Positive

Positive"

Function

Acquisition of iron from serum and ferritin Acquisition of iron from precipitated or colloidal forms Ability to supply iron to the bacteria for growth

From references 6 and 9.

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nism-directed response since the same removal of mycobactin by Tween 80 can occur using killed cells (C. Ratledge, unpublished work). Thus, under these conditions of growth, Kochan would indeed have detected mycobactin in the culture filtrates, but this would still be in association with the exochelins whose production is not affected by Tween 80. Even under these circumstances, however, the concentration of mycobactin in the medium (at about 0.75 ,ug/ml) is much less than that of the exochelins, which are up to 20 ug/ml. Though mycobactin can occur extracellularly when cells are grown in the presence of a surfactant, mycobactin cannot fulfil all the functions required of an iron-solubilizing agent (see Table 2). It cannot readily solubilize inorganic precipitated iron (17), though it can and does abstract iron from compounds such as ferritin and, presumably, transferrin as well (6, 10). Therefore, under appropriate conditions, it can be shown to reverse the tuberculostatic effect of serum on the growth of mycobacteria (10). But this is an artificially contrived situation where mycobactin is deliberately added exterior to the cells at the onset of the incubation period. Another but perhaps less serious objection to mycobactin being involved in extracellular iron acquisition is the inability of its ferri-form to be freely diffusible. This is clearly a process which a pathogen must accomplish for successful multiplication in vivo since the source of iron may be within an organelle separate from that which contains the bacterium. Mycobactin would appear to form micelles which are too large, even in the presence of a surfactant, to be able to pass through a dialysis membrane. Such a lack of diffusibility, in an in vivo situation, would preclude its function in an extracellular capacity. The physicochemical properties of mycobactin suggest that it is not an extracellular ironbinding compound. Its long alkyl chain, which contributes nothing to the affinity of the molecule for iron, serves to make it highly lipophilic. Although a low water solubility of between 5 to 15 A.g/ml has been estimated for the mycobactins (20), the growing culture is essentially a biphasic system: an aqueous phase (the medium) and a lipid phase (the bacterial outer layers). Consequently, mycobactin partitions exclusively into the lipid. Within the outer layers of the mycobacterial cell, the lipophilic nature of mycobactin permits it to fulfil its role as an iron ionophore, as has already been demonstrated (16). The recognition of the exochelins as the sole iron-binding agents in culture filtrates of BCG and M. smegmatis now explains how iron is

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acquired by mycobacteria growing in vitro and is strongly indicative of how iron acquisition in vivo is accomplished. There is no need with the exochelins to postulate the occurrence of surfactant compounds in the in vivo situation as Kochan et al. were obliged to do to account for the release of mycobactin. Exochelins are produced irrespective of the presence of such agents; the only stimulus needed is a deprivation of iron, which many workers now agree is likely to pertain for a pathogen growing in a host organism (4, 22). Mycobactin is produced in response to the same stimulus (18); the observation by Kochan et al. (9) of finding mycobactin in iron-sufficient (Tween-containing) cultures is due to using nonshaken conditions which leads to the occurrence of iron-deficient conditions in those cells which are growing on the upper surface of the pellicle (16). The exochelins hold iron in solution at physiological pH values, can abstract iron from ferritin, and are freely diffusible in both the desferriand ferri-forms. They can reverse the inhibitory effect of serum on the growth of mycobacteria, which is clearly related to their ability to acquire iron from transferrin and ferritin. Serum cannot prevent the transport of iron from ferriexochelin once formed to the bacteria. Their occurrence accounts for the earlier observation made by Kochan et al., which elicited their surprise, that culture filtrates of BCG and H37Ra, grown without a surfactant, could reverse tuberculostasis (6). Exochelins are not precursors or breakdown products of mycobactin. Preliminary chemical investigations (L. P. Macham, unpublished work) show exochelin MB and exochelin MS to be structurally similar molecules: both are peptides containing 3 mol of E-N-hydroxylysine and 1 mol of threonine, thus eliminating them as breakdown products of mycobactin, which contains only 2 mol of the former amino acid. Exochelin MS also contains ,3-alanine, whereas exochelin MB contains salicylic acid. The molecular weight of exochelin MS is between 750 and 800. Further studies on their structures are in progress. The ability of the exochelins to transfer iron to cells is undoubtedly aided, in the case of exochelin MB, by the increased lipophilicity of the ferri complex as compared to the desferri material (L. P. Macham, unpublished work). Ferriexochelin MB thus can readily approach and associate with the mycobacterial cell surface where the transference of iron to mycobactin will occur (Fig. 6). The necessity for two distinct iron transport compounds in mycobacteria (most other bacteria elaborate only one such compound) may

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|SOURCE OF

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MACHAM, RATLEDGE, AND NOCTON

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tion, and their discovery opens up another aspect of iron metabolism in the mycobacteria. e

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FIG. 6. Proposed scheme for iron mycobacteria.

transport in the

well be related to the thick, lipid-rich boundary layers of these organisms. These would act as a barrier to a hydrophilic iron transport molecule, such as is found with other bacteria, and may have resulted in the evolution of specific lipophilic carriers-the mycobactins. Notably the related group of organisms, the nocardia, which also have thick lipoidal cell walls, similarly elaborate two distinct types of iron carriers. One is lipophilic and structurally similar to mycobactins (14, 19) and the other is hydrophilic but structurally distinct from the exochelins (P. V. Patel, personal communication). Our investigations into the exochelins of both M. smegmatis and BCG have been primarily directed at establishing their function. As their roles for both organisms seem to be essentially similar, they have been given the same trivial group name. The agar well experiments showed that ferriexochelin MB could donate iron to M. smegmatis as well as BCG, but that ferriexochelin MS was specific for M. smegmatis. It will be of obvious interest to know if the very hydrophilic molecules (e.g., exochelin MS) will be confined to the nonpathogenic strains, whereas the more "amphiphilic" types (e.g., exochelin MB) will be found only in the pathogenic organisms. Their relevance in the in vivo development of pathogenic strains of mycobacteria would seem clear in view of current evidence linking pathogenicity to the iron-sequestering ability of the invading microorganism (4, 8, 21). We propose the iron transport system as represented in Fig. 6. We cannot support the view that mycobactin fulfills anything other than an intracellular role, although we do not disagree with its ability to perform various functions, such as the reversal of tuberculostasis, if the appropriate conditions are contrived in model experiments. The exochelins, however, possess all the properties that are necessary for a compound to carry out extracellular iron sequestra-

ACKNOWLEDGMENTS We thank the Science Research Council (U.K.) for the award of a grant (B/RG/2256) in support of this work and a postdoctoral fellowship for L.P.M. LITERATURE CITED 1. Armstrong, B. A., and C. P. Sword. 1966. Electron microscopy of Listeria monocytogenes-infected mouse spleen. J. Bacteriol. 91:1346-1355. 2. Beamish, M. R., G. M. Addison, P. Llewellin, M. Hodgkins, C. N. Hales, and A. Jacobs. 1972. Ferritin estimation by a radiommunoassay technique: serum levels in normal subjects and patients with blood disorders. Br. J. Haematol. 22:637. 3. Brown, K. A., and C. Ratledge. 1975. Iron transport in Mycobacterium smegmatis: ferrimycobactin reductase (NAD(P)H:ferrimycobactin oxidoreductase), the enzyme releasing iron from its carrier. FEBS Lett. 53:262-266. 4. Bullen, J. J., H. J. Rogers, and E. Griffiths. 1974. Bacterial iron metabolism in infection and immunity, p. 517-551. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 5. Dognin, J., J. L. Girardet, and Y. Chapron. 1973. Etude polarographique de la mobilisation du fer de la ferritine. Biochim. Biophys. Acta 297:276-284. 6. Golden, C. A., I. Kochan, and D. R. Spriggs. 1974. Role of mycobactin in the growth and virulence of tubercule bacilli. Infect. Immun. 9:34-40. 7. Keller-Schierlein, W., V. Prelog, and H. Zahner. 1964. Siderchrome (Naturliche Eisen (III)-trihydromateKomplexe). Fortschr. Chem. Org. Naturst. 22:279322. 8. Kochan, I. 1973. The role of iron in bacterial infections, with special consideration of host-tubercule bacillus interaction. Curr. Top. Microbiol. Immunol. 60: 1-30. 9. Kochan, I., D. L. Cahall, and C. A. Golden. 1971. Employment of tuberculostasis in serum-agar medium for the study of production and activity of mycobactin. Infect. Immun. 4:130-137. 10. Kochan, I., N. R. Pellis, and C. A. Golden. 1971. Mechanism of tuberculostasis in mammalian serum. III. Neutralization of serum tuberculostasis by mycobactin. Infect. Immun. 3:553-558. 11. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331. 12. Macham, L. P., and C. Ratledge. 1975. A new group of water-soluble iron-binding compounds from mycobacteria: the exochelins. J. Gen. Microbiol. 89:379-382. 13. Neilands, J. B. 1973. Microbial iron transport compounds (siderochromes), p. 167-202. In G. L. Eichhorn (ed.), Inorganic biochemistry, vol. 1. Elsevier Scientific Publishing Co., Amsterdam. 14. Patel, P. V., and C. Ratledge. 1973. Isolation of lipidsoluble compounds that bind ferric ions from Nocardia species. Biochem. Soc. Trans. 1:886-888. 15. Pirt, S. J. 1971. The diffusion capsule, a novel device for the addition of a solute at a constant rate to a liquid medium. Biochem. J. 121:293-297. 16. Ratledge, C., and M. J. Hall. 1971. Influence of metal ions on the formation of mycobactin and salicylic acid in Mycobacterium smegmatis grown in static culture. J. Bacteriol. 108:314-319. 17. Ratledge, C., L. P. Macham, K. A. Brown, and B. J. Marshall. 1974. Iron transport in Mycobacterium smegmatis: a restricted role for salicylic acid in the extracellular environment. Biochim. Biophys. Acta 372:39-51.

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18. Ratledge, C., and B. J. Marshall. 1972. Iron transport in Mycobacterium smegmatis: the role of mycobactin. Biochim. Biophys. Acta 279:58-74. 19. Ratledge, C., and G. A. Snow. 1974. Isolation and structure of nocobactin NA, a lipid-soluble iron-binding compound from Nocardia asteroides. Biochem. J. 139:407-413. 20. Ratledge, C., and F. G. Winder. 1962. The accumulation of salicylic acid by mycobacteria during growth

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on an iron-deficient medium. Biochem. J. 84:501-506. 21. Snow, G. A. 1970. Mycobactins: iron-chelating growth factors from mycobacteria. Bacteriol. Rev. 34:99-125. 22. Sussman, M. 1974. Iron and infection, p. 649-679. In A. Jacobs and M. Worwood (ed.), Iron in biochemistry and medicine. Academic Press Inc., New York. 23. White, A. J., and G. A. Snow. 1969. Isolation of mycobactins from various mycobacteria. The properties of mycobactins S and H. Biochem. J. 111:785-792.