Eur. J. Biochem. 178,477-481 (1988) FEBS 1988

Domain preference in iron removal from human transferrin by the bacterial siderophores aerobactin and enterochelin Steven FORD', Ronald A. COOPER', Robert W. EVANS', Robert C. HIDER3 and Peter H. WILLIAMS4 ' Department of Biochemistry, University of Leicester * Division of Biochemistry, University Medical and Dental School, Guys Hospital, London

Department of Pharmacy, King's College, University of London Department of Genetics, University of Leicestcr

(Received August 3, 1988) - EJB 88 0938

The ability of the siderophores aerobactin and enterochelin to remove iron from transferrin is reported. Aerobactin removes iron from both high-affinity sites on the transferrin molecule, but shows a marked preference for the C-terminal site. This preference is different to that of many iron chelators. Enterochelin removes iron perferentially from the N-terminal site. No evidence for synergism between aerobactin and bidentate ligands could be detected.

The transferrins, a family of iron-binding proteins found in blood, secretions and egg white, comprise a single polypep- tide chain (molecular masses in the range 76 - 81 kDa) with two similar but not identical iron-binding sites in separate domains. Studies on several transferrins indicate that the bind- ing constants for iron are different for the two sites [l]; there- fore they differ in their ability to sequester iron from iron- chelator complexes and to release iron to other chelators [2 - 41. The physiological significance of such observed differences, however, remains a matter for speculation, although from a practical point of view the fact that, in normal healthy ani- mals, transferrins are only partially iron-saturated implies that a knowledge of domain preferences of natural and synthetic compounds may be of crucial importance in the design of chelators for the therapeutic treatment of iron overload and other diseases of iron imbalance [5].

Similarly, in considering bacterial diseases, where compe- tition for iron between host transferrins and microbial siderophores is a critical feature of the host/pathogen interac- tion [6], a knowledge of siderophore site-preference is neces- sary in determining the availability of iron to the infecting organism and assessing possible methods of control. In this paper we demonstrate differential iron removal from the indi- vidual binding sites of diferric human serum transferrin by the bacterial siderophores enterochelin and aerobactin, and the effects of possible mediators on this process.

MATERIALS AND METHODS Siderophores and related compounds

Hydroxamates. Acetohydroxamic acid was obtained from Aldrich Chemical Co. Ltd. Aerobactin and N6-acetyl-N6-

Correspondence to R. C. Hider, Department of Pharmacy, King's College, London University, Manresa Road, London SW3 6LX, England

Ahhreviutions. Fez-Tf, Diferric transferrin; Tf, apotransferrin; Fe(N)-Tf, monosubstituted transferrin(N-terminal); Fe(C)-Tf, mono- substituted transferrin(C-terminal); Bz(OH),Ser, 2,3-dihydroxy- ben7oylscrine; LICAMS, 1,5,10,14-tetrakis(2,3-dihydroxy-5-sulfo- benzoy1)- 1,5,10,14-tetraazatetradecane.

hydroxylysine were prepared by Dowex-1 (C1 F) chroma- tography of culture supernatants of Aerobacter aerogenes 62- I, essentially as previously described by Gibson and Magrath [7] and de Lorenzo et al. [8], respectively.

Catecholates. 2,5- and 2,3-Dihydroxybenzoic acid were purchased from Aldrich Chemical Co. Ltd; 2,3-dihydroxy-N- benzoylserine [ B Z ( O H ) ~ S ~ ~ ] was prepared by the reaction of the N-hydroxysuccinimide ester of 2,3-dimethoxybenzoic acid with 0-benzylserine in anhydrous dimethylformamide. Sub- sequent treatment of the resulting amide with boron triflu- oride lead to the simultaneous deprotection of the catechol and hydroxyl functions. Chloroform/water extraction lead to the isolation of B ~ ( 0 H ) ~ s e r in over 70% yield.

Enterochelin was purified from bacterial culture super- natants by a modification of the method of Young [9]. Briefly, Escherichia coli strain LG1522, which produces enterochelin but is unable to utilise it, was grown in M9 minimal medium, and production of enterochelin was monitored by the method of Arnow [lo]; when enterochelin synthesis had ceased, bac- terial cells were removed from the culture fluid by centrifuga- tion. A portion (100 ml) of supernatant was acidified to pH l with concentrated H2S04 and extracted three times with 10-ml volumes of ethyl acetate. Pooled extracts were washed with an equal volume of 0.1 M Tris/HCl buffer, pH 7.0 and ethyl acetate was removed by evaporation to leave an aqueous residue, which was adjusted to pH 7.4 with NaOH before use. The enterochelin concentration was measured by the Arnow reaction. A similar preparation from a culture to which 10 pM FeCl, was added to repress enterochelin production was used in control experiments.

Transjerr in

Human serum transferrin was isolated from fresh plasma by immunoaffinity chromatography [I 11. Samples of protein in 0.1 M NaHC03 were iron-saturated by addition of excess iron nitrilotriacetate; unbound iron was removed by gel filtration on Sephadex G-25 equilibrated with 50 mM NH4HC03 and the protein (Fez-Tf) was freeze-dried. Trans-

478

loo1 b

Incubation time, h

Fig. 1 . Removal of' iron ,from [ransferrin by aerohacfin. (a) Urea/polyacrykimide gel electrophoresis of human diferric transferrin (Fe2-Tf) incubated at 37"C in the presence of 9 mM aerobactin. Samples were removed from the reaction mixture at the times indicated and applied directly to the gel. Marker proteins (M) were apotransferrin (TO, monoferric transferrins with iron in either the C-terminal [Fe(C)-Tfl or N-terminal [Fe(N)-Tfl binding sites and difcrric transferrin (Fe,-Tf). (b) Graphic representation of the kinetics of iron removal from human transferrin by aerobactin. Data points were derived by quantification of densitometer tracings of lanes of the gel shown; (0) Tf; (V) Fe(N)- _ . Tf; ( A ) Fe(C)-TF; (0) Fe2-Tf

ferrin containing iron predominantly in the C-terminal site [Fe(C)-Tfl was prepared as described by Cowart et al. [12] and the method of Baldwin [13] was used to prepare monoferric transferrin with iron predominantly located in the N-terminal site.

Monoferric transferrin preparations were prepared in such a manner that apotransferrin (TO formed approximately 50% of the total protein, thereby ensuring that levels of Fe2-Tf were kept to a minimum.

Removal of iron f rom human transferrin by siderophores and related compounds

Human Fe2-Tf (31.25 pM) was incubated at 37°C in 100 mM Tris/HCI (pH 7.4) containing glycerol (loo/, vol./ vol.) with various compounds as detailed in Results. Samples were taken at intervals and applied directly to polyacrylamide gels (6.5%) containing 6M urea [ 5 ] . After electrophoresis at 100 V for 18 h, gels were stained with PAGE blue 83 (BDH), destained and scanned at 570 nm on a Camlab EC910 trans- mission densitometer. Relative amounts of each of the four transferrin bands were estimated by excising and determining the mass of peaks from the densitometer traces. Removal of iron from Fe(N)-Tf and Fe(C)-Tf was followed similarly.

RESULTS

Remo vnl of'iron.from transferrin by aerobactin and enterochelin

Aerobactin sequestered iron from both binding sites of human Fe2-Tf (Fig. 1); during incubation at 37 OC with 9 mM aerobactin, Tfappeared in the reaction mixture within 90 min and represented about 40% of the total protein after 6 h.

Fig. 2. Removal of iron from transferrin by hydroxamate and catechol chelators. Human diferic transferrin (Fe,-Tf) was incubated at 37 ~ C for 24 h in the presence of (1) 9 mM aerobactin, (2) 36.3 rnM N 6 - acctyl-N'-hydroxylysine, (3) 4.5 mM acetohydroxamic acid and (4) 9 mM 2,3-dihydroxybenzoic acid, and analysed by urea/poly- acrylamide gel electrophoresis. Marker proteins (M) werc apo- transferrin (Tf), and monoferric transferrins with iron in either the C- terminal [Fe(C)-Tfl or N-terminal (Fe(N)-Tf] binding sites

Similarly, at 20°C about 50% of the Fe2-Tf had been convert- ed to the iron-free form after incubation with aerobactin for 24 h (data not shown). However, removal was clearly not random in that at either temperature there was a marked preference for removal of iron from the C-terminal site, leav- ing transferrin with iron predominantly in the N-terminal site [Fe(N)-Tfj (Fig. 1). Aerobactin removed 93% of the iron from

479

I I I 1 0 1 2 3 4 5 6

Incubation time, h Fig. 3 . Transjerrin iron-binding site prejkrence fiw iron removal by nerobactin. Human monoferric transfcrrins with iron in cither the N- terminal (0) or the C-terminal ( 0 ) binding domains were incubated at 37"C with 9 mM aerobactin. Samples were removed from reaction mixtures at intervals, and analysed by urea/polyacrylamide gel electrophoresis. Data points represent levels of monoferric transferrin remaining in the reaction mixture and were derived by quantification of densitonieter tracings or gel lanes

Fez-Tf in 24 h at 37°C (Fig. 2, lane 1) and a similar fraction in 48 h at 20°C; in each case the residual traces of iron were located in the N-terminal site. The preference of aerobactin for iron in the C-terminal binding site of iron-saturated trans- ferrin was confirmed in experiments with monoferric proteins (Fig. 3) in which initial rates of iron removal at 37 "C of 13.1 % h- ' and 1.2% h-' from the C-terminal and N-terminal iron- binding sites, respectively, were observed. Almost four times more iron was sequestered from Fe(C)-Tf than from the N- terminal monoferric transferrin during a 6-h incubation. After 24 h, when all the iron had been removed from the C-terminal site, 7% of the original N-terminal site iron remained bound.

Enterochelin was found to remove iron virtually exclu- sively from the N-terminal site of Fez-Tf (Fig. 4a). At an enterochelin concentration of 47 pM, approximately half the total amount of iron was removed in 3 h at 37°C.

Facilitution of iron removal from trunsferrin by siderophore precursors and metabolites

Konopka et al. have reported the existance of a synergistic cffect between dihydrobenzoic acid and aerobactin [14]. In order to investigate this phenomenon in more detail it was decided to assess the ability of various siderophore precursors and metabolites to remove iron from transferrin at 37°C.

In contrast to the earlier report we found that 2,3- dihydroxybenzoic acid was able to remove iron from transfer- ring, albeit rather slowly (Table 1). Indeed 2,3-dihydroxy- benzoic acid showed a marked preference for the N-terminal site (Fig. 2, lane 4). However this apparent disparity with the data of Konopka et al. [I41 results from the different

temperatures employed. In a direct comparative study using the two temperatures 20°C and 37"C, a marked differencc in the ability of dihydroxybenzoic acid to mobilise iron was observed. At 20"C, virtually no iron was removed from either site of the transferrin molecule after 24-h incubation (data not shown).

In contrast to dihydroxybenzoic acid, another entero- chelin degradation product, Bz(OH),Ser, was highly effective at iron sequestration (Table 1). For instance, at 9 mM it re- moved over 90% of the iron from both domains of Fez-Tf in 1 h at 37°C. A slight preference for the N-terminal domain is demonstrated (Fig. 4b).

No synergism between either of these ligands and aerobactin was observed at 37°C. Thus, the quantity of iron removed from transferrin by a mixture of aerobactin (9 mM) and 2,3-dihydroxybenzoic acid (9 mM or 0.9 mM) was iden- tical to the sum of the amounts mobilised by the hgdnds when incubated separately. Similarity, there was no synergism observed between aerobactin and B ~ ( 0 H ) ~ s e r .

Clinical isolates of E. coli that make aerobactin also ex- crete various amounts of the biosynthetic intermediate NO- acetyl-N6-hydroxylysine [15, 161. In some cases equimolar amounts of the intermediate and aerobactin arc produced. This compound was found to remove iron quite slowly from Fe2-Tf (Table l), but, unlike aerobactin, it possesses a clear preference for the N-terminal domain (Fig. 2, lane 2). A simi- lar preference was observed for the simple bidentate mono- hydroxamate ligand acetohydroxamic acid (Fig. 2, lane 3) a compound previously reported to mobilise iron from trans- ferrin [17]. When either acetohydroxamate (4.5 mM), or Nh- acetyl-N6-hydroxylysine (36 mM) were included in incubation mixtures with aerobactin (9 mM) and Fez-Tf (65 pM), no effect on iron removal was observed over and above the addi- tive effect of the individual chelators concerned.

DISCUSSION For bacterial pathogens attempting to establish infcction

in an animal body, the ability to adapt rapidly to iron limi- tation is an essential virulence determinant. Although the iron content of body fluids is high (approximatcly 20 pM), levels of free iron are of the order of only 10- M due to the partial saturation of the iron-binding glycoproteins transferrin, in serum and lactoferrin, in secretions. In order to sequester iron from such complexed states in the mammalian body, pathogenic bacteria have evolved a variety of active iron- uptake mechanisms, many of which involve low-molecular- mass iron chelators called siderophores.

Most strains of enteric bacteria carry the genetic determi- nants required for the biosynthesis and transport of the siderophore enterochelin 118, 191, a catecholate compound whose affinity for iron is higher than that of any known natural iron chelator [20]. However, there is no clear evidence that the specific ability to utilise enterochelin enhances bac- terial virulence. The hydroxamate siderophore aerobactin, on the other hand, has been clearly demonstrated to be an important virulence determinant in some strains of E. coli, particularly those associated with extra-intestinal infections of man and domestic animals [21]. Aerobactin was first iden- tified in culture supernatants of A . aerogenes 62-1 [6], and is also produced by a range of other pathogenic members of the Enterobacteriaceae including some Shigella, Salrnonc4lu, Yersinia, and Enterohacter species [22 - 241.

Both siderophores are able to sequester ferric iron effectively from its bound state in transferrin. In the case

480

loor

80 i a

r b

/

I I 1 O 1 , 60 120 180 0 20 40 60

Incubation time, rnin Fig. 4. Kinetics of iron removal from Irunsferrin by the catechol siderophore enterochelin and its degradation product B z ( 0 H ) zSer. Human diferric transferrin was incubated at 37°C in the presence of either (a) 47 FM enterochelin or (b) 9 mM Bz(OH)2Ser; samples were removed at intervals and analysed by urea/polyacrylamide gel electrophoresis. Data points were derived by quantification of densitometer tracings of gel lanes; (0) Tf; ( A ) Fe(N)-Tf; (V) Fe(C)-Tf; (0 ) Fe2-Tf

Table 1. Removal qf iron from suturuted transferrin by siderophore precursors and degrudution products

Competing ligand Concn Iron removed in

enterochelin [14]. Thus, it is clear that structural consider- ations, rather than simply the stability constant of a compet- ing ligand, are important in determining the rate of iron removal from transferrin.

5 h 24 h

mM %

2,3-Dihydroxybenzoic acid 0.9 < 2 -

2,5-Dihydroxybenzoic acid 9 <1 < I 9 10 21

- Bz(OH)*Ser 0.9 70 9 > 95

Acetohydroxamate 4.5 15

-

-

45 YO -

Aero bactin 9 53 93 N6-Acetyl-N6-hydroxylysine 36.3 2 20

of enterochelin, and various other natural and synthetic tricatecholate ligands [25], iron transfer occurs from trans- ferrin to the siderophore without appreciable accumulation of kinetic intermediates. In contrast, the reaction between transferrin and aerobactin at 20°C is reported to proceed via the formation of a relatively stable ternary complex in which the ferric ion is bound to both transferrin and aerobactin [14]. Aerobactin removes iron from saturated human transferrin in buffered (pH 7.4) solution at a rate significantly greater than that shown by other hydroxamate chelators with appar- ently superior affinities for iron [14]. Aerobactin is also able to supply iron from transferrin to E. coli cells expressing the appropriate receptor in defined minimal medium or in inactivated serum; in the latter case more efficiently even than

Transferrin site preference f o r aerobactin

Aerobactin exhibits a marked preference for iron bound to the C-terminal site, which is thermodynamically the more stable of the two [l, 21. Aerobactin will also scavenge iron from the N-terminal site but at a much slower rate (Fig. 1). The two binding sites apparently behave independently with respect to their interaction with aerobactin. Iron is removed from either monoferric form of transferrin, but more rapidly from the form with the C-terminal site occupied (Fig. 3).

The activation enthalpy for iron release from the N-ter- minal site of transferrin, in the presence of the synthetic catecholate LICAMS, changes in the vicinity of 20' C from 88 kJ mol-' (21 kcal mol-') to 63 kJ mol-' (15 kcal mol-') [26]. In contrast, the activation enthalpy for iron release from the C-terminal domain remains constant, at a value of 84 kJ mol- (20 kcal mol- '). Kretchmar and Raymond [26] suggest that the N-terminal site undergoes a conformational change which results in more facile iron release at 37°C. Thus, for both thermodynamic and kinetic reasons, iron removal from the N-terminal site is predicted to be easier than from the C-terminal site at 37 "C. Significantly, most iron chelators preferentially remove iron from the N-terminal site, for in- stance, mimosine [5] , LICAMS [26], pyrophosphate [27], enterochelin (Fig. 4), dihydroxybenzoic acid and N6-aceteyl- N6hydroxylysine (Fig. 2). In contrast, aerobactin and 1,2- dimethyl-3-hydroxypyridin-4-one [5] preferentially remove iron from the C-terminal domain. It is clear therefore, that with some ligands, a favourable transferrin complex is formed

48 1

with the C-terminal site and that this leads to the facile re- moval of iron. It is conceivable that the design of aerobactin has evolved in order to optimise such an interaction.

The recent publications reporting the crystal structures of human lactoferrin [28] and rabbit serum transferrin [29] will facilitate molecular graphics-aided design of synthetic chelators with selectivity for either the N- or C-terminal do- mains.

Iron-scavenging efficiency of'siderophores under physiological conditions

Enterochelin is extremely efficient at removing iron from transferrin. However in the presence of plasma proteins it largely partitions with albumin which reduces its iron-scav- enging capability [14]. In contrast, aerobactin is highly effec- tive under these conditions [14]. The presence of bidentate iron chelators, for instance dihydroxybenzoic acid and Bz(OH)2Ser [30], does not enhance the efficiency of aero- bactin.

In the serum of normal humans, transferrin is only about 30% iron-saturated, and the distribution of iron is unequal between the two bindings domains, iron being found pre- dominantly in the N-terminal site [31], where it is apparently less readily available to aerobactin. Nevertheless it is possible that there is, enough iron present in the C-terminal domain to support an infection such as septicaemia. Furthermore, iron may also be obtained from the N-terminal site. Although this is a slower process, the growth rate of bacteria in the initial stages of infection may be so slow (of the order of 0.02-0.05 h- ' rather than 2-3 h-' typically observed in laboratory culture [32, 331) that access to the N-terminal iron may not be rate-limiting.

An attractive alternative possibility is that the distribution of iron between the binding sites of transferrin may change during an infection. As the total iron in the circulatory system is reduced during the hypoferraemic response to microbial attack [34], it may be that the remaining iron is more abundant in the preferred C-terminal binding site of serum transferrin. This would render the iron less available to most chelators, but relatively more available to aerobactin. Experiments to test this possibility have been initiated in our laboratories.

4. Morgan, E. H. (1979) Biochim. Biophys. Actu 580,312-326. 5. Kontoghiorges, G. J. & Evans, R. W. (1985) FEBS Lett. 189.

6. Weinberg, E. D. (1978) Microhiol. Rev. 42, 45-66. 7. Gibson, F. & Magrath, D. I. (1969) Biochim. Biophys. Acta 192,

8. de Lorenzo, V., Bindereif, A., Paw, B. H. & Neilands, J. B. (1 986)

9. Young, I. G. (1976) Prep. Biochem. 6, 123-131. 10. Arnow, L. E. (1937) J . Bid. Chem. 118, 531-537. 11. Evans, R. W., Williams, J . & Morcton, K . (1982) Biochem. J .

12. Cowart, R. E., Swote, S . , Loh, T. T., Chasteen, N. D. & Bates,

13. Baldwin, D. A. & De Sousa, I). M. R. (1981) Biochem. Biophys.

14. Konopka, K., Bindereif, A. & Ncilands, J. B. (1982) Biochmzistry

15. Neilands. J. B. (1983) Microbiology 1983 (Schlessingcr. D., ed.) pp. 284 -287, Amcr. Soc. Microbiol.. Washington, USA.

16. Linggood, M. A., Roberts, M., Ford, S. , Parry, S . H. &Williams. P. H. (1987) J . Gen. Microhiol. 133, 835-842.

17. Cowart, R. E., Kojima, N. & Bates, Ci. W. (1982) 1. Bid. Chem.

18. Miles, A. A. & Khimji, P. L. (1975) J . Med. Microhiol. 8, 477-

19. Neilands, J . €3. (1982) Annu. Rev. Microhiol. 36, 285-309. 20. Harris, W. R., Carrano, C . J., Cooper, S. R., Sofen, S. R., Ardeef,

A. E., McArdle, J. V. & Raymond, K. N. (1979) J. Am. Chem.

21. Warner, P. J., Williams, P. H., Bindereif, A. B Neilands, J. B.

22. Lawlor, K. M. & Payne, S. M. (1984) J . Bacteriol. 160,266-272. 23. Stuarl, S. J., Prpic, J. K. & Robins-Brownc, R. M. (1986) J .

Bacteriol. 166, 1131 -1133. 24. Van Tiel-Menkveld, G. J., Mentjox-Vervuurt, J . M., Oudega,

B. & De Graaf, F. K. (1982) J . Bacteriol. /SO, 490-497. 25. Carrano, C. J. & Raymond. K. N. (1979) J . Am. C'hem. Soc. 101,

26. Kretchmar, S. A. & Raymond, K. N. (1986) J . Am. Chem. SOC.

27. Chcuk, M. S., Keung, W. M . & Loh, T. T. (1987) J . Inoig. Biochem. 30, 121 -131.

28. Anderson, B. F., Baker, H. M., Dodson, E. J. , Norris, G. E., Rumball, S. V., Waters, J. M. & Baker, E. N. (1987) Proc. Natl Acad. Sci. U S A 84,1768 - 1774.

29. Bailey, S., Evans, R. W., Garratt, R. C., Gorinsky, B., Hasnain.

141 - 144.

175-184.

J . Bacteriol. 165, 570-578.

201,19-26.

G. W. (1986) J . Biol. Chem. 261,4607-4614.

Res. Commun. 99, 11 01 - 1107.

21,6503 - 6508.

257,7560- 7565.

490.

SOC. 101,6097-6104.

(1981) Infect. Immun. 33, 540-545.

5401 - 5404.

108,6212-6218.

S. S., Horsburgh, C . , Jhoti, H., Lindley, P. F., Mydin, A,, Sarro, R. &Watson, J. L. (1988) Biochemistry 27, 5804-5812.

30. Perry, R. D. & San Clemente, C. L. (1979) J . Bacteriol. 140,

We thank thc Wellcome Trust for financial support of part of this work (grant 12289/1.5).

l i29- 11 32. 31. Lcibman, A. & Aisen, P. (1979) B/ood53, 1058-1065. 32. Meynell, G. G. & Subbiah, T. V. (1963) Br. J . Exp. Puthul. 4 4 ,

33. Eudy, W. W. & Burroughs, S. E. (1973) Chemotherapy 19, 161 -

34. Bullen, J. J . & Grifiths, E. (1987) Iron and infection, John Wiley.

REFERENCES

1930- 1937. 1. Aiscn, P., Leibman, A. & Zweier, J . (1978) J . Biol. Chem. 252, 197 - 219.

2. Evans, R. W. & Williams, J. (1978) Biochem. J. 173, 543-552. 3. Zapolski, E. J. & Princiotto, J. V. (1979) Biochemi.stry I Y , 3599-

170.

3603. Chichester.