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Active transport of siderophore-mimicking antibacterials across the outer membrane
Active antibiotic transport
Active transport of siderophore-mimicking antibacterials across the outer membrane Volkmar Braun Mikrobiologie/Membranphysiologie, Universität Tübingen,Auf der Morgenstelle 28, D-72076 Tübingen, Germany
Abstract The outer membrane of gram-negative bacteria forms a permeability barrier that usually reduces antibiotic access to intracellular targets and renders gram-negative bacteria less susceptible to antibiotics than gram-positive bacteria, which lack an outer membrane. However, gramnegative bacteria become highly susceptible to antibiotics that are actively transported across the outer membrane. Some antibiotics use active transport systems of substrates with which they share structural features. Examples are naturally occurring sideromycins and synthetic derivatives of Fe3+siderophores, which are taken up across the outer membrane by transport systems for Fe3+-siderophores.A well-studied example is albomycin, which has structural similarities to the natural substrate ferrichrome; albomycin and ferrichrome are both transported by the FhuA protein.A semisynthetic rifamycin derivative, CGP 4832, is also taken up by the FhuA transport protein, although its structure is completely different from that of ferrichrome.The crystal structures of FhuA with bound ferrichrome, albomycin, or rifamycin CGP 4832 reveal that the three compounds occupy the same site on FhuA; this site is accessible from the growth medium by a surface cavity that accommodates the antibiotic moieties.There is a rather strict stereochemical requirement for the portion that fits into the active site of FhuA, but a rather large tolerance regarding the portion that is located in the cavity.These data provide precise structural information for the design of highly active antibiotics composed of an antibiotically active moiety connected by a linker to a transported carrier.A number of Fe3+-siderophore carriers of the hydroxamate and catechol type linked to antibiotics have been isolated from microbes and synthesized; their superior efficacy has been demonstrated in vitro and in mice.Although none have been therapeutically employed, it is proposed that this alternative method of synthesizing useful antibiotics should be tested in light of the increasing problem of resistant pathogens. © 1999 Harcourt Publishers Ltd
SUBSTRATE TRANSLOCATION ACROSS THE OUTER MEMBRANE
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ram-negative bacteria are surrounded by an outer membrane that consists of a lipid bilayer into which proteins are inserted. The proteins determine the permeability of the outer membrane to hydrophilic compounds and are grouped into three functional classes with respect to the uptake of substrates.
The first class of outer-membrane proteins, the porins, form permanently open, water-filled channels through which compounds not larger than 600 Da freely diffuse along their concentration gradient. The porins (OmpF, OmpC) do not recognize the compounds that flow through.1,2 The second class of proteins also forms pores, similar to those formed by porins,3 except that they recognize their substrates. Maltodextrins bind to the LamB protein, sucrose binds to the ScrY protein, nucleosides and deoxynucleosides interact with the Tsx protein, and organic and inorganic phosphates bind to the PhoE protein.2 Movement of these substrates across the outer membrane follows their concentration gradient and does not consume energy. Binding to the transport proteins accelerates the rate of diffusion, and the substrates can be larger than those tolerated by the porins. For example, maltose can easily diffuse through the porins and does not require LamB. However, at low, micromolar maltose concentrations, growth is supported by facilitated diffusion through LamB and is no longer supported by diffusion through the porins. Uptake of maltotriose is more strongly dependent on LamB, and uptake of the larger homologs (maltotetraose to maltoheptaose) depends completely on LamB. The third class of proteins is involved in the uptake of ferric siderophores and vitamin B12.4 The molecular mass of ferric siderophores is usually in the range of 700 Da and above; therefore, diffusion through the porins is not fast enough to support growth. The concentration of the ferric siderophores is also too low to support growth by diffusion through the outer membrane since the siderophores are released and are diluted in the growth medium, where they complex the rare iron, often in competition with other strong iron scavengers, such as human transferrin and lactoferrin. Whenever ferric siderophores encounter a cell, the siderophores bind to transport proteins; this binding greatly increases the efficiency of iron uptake. The insolubility of Fe3+ requires the formation of soluble iron complexes in order for iron to be transported by bacteria; therefore, Fe3+ is not taken up as a metal ion, but is bound to low-molecular-weight siderophores.5 The Fe3+-siderophores bind to highly specific outer membrane proteins with Kd values in the nanomolar range and are transported across the outer membrane at the expense of cellular energy. In the periplasm, the Fe3+-siderophores are passed to binding proteins that deliver them to ABC transporters in the cytoplasmic membrane. Energy is provided by ATP, and the transporters contain ATP-binding sites and are therefore collectively termed ABC (ATP-binding cassette) transporters.6 Gram-positive bacteria also contain binding proteins that are anchored to the outer surface of the cytoplasmic membrane, recognize ferric siderophores, and transmit them to ABC transporters in the cytoplasmic membrane.7 THE CRYSTAL STRUCTURE OF FHUA PROVIDES THE BASIS FOR THE UNDERSTANDING OF FHUA AS A TRANSPORTER FhuA serves as transporter for ferrichrome (Fig. 1), a Fe3+hydroxamate synthesized by the fungus Ustilago sphaerogena, which is released into the growth medium and can be 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 363–369 DOI: 10.1054/drup.1999.0107, available online at http://www.idealibrary.com on
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Braun used by E. coli and many other gram-negative and gram-positive bacteria as an iron source. FhuA also transports the antibiotic albomycin (Fig. 1), a structural analogue of ferrichrome and the rifamycin antibiotic CGP 4832 (Fig. 1), which has no structural similarity to ferrichrome. FhuA deletion mutants no longer transport ferrichrome, are resistant to albomycin, and are as sensitive to rifamycin CGP 4832 as to unmodified rifamycin (Rifampicin). In 1998 the crystal structures of FhuA and of FhuA loaded with ferrichrome were published at 2.7 Å.resolution.8,9 FhuA
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consists of 22 antiparallel β-strands that form a β-barrel (Fig. 2), similar to the porins which form β-barrels of 16 or 18 strands. In contrast to the porins, the FhuA β-barrel is completely closed by residues 19 to 159, which form a globular structure that enters the β-barrel from the periplasmic side and is for this reason designated as the cork or plug. Ferrichrome binds in a pocket that is exposed to the cell surface above the external outer membrane interface (Fig. 2). Binding of ferrichrome causes movement of the cork by 1.7 Å towards ferrichrome and a large structural transition in the
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Fig. 1 Structures of the FhuA ligands ferrichrome, albomycin, and rifamycin CGP 4832 compared with the unmodified drug Rifampicin® which is the trade name of rifamycin.
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Active antibiotic transport the cytoplasmic membrane may release ferrichrome from the FhuA binding site and open the channel in the FhuA βbarrel, resulting in translocation of ferrichrome through FhuA into the periplasm, where it binds to the FhuD protein. FHUA AS AN ACTIVE ANTIBIOTIC TRANSPORTER Most antibiotics diffuse into bacteria.Their efficiency, as measured by the minimal inhibitory concentration (MIC), is determined by the diffusion rate and the activity at the target sites.Additional and specific mechanisms confer resistance15 and will not be discussed here. Gram-negative bacteria are usually less sensitive to antibiotics than gram-positive bacteria because they contain an outer membrane that functions as a permeability barrier. However, if antibiotics are actively transported across the outer membrane, their MIC may be lower in gram-negative than in gram-positive bacteria because the antibiotic is accumulated in the periplasm and forms a steep concentration gradient into the cytoplasm, thereby enhancing the diffusion rate, or the antibiotic may even be actively transported across the cytoplasmic membrane.
Fig. 2 Crystal structure of the outer membrane FhuA transport protein with bound ferrichrome.The arrow indicates the movement of the glutamate residue 19 (E19) upon binding of ferrichrome.
periplasmically exposed portion, where a short α-helix unwinds and Glu-19 moves 17.3 Å from its former α-carbon position. The ferrichrome-induced movement through the FhuA molecule and across the entire outer membrane does not open the channel of the β-barrel.This is thought to occur by input of energy from the cytoplasmic membrane, mediated by the Ton complex composed of the proteins TonB, ExbB, and ExbD.10,11 Energization of transport across the outer membrane occurs by the proton-motive force of the cytoplasmic membrane. Energy transfer could be achieved by a conformational change in TonB that is transmitted to FhuA. It has been shown that TonB binds to FhuA12,13 and that the TonB box (residues 7–11 of mature FhuA) interacts with residue 160 of TonB.14 It is thought that TonB, with the help of ExbB and ExbD, assumes an energized conformation that activates FhuA.The active conformation of FhuA translocates ferrichrome. Anchoring of TonB and ExbD by their N-terminal ends in the cytoplasmic membrane and the extension of the proteins into the periplasm make them physically suitable for conferring the energy transduction between the two membranes. The TonB box is exposed to the periplasm, but it cannot be identified in the ferrichrome-loaded and unloaded crystal structure since it is disordered. It is attractive to assume that the large, ferrichrome-induced structural change in the periplasmic pocket of FhuA exposes the TonB box and facilitates its interaction with the TonB protein. Energy input from
FhuA-albomycin: the first crystal structure of an antibiotic protein transporter Albomycin is a broad-spectrum antibiotic with excellent activity toward gram-positive and gram-negative bacteria.The minimal inhibitory concentration for E. coli K-12 is 100-fold lower than that of ampicillin.Albomycin belongs to the class of sideromycins which contain Fe3+. The albomycin-producing strain Streptomyces spec WS116 synthesizes three derivatives that differ in the pyrimidine side chains (Fig. 1). The chemical structure of the albomycins has been determined by 1H and 13C nuclear magnetic resonance spectroscopy, UV spectroscopy, and mass spectroscopy of the chemical degradation products.Albomycin is composed of a trihydroxamate that binds Fe3+, a peptide linker, and a thioribosyl pyrimidine moiety that confers the antibiotic activity.16 The high specific activity of albomycin comes from the active transport across the outer membrane and the cytoplasmic membrane into bacteria via the transport system of the structural analogue ferrichrome. Albomycin, like ferrichrome, is transported across the outer membrane by the FhuA protein. In the periplasm, albomycin binds to the FhuD protein, which then donates albomycin to the FhuB protein in the cytoplasmic membrane.Transport across the cytoplasmic membrane is energized by ATP hydrolysis catalyzed by the FhuC protein, which is associated with FhuB at the cytoplasmic side of the cytoplasmic membrane. The moiety of albomycin that is analogous to ferrichrome (Fig. 1) serves as the carrier of the antibiotically active thioribosyl pyrimidine group. After transport into the cytoplasm, iron is released from albomycin, and the thioribosyl pyrimidine group has to be cleaved from the carrier to be inhibitory; in E. coli, this is mainly achieved by peptidase N.17,18 Mutants devoid of peptidase N activity are resistant to albomycin, and albomycin then serves only as an iron carrier. Most of the thioribosyl pyrimidine moiety remains inside the cell, whereas the carrier is released into the culture medium.Albomycin is one of the very few antibiotics for which transport and intracellular 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 363–369
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activation have been characterized. The intracellular target has not been identified.The lack of albomycin-resistant target mutants suggest several targets and/or essential functions of the targets. It is likely that albomycin interferes with nucleic acid metabolism and/or with related functions, such as protein biosynthesis. Albomycin has been co-crystallized with FhuA to determine whether it binds to the ferrichrome binding site of FhuA, how it fits in the binding site, whether the thioribosyl pyrimidine moiety sterically hinders access to the ferrichrome binding site, and where this bulky side chain is located in FhuA (Fig. 2). In addition, the extra binding sites on FhuA could result in a stronger binding of FhuA and a lower transport rate of albomycin. The crystal structure reveals that the Fe3+-hydroxamate portion of albomycin occupies the same site on FhuA and interacts with the same amino acid chains as ferrichrome.19 The predominant binding sites are aromatic residues (69%). The thioribosyl pyrimidine moiety binds in the external pocket and five residues are involved; these residues are not involved in ferrichrome binding. These additional binding sites do not prevent release of albomycin from FhuA and transport through FhuA.The albomycin transport rate is half the transport rate of ferrichrome, but it is not clear whether the lower transport rate is through FhuA or through FhuD/FhuB/FhuC across the cytoplasmic membrane. The structure of the FhuA albomycin co-crystal has also revealed the hitherto unknown conformation of albomycin and the conformation in the transport-competent form.The most unexpected result was the existence of two albomycin conformations in the crystal: an extended and a compact conformation. Both conformations fit into the external cavity of FhuA and occupy seven different amino acid ligands.The solvent-exposed external cavity of FhuA is sufficiently large to accommodate the voluminous side chain of albomycin. With the modular composition of albomycin, in which the iron carrier is connected by a peptide linker to the antibiotically active thioribosyl pyrimidine, nature provides a clue of how to design highly efficient antibiotics that can be actively transported into bacteria. Such antibiotics could be synthetically assembled from Fe3+-hydroxamates, which fit into the active center of the transporters, and from an antibiotic that diffuses too slowly into cells to be useful by itself as a drug.The FhuA–albomycin structure demonstrates that the water-filled cavities in transporters can tolerate rather large antibiotics that are structurally unrelated to the carrier.This tolerance is not confined to FhuA since albomycin is transported very well also across the cytoplasmic membrane and is in this process recognized by the FhuD and the FhuB proteins.
the proteins required for active transport of ferrichrome across the cytoplasmic membrane display unaltered CGP 4832 sensitivity.20 Our attempts to find out whether CGP 4832 is also actively transported across the cytoplasmic membrane yielded mutations only in the fhuA, tonB, exbB, and exbD genes required for transport across the outer membrane, which suggests that CGP 4832 crosses the cytoplasmic membrane by diffusion rather than by transport.The use of FhuA as transporter for CGP 4832 was surprising since CGP 4832 does not contain iron and has no structural resemblance to ferrichrome (Fig. 1). Therefore, it was particularly attractive to determine the crystal structure of FhuA loaded with CGP 4832. Analysis of the X-ray diffraction data revealed that CGP 4832 largely occupies the site in FhuA that is also used by ferrichrome.21 Interestingly, of 16 amino acid residues of FhuA that bind CGP 4832, 5 residues recognize those side chains of CGP 4832 in which it differs from unmodified rifamycin. Nine residues that bind CGP 4832 also bind ferrichrome. Two additional amino acid residues specifically bind the unique CGP 4832 side chains, whereas the other residues bind to sites which CGP 4832 shares with rifamycin.The latter residues seem to confer a higher sensitivity to rifamycin to E. coli carrying FhuA than to E. coli lacking FhuA. In contrast to ferrichrome and albomycin, CGP 4832 in the crystal does not cause the large structural change in the periplasmically oriented pocket of FhuA.This does not seem to be caused by restriction of FhuA movements since binding of CGP 4832 to FhuA in solution does not result in intrinsic FhuA tryptophan fluorescence quenching, which is observed when FhuA binds ferrichrome.This finding has an impact on the concept of how FhuA interacts with TonB since, as discussed above, the large structural transition is thought to facilitate interaction of FhuA with TonB.Transport of CGP 4932 depends on TonB; therefore, interaction of TonB with FhuA can also occur in the absence of the structural change. This conclusion is supported by results obtained with a mutant, FhuA∆5–160, in which the entire cork including the TonB box has been deleted by genetic engineering.22 At high concentrations, ferrichrome passively diffuses through FhuA∆5–160, but at low concentrations, ferrichrome is actively transported by FhuA∆5–160. The deletion mutant is highly sensitive to albomycin and CGP 4832; the sensitivity is conferred by FhuA∆5–160. Apparently TonB not only interacts via the TonB box with FhuA, but binds to additional sites of FhuA in a function-relevant manner.
Crystal structure of FhuA with bound rifamycin CGP 4832 In 1987, a group from Ciba-Geigy reported on a semisynthetic rifamycin derivative, CGP 4832, with an activity against many gram-negative bacteria 200-fold higher than that of unmodified rifamycin.20 It was then shown with mutants that rifamycin CGP 4832 is transported by FhuA across the outer membrane of E. coli and that the TonB activity is required. Mutants in the fhuBCD genes, which encode
A number of sulfonamides conjugated to the Fe3+siderophores ferricrocin, a ferrichrome derivative, and ferrioxamine B have been synthesized in the first attempt to examine the efficacy of synthetic antibiotics that enter bacterial cells via Fe3+-siderophore transport systems.23 A sulfanilamido-nicotinic acid ferricrocinyl ester and a sulfanilamido-nicotinic acid ferrioxamine B conjugate display antibiotic activity toward S. aureus, but not toward E. coli; in the latter case, the conjugates could be antagonized by the
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FE3+-HYDROXAMATE-SULFONAMIDE CONJUGATES
Active antibiotic transport Fe3+-siderophores, which points toward competition for the transport systems. However, the activity of the conjugates was too low for their use as therapeutic antibiotics.
FE3+-HYDROXAMATE-Β-LACTAM CONJUGATES Iron-transport-mediated drug delivery has also been investigated by chemical synthesis of the trihydroxamate (N5acetyl-N5-hydroxy-L-ornithine)3 linked to β-lactams. 24,25 However, these compounds, instead of inhibiting growth, promoted growth of strains of E. coli, Shigella flexneri, and Salmonella under iron-limiting conditions in that they served as iron siderophores and not as antibiotics.The β-lactam-hypersensitive E. coli X580 and the Salmonella X514 strain exhibited an increased sensitivity to the β-lactam conjugates as compared to the unmodified β-lactams. Pseudomonas, Staphylococcus aureus, and Streptococcus pneumoniae were resistant. FE3+-CATECHOLATE-CEPHALOSPORIN CONJUGATES Catechol-substituted cephalosporins represent the largest group of antibiotics that have been linked to Fe3+siderophores.27–34 Their MIC values are frequently below 1 µg/ml, particularly against gram-negative bacteria, including P. aeruginosa. Their antimicrobial activities may exceed the activity of the unsubstituted cephalosporins more than 100-fold.The highly active cephalosporin derivatives contain a catechol group, as does the E. coli enterobactin Fe3+ siderophore. Removal of one or both vicinal hydroxyl groups, which bind iron, strongly reduces the activities of the catechol cephalosporins.Their high activity is related to their active transport into the periplasm. For example, resistant mutants of five sensitive E. coli strains are 500-fold less sensitive to the cephalosporin derivative E-0702 than the wild-type strains. The resistant strains are mutated in the tonB gene.27 Since TonB is involved in iron transport, it was determined whether the cephalosporin derivatives bind iron. Specific iron chelation was demonstrated spectroscopically.27 Moreover, the activity of the antibiotics depends on the iron supply of the cells and is high under iron-limiting conditions and low under iron-replete conditions. Lack or substitution of the iron-chelating hydroxyl groups reduces the activity of catechol cephalosporins to that of unsubstituted cephalosporins. Other studies have demonstrated the involvement of the Fe3+-catechol receptor proteins Fiu and Cir26 in the transport of the catechol cephalosporins into E. coli. Iron limitation increases the susceptibility of E. coli strains to E-0702 (Fig. 3) 4000-fold over that of a transportnegative tonB mutant and 2000-fold over that of a transportnegative cir fiu double mutant. The minimal inhibitory concentration for these mutants lacking the Fur iron repressor is even more reduced (8000-fold and 4000 fold, respectively). The cephalosporin derivatives bind to penicillin binding protein 3 with an affinity (I50 0.03–0.13 µg/ml) similar to that of the unsubstituted cephalosporins; this clearly demonstrates that the high activity of catechol-substituted cephalosporins is caused by their active transport. Determination of the rate of iron-free E-0702 hydrolysis in
Fig. 3 Examples of cephalosporin catecholates, E-0702,27 catechol-cephalosporins,28 GR69153,30 L-658,310,31 and KP-736,34 which are actively transported into the periplasm of E. Coli by the Cir and Fiu proteins and the Ton energizing system.
the periplasm by the TEM β-lactamase as a measure of the E-0702 entry into the periplasm has clearly revealed a dependence on Fiu and Cir and suggests that iron-free E-0702 is in fact transported.29 Since the cephalosporins only have to enter the periplasm, where their target site is located, active transport across the outer membrane is sufficient to enhance their antibiotic activity greatly. The efficacy of the catecholate cephalosporin L-658, 310 alone and in combination with gentamicin against P. aeruginosa has been determined in mice.32,33 The median effective dose of L-658,310 is 30.4 mg/kg, that of gentamicin 63.3 mg/kg, and that of the combined antibiotics 2 mg/kg. The challenge contained 32 LD50 doses, and the antibiotics were administered subcutaneously 0 and 6 h after infection. 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 363–369
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Braun The number of survivors was measured after 7 days observation. FERRIMYCIN Ferrimycins are among the first sideromycins discovered. The structure of ferrimycin A1 is shown in Figure 4.Although it inhibits incorporation of amino acids into proteins of Staphylococcus aureus SG511, it does not inhibit, but rather enhances polyphenylalanine synthesis in an in vitro S-30 polyU-directed protein synthesis system of S. aureus. The action of ferrimycins is antagonized by ferroxamine B, which competes for ferrimycin uptake.35 SALMYCINS Salmycins have been isolated from Streptomyces violaceus 37290 (DSM 8286) and are active against staphylococci and streptococci (MIC 10 µg/ml).36 Salmycins consist of an Fe3+siderophore of the ferrioxamine group and an antibiotically active aminodisaccharide, which in salmycin B consists of a 2-ketoglucose linked to the 2-position of a 6-methylaminoheptopyranase (Fig. 4).The salmycin structures are similar to those of danomycin and A-22765, whose complete structures have not been determined. It is assumed that the aminodisaccharide is released from the carrier by cleavage of the ester bond. Like danomycin and A-22765, salmycins inhibit protein synthesis, and how this is achieved by a disaccharide is an interesting, open question. RESISTANCE TO IRON-CARRIER ANTIBIOTICS In the laboratory, resistant bacteria emerge on each nutrient agar plate seeded with sensitive bacteria and antibiotics that are carried into the bacteria by active Fe3+-siderophore transport systems – the higher the number of genes involved in a
particular transport system, the higher the frequency of resistance. However, when two transport systems are used by an antibiotic, for example Cir and Fiu for catechol cephalosporins, the frequency of resistant mutants is low. Although the high resistance frequency seems to prevent development of such antibiotics as antibacterial drugs, the in vivo situation might be quite different. In the few unpublished attempts to evaluate the in vivo efficacy of iron-carrier antibiotics known to the author, resistance in mice was not a problem (see also references 32 and 33). In cases where an iron-transport system is important for the proliferation of the pathogenic bacteria, loss of the iron transport system is detrimental. Even when several iron transport systems exist and only one is inactivated by the antibiotic, the inactivated one may be the transport system that is essential for the bacteria to survive and multiply at the site of infection in the human host. Under these circumstances, it does not matter whether the number of bacteria are reduced by the antibiotic or by the loss of the iron supply since under both conditions the immune defense system gains time to cope with the infection. OUTLOOK The emergence of resistant bacteria is an increasing problem; therefore, the possibility of developing antibiotics which are carried into bacterial cells by iron-transport systems should not be ignored. Active transport reduces the minimal inhibitory concentration by more than 100-fold and thus lowers the risk of antibiotic toxicity. Bacteria are also killed faster because the antibiotic attains intracellular inhibitory concentration sooner. The crystal structures of the FhuA transport protein loaded with the antibiotic albomycin or rifamycin CGP 4832 provides the first insights into the high degree of tolerance to distinct structures that are accepted by the transport protein and strongly supports the proposal for the semisynthesis of antibiotics hooked onto Fe3+-siderophore carriers to facilitate their entry into pathogens. Acknowledgements
The author would like to thank Michael Braun for the preparation of Figures 1 and 2 and Karen A. Brune for critically reading the manuscript. The author’s work was supported by the Deutsche Forschungsgemeinschaft (SFB 323, Graduiertenkolleg Mikrobiologie, Br330/14–2) and the Fonds der Chemischen Industrie. Received 26 October 1999; Revised 8 November 1999; Accepted 8 November 1999 Correspondence to: Volkmar Braun, Mikrobiologie/Membranphysiologie, Universität Tübingen,Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Tel: +49 7071 2972096; Fax: +49 7071 29 5843; E-mail [email protected]
Fig. 4 Examples of antibiotic hydroxamates, ferrimycin A (top) and salmycin (bottom), which are structurally related to ferrioxamine B,5,35, in contrast to albomycin (Fig. 1), which belongs to the antibiotic hydroxamates of the ferrichrome type.
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References 1. Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 1994; 264: 382–388.
Active antibiotic transport 2. Benz R. Uptake of solutes through bacterial outer membranes. In: Ghuysen J-M, Hakenbeck R, eds. Bacterial cell wall.Amsterdam: Elsevier, 1994: 397–423. 3. Schirmer T, Keller TA,Wang Y-F, Rosenbusch JP. Structural basis for sugar translocation through the maltoporin channels at 3.1 Å resolution. Science 1995; 276: 512–514. 4. Braun V, Hantke K, Köster W. Bacterial iron transport: mechanisms, genetics, and regulation. In: Sigel A, Sigel H, eds. Metal ions in biological systems. New York: Marcel Dekker. 1998; 35: 67–145. 5. Drechsel H,Winkelmann G. Iron chelation and siderophores. In: Winkelmann G, Carrano CJ, eds.Transition metals in microbial metabolism.Amsterdam: Harwood Academic Publishers, 1997: 1–49. 6. Higgins CF.ABC transporters from microorganisms to man.Ann Rev Cell Biol 1992; 8: 67–113. 7. Schneider R, Hantke K. Iron hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding proteindependent transport system. Mol Microbiol 1993; 8: 111–21. 8. Ferguson AD, Hofmann E, Coulton JW, Diederichs K,Welte W. Structural basis for siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 1998; 282: 2215–2220. 9. Locher K, Rees B, Koebnik R et al.Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 1998; 95: 771–778. 10. Braun V. Pumping iron through cell membranes. Science 1998; 282: 2202–2203. 11. Braun V. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS Microbiol Rev 1995; 16: 295–307. 12. Günter K, Braun V. In vivo evidence for FhuA outer membrane interaction with the TonB inner membrane protein TonB. FEBS Lett 1990; 274: 85–88. 13. Moeck GS, Coulton JW, Postle K. Cell envelope signaling in Escherichia coli. Ligand binding to the ferrichrome-iron receptor FhuA promotes interaction with the energy-transducing protein TonB. J Biol Chem 1997; 272: 28391–28397. 14. Schöffler H, Braun V.Transport across the outer membrane of Escherichia coli via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane. Mol Gen Genet 1989; 217: 378–383. 15. Nikaido H.The role of outer membrane and efflux pumps in the resistance of gram-negative bacteria. Can we improve drug access? Drug Resistance Updates 1998; 1: 93–98. 16. Benz G, Schröder T, Kurz J,Wünsche C, Karl W, Steffens G, Pfitzne, J, Schmidt D Konstitution der Desferriform der Albomycine ∂1, ∂2, ε.Angew Chem Suppl 1982; 1322–1335. 17. Hartmann A, Fiedler H-P, Braun V Uptake and conversion of the antibiotic albomycin by Escherichia coli K-12. Eur J Biochem 1979; 99: 517–524. 18. Braun V, Günther K, Hantke K, Zimmermann L. Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium. J Bacteriol 1983; 156: 308–315. 19. Ferguson AD, Braun V, Fiedler H-P, Coulton JW, Diederichs K, Welte W. Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA. 2000; submitted 20. Pugsley PA, Zimmermann W,Wehrli W. Highly efficient uptake of a rifamycin derivative via FhuA-TonB-dependent uptake route in Escherichia coli. J Gen Microbiol 1987; 133: 3505–3511.
21. Ferguson AD, Ködding J,Walker G et al. Crystal structure of a semisynthetic rifamycin derivative in complex with the active outer membrane transporter FhuA from Escherichia coli K-12. 2000; submitted. 22. Braun M, Killmann H, Braun V.The β-barrel domain of FhuA∆5–160 is sufficient for TonB-dependent FhuA activities of Escherichia coli. Mol Microbiol 1999; 33: 1037–1049. 23. Zähner H, Diddens H, Keller-Schierlein W, Nägeli H-U. Some experiments with semisynthetic sideromycins. Jap J Antibiot Suppl 1977; 30: 201–206. 24. Dolence EK, Minnick AA, Lin C-E, Miller M. Synthesis and siderophore and antibacterial activity of N5-acetyl-N5-hydroxy-Lornithine-derived siderophore-β-lactam conjugates: iron transport-mediated drug delivery. J Med Chem 1991; 34: 868–978. 25. Diarra MS, Lavoie MC, Jacques M et al. Species selectivity of new siderophore-drug conjugates that use specific iron uptake for entry into bacteria.Antimicrobial Agents Chemother 1996; 40: 2610–2617. 26. Hantke K. Dihydroxybenzoylserine—a siderophore for E. coli. FEMS Microbiol Lett 1990; 67: 5–8. 27. Watanabe N-A, Nagasu T, Katsu K, Kitoh K. E-0702, a new cephalosporin, is incorporated into Escherichia coli cells via the tonB-dependent iron transport system.Antimicrobial Agents Chemother 1987; 31: 497–504. 28. Curtiss NAC, Eisenstadt RL, East SJ et al. Iron regulated outer membrane proteins of Escherichia coli K-12 and mechanisms of action of catechol-substituted cephalosporins.Antimicrobial Agents Chemother 1988; 32: 1879–1886. 29. Nikaido H, Rosenberg EY. Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with β-lactam antibiotics containing catechol and analogous groups. J Bacteriol 1990; 172: 1361–1367. 30. Silley P, Griffith JW, Monsey D, Harris AM. Mode of action of GR69153, a novel catechol-substituted cephalosporin, and its interaction with the tonB-dependent iron transport system. Antimicrobial Agents Chemother 1990; 34: 1806–1808. 31. Weissberger BA,Abruzzo GK, Fromtling RA et al. L-658–310, a new injectable cephalosporin. I. In vitro antibacterial properties. J Antibiotics 1989; 42: 795–806. 32. Valiant ME Gilfillan EC, Gadebusch HH, Pelak BA. L-658–310 a new injectable cephalosporin. II. In vitro and in vivo interactions between L-658, 310 and various amino glycosides or ciprofloxacin versus clinical isolates of Pseudomonas aeruginosa. J Antibiotics 1989; 42: 807–814. 33. Gilfillan EC, Pelak BA,Weissberger BA et al. L-658–310, a new injectable cephalosporin. III. Experimental chemotherapeutics and pharmacokinetics in laboratory animals. J Antibiotics 1989; 42: 815–822. 34. Tatsumi Y, Mejima T, Mitsuhashi S. Mechanism of tonB-dependent transport of KP6736, a 1,5-dihydroxy-4-pyridone-substituted cephalosporin, into Escherichia coli K-12 cells.Antimicrobial Agents Chemother 1995; 39: 613–619. 35. Knüsel F, Zimmermann W. Sideromycins. In: Corcoran JW, Hahn FE, eds.Antibiotics III. Mechanisms of action of antimicrobial and antitumor agents. Berlin, Springer 1975: 653–667. 36. Vertesy L,Aretz W, Fehlhaber H-W, Kogler H. Salmycins A-D, antibiotics from Streptomyces violaceus DSM 8286 having a siderophore-aminoglycoside structure. Helv Chim Acta 1995; 78: 46–60.
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Active transport of siderophore-mimicking antibacterials across the outer membrane Volkmar Braun Mikrobiologie/Membranphysiologie, Universität Tübingen,Auf der Morgenstelle 28, D-72076 Tübingen, Germany
Abstract The outer membrane of gram-negative bacteria forms a permeability barrier that usually reduces antibiotic access to intracellular targets and renders gram-negative bacteria less susceptible to antibiotics than gram-positive bacteria, which lack an outer membrane. However, gramnegative bacteria become highly susceptible to antibiotics that are actively transported across the outer membrane. Some antibiotics use active transport systems of substrates with which they share structural features. Examples are naturally occurring sideromycins and synthetic derivatives of Fe3+siderophores, which are taken up across the outer membrane by transport systems for Fe3+-siderophores.A well-studied example is albomycin, which has structural similarities to the natural substrate ferrichrome; albomycin and ferrichrome are both transported by the FhuA protein.A semisynthetic rifamycin derivative, CGP 4832, is also taken up by the FhuA transport protein, although its structure is completely different from that of ferrichrome.The crystal structures of FhuA with bound ferrichrome, albomycin, or rifamycin CGP 4832 reveal that the three compounds occupy the same site on FhuA; this site is accessible from the growth medium by a surface cavity that accommodates the antibiotic moieties.There is a rather strict stereochemical requirement for the portion that fits into the active site of FhuA, but a rather large tolerance regarding the portion that is located in the cavity.These data provide precise structural information for the design of highly active antibiotics composed of an antibiotically active moiety connected by a linker to a transported carrier.A number of Fe3+-siderophore carriers of the hydroxamate and catechol type linked to antibiotics have been isolated from microbes and synthesized; their superior efficacy has been demonstrated in vitro and in mice.Although none have been therapeutically employed, it is proposed that this alternative method of synthesizing useful antibiotics should be tested in light of the increasing problem of resistant pathogens. © 1999 Harcourt Publishers Ltd
SUBSTRATE TRANSLOCATION ACROSS THE OUTER MEMBRANE
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ram-negative bacteria are surrounded by an outer membrane that consists of a lipid bilayer into which proteins are inserted. The proteins determine the permeability of the outer membrane to hydrophilic compounds and are grouped into three functional classes with respect to the uptake of substrates.
The first class of outer-membrane proteins, the porins, form permanently open, water-filled channels through which compounds not larger than 600 Da freely diffuse along their concentration gradient. The porins (OmpF, OmpC) do not recognize the compounds that flow through.1,2 The second class of proteins also forms pores, similar to those formed by porins,3 except that they recognize their substrates. Maltodextrins bind to the LamB protein, sucrose binds to the ScrY protein, nucleosides and deoxynucleosides interact with the Tsx protein, and organic and inorganic phosphates bind to the PhoE protein.2 Movement of these substrates across the outer membrane follows their concentration gradient and does not consume energy. Binding to the transport proteins accelerates the rate of diffusion, and the substrates can be larger than those tolerated by the porins. For example, maltose can easily diffuse through the porins and does not require LamB. However, at low, micromolar maltose concentrations, growth is supported by facilitated diffusion through LamB and is no longer supported by diffusion through the porins. Uptake of maltotriose is more strongly dependent on LamB, and uptake of the larger homologs (maltotetraose to maltoheptaose) depends completely on LamB. The third class of proteins is involved in the uptake of ferric siderophores and vitamin B12.4 The molecular mass of ferric siderophores is usually in the range of 700 Da and above; therefore, diffusion through the porins is not fast enough to support growth. The concentration of the ferric siderophores is also too low to support growth by diffusion through the outer membrane since the siderophores are released and are diluted in the growth medium, where they complex the rare iron, often in competition with other strong iron scavengers, such as human transferrin and lactoferrin. Whenever ferric siderophores encounter a cell, the siderophores bind to transport proteins; this binding greatly increases the efficiency of iron uptake. The insolubility of Fe3+ requires the formation of soluble iron complexes in order for iron to be transported by bacteria; therefore, Fe3+ is not taken up as a metal ion, but is bound to low-molecular-weight siderophores.5 The Fe3+-siderophores bind to highly specific outer membrane proteins with Kd values in the nanomolar range and are transported across the outer membrane at the expense of cellular energy. In the periplasm, the Fe3+-siderophores are passed to binding proteins that deliver them to ABC transporters in the cytoplasmic membrane. Energy is provided by ATP, and the transporters contain ATP-binding sites and are therefore collectively termed ABC (ATP-binding cassette) transporters.6 Gram-positive bacteria also contain binding proteins that are anchored to the outer surface of the cytoplasmic membrane, recognize ferric siderophores, and transmit them to ABC transporters in the cytoplasmic membrane.7 THE CRYSTAL STRUCTURE OF FHUA PROVIDES THE BASIS FOR THE UNDERSTANDING OF FHUA AS A TRANSPORTER FhuA serves as transporter for ferrichrome (Fig. 1), a Fe3+hydroxamate synthesized by the fungus Ustilago sphaerogena, which is released into the growth medium and can be 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 363–369 DOI: 10.1054/drup.1999.0107, available online at http://www.idealibrary.com on
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Braun used by E. coli and many other gram-negative and gram-positive bacteria as an iron source. FhuA also transports the antibiotic albomycin (Fig. 1), a structural analogue of ferrichrome and the rifamycin antibiotic CGP 4832 (Fig. 1), which has no structural similarity to ferrichrome. FhuA deletion mutants no longer transport ferrichrome, are resistant to albomycin, and are as sensitive to rifamycin CGP 4832 as to unmodified rifamycin (Rifampicin). In 1998 the crystal structures of FhuA and of FhuA loaded with ferrichrome were published at 2.7 Å.resolution.8,9 FhuA
H
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H N H 3C C
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consists of 22 antiparallel β-strands that form a β-barrel (Fig. 2), similar to the porins which form β-barrels of 16 or 18 strands. In contrast to the porins, the FhuA β-barrel is completely closed by residues 19 to 159, which form a globular structure that enters the β-barrel from the periplasmic side and is for this reason designated as the cork or plug. Ferrichrome binds in a pocket that is exposed to the cell surface above the external outer membrane interface (Fig. 2). Binding of ferrichrome causes movement of the cork by 1.7 Å towards ferrichrome and a large structural transition in the
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OH CH3
O
Albomycin δ 2
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CH3COO CH3O
R= O
O
N
O
CO
O
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CH3 CGP 4832
Fig. 1 Structures of the FhuA ligands ferrichrome, albomycin, and rifamycin CGP 4832 compared with the unmodified drug Rifampicin® which is the trade name of rifamycin.
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Active antibiotic transport the cytoplasmic membrane may release ferrichrome from the FhuA binding site and open the channel in the FhuA βbarrel, resulting in translocation of ferrichrome through FhuA into the periplasm, where it binds to the FhuD protein. FHUA AS AN ACTIVE ANTIBIOTIC TRANSPORTER Most antibiotics diffuse into bacteria.Their efficiency, as measured by the minimal inhibitory concentration (MIC), is determined by the diffusion rate and the activity at the target sites.Additional and specific mechanisms confer resistance15 and will not be discussed here. Gram-negative bacteria are usually less sensitive to antibiotics than gram-positive bacteria because they contain an outer membrane that functions as a permeability barrier. However, if antibiotics are actively transported across the outer membrane, their MIC may be lower in gram-negative than in gram-positive bacteria because the antibiotic is accumulated in the periplasm and forms a steep concentration gradient into the cytoplasm, thereby enhancing the diffusion rate, or the antibiotic may even be actively transported across the cytoplasmic membrane.
Fig. 2 Crystal structure of the outer membrane FhuA transport protein with bound ferrichrome.The arrow indicates the movement of the glutamate residue 19 (E19) upon binding of ferrichrome.
periplasmically exposed portion, where a short α-helix unwinds and Glu-19 moves 17.3 Å from its former α-carbon position. The ferrichrome-induced movement through the FhuA molecule and across the entire outer membrane does not open the channel of the β-barrel.This is thought to occur by input of energy from the cytoplasmic membrane, mediated by the Ton complex composed of the proteins TonB, ExbB, and ExbD.10,11 Energization of transport across the outer membrane occurs by the proton-motive force of the cytoplasmic membrane. Energy transfer could be achieved by a conformational change in TonB that is transmitted to FhuA. It has been shown that TonB binds to FhuA12,13 and that the TonB box (residues 7–11 of mature FhuA) interacts with residue 160 of TonB.14 It is thought that TonB, with the help of ExbB and ExbD, assumes an energized conformation that activates FhuA.The active conformation of FhuA translocates ferrichrome. Anchoring of TonB and ExbD by their N-terminal ends in the cytoplasmic membrane and the extension of the proteins into the periplasm make them physically suitable for conferring the energy transduction between the two membranes. The TonB box is exposed to the periplasm, but it cannot be identified in the ferrichrome-loaded and unloaded crystal structure since it is disordered. It is attractive to assume that the large, ferrichrome-induced structural change in the periplasmic pocket of FhuA exposes the TonB box and facilitates its interaction with the TonB protein. Energy input from
FhuA-albomycin: the first crystal structure of an antibiotic protein transporter Albomycin is a broad-spectrum antibiotic with excellent activity toward gram-positive and gram-negative bacteria.The minimal inhibitory concentration for E. coli K-12 is 100-fold lower than that of ampicillin.Albomycin belongs to the class of sideromycins which contain Fe3+. The albomycin-producing strain Streptomyces spec WS116 synthesizes three derivatives that differ in the pyrimidine side chains (Fig. 1). The chemical structure of the albomycins has been determined by 1H and 13C nuclear magnetic resonance spectroscopy, UV spectroscopy, and mass spectroscopy of the chemical degradation products.Albomycin is composed of a trihydroxamate that binds Fe3+, a peptide linker, and a thioribosyl pyrimidine moiety that confers the antibiotic activity.16 The high specific activity of albomycin comes from the active transport across the outer membrane and the cytoplasmic membrane into bacteria via the transport system of the structural analogue ferrichrome. Albomycin, like ferrichrome, is transported across the outer membrane by the FhuA protein. In the periplasm, albomycin binds to the FhuD protein, which then donates albomycin to the FhuB protein in the cytoplasmic membrane.Transport across the cytoplasmic membrane is energized by ATP hydrolysis catalyzed by the FhuC protein, which is associated with FhuB at the cytoplasmic side of the cytoplasmic membrane. The moiety of albomycin that is analogous to ferrichrome (Fig. 1) serves as the carrier of the antibiotically active thioribosyl pyrimidine group. After transport into the cytoplasm, iron is released from albomycin, and the thioribosyl pyrimidine group has to be cleaved from the carrier to be inhibitory; in E. coli, this is mainly achieved by peptidase N.17,18 Mutants devoid of peptidase N activity are resistant to albomycin, and albomycin then serves only as an iron carrier. Most of the thioribosyl pyrimidine moiety remains inside the cell, whereas the carrier is released into the culture medium.Albomycin is one of the very few antibiotics for which transport and intracellular 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 363–369
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activation have been characterized. The intracellular target has not been identified.The lack of albomycin-resistant target mutants suggest several targets and/or essential functions of the targets. It is likely that albomycin interferes with nucleic acid metabolism and/or with related functions, such as protein biosynthesis. Albomycin has been co-crystallized with FhuA to determine whether it binds to the ferrichrome binding site of FhuA, how it fits in the binding site, whether the thioribosyl pyrimidine moiety sterically hinders access to the ferrichrome binding site, and where this bulky side chain is located in FhuA (Fig. 2). In addition, the extra binding sites on FhuA could result in a stronger binding of FhuA and a lower transport rate of albomycin. The crystal structure reveals that the Fe3+-hydroxamate portion of albomycin occupies the same site on FhuA and interacts with the same amino acid chains as ferrichrome.19 The predominant binding sites are aromatic residues (69%). The thioribosyl pyrimidine moiety binds in the external pocket and five residues are involved; these residues are not involved in ferrichrome binding. These additional binding sites do not prevent release of albomycin from FhuA and transport through FhuA.The albomycin transport rate is half the transport rate of ferrichrome, but it is not clear whether the lower transport rate is through FhuA or through FhuD/FhuB/FhuC across the cytoplasmic membrane. The structure of the FhuA albomycin co-crystal has also revealed the hitherto unknown conformation of albomycin and the conformation in the transport-competent form.The most unexpected result was the existence of two albomycin conformations in the crystal: an extended and a compact conformation. Both conformations fit into the external cavity of FhuA and occupy seven different amino acid ligands.The solvent-exposed external cavity of FhuA is sufficiently large to accommodate the voluminous side chain of albomycin. With the modular composition of albomycin, in which the iron carrier is connected by a peptide linker to the antibiotically active thioribosyl pyrimidine, nature provides a clue of how to design highly efficient antibiotics that can be actively transported into bacteria. Such antibiotics could be synthetically assembled from Fe3+-hydroxamates, which fit into the active center of the transporters, and from an antibiotic that diffuses too slowly into cells to be useful by itself as a drug.The FhuA–albomycin structure demonstrates that the water-filled cavities in transporters can tolerate rather large antibiotics that are structurally unrelated to the carrier.This tolerance is not confined to FhuA since albomycin is transported very well also across the cytoplasmic membrane and is in this process recognized by the FhuD and the FhuB proteins.
the proteins required for active transport of ferrichrome across the cytoplasmic membrane display unaltered CGP 4832 sensitivity.20 Our attempts to find out whether CGP 4832 is also actively transported across the cytoplasmic membrane yielded mutations only in the fhuA, tonB, exbB, and exbD genes required for transport across the outer membrane, which suggests that CGP 4832 crosses the cytoplasmic membrane by diffusion rather than by transport.The use of FhuA as transporter for CGP 4832 was surprising since CGP 4832 does not contain iron and has no structural resemblance to ferrichrome (Fig. 1). Therefore, it was particularly attractive to determine the crystal structure of FhuA loaded with CGP 4832. Analysis of the X-ray diffraction data revealed that CGP 4832 largely occupies the site in FhuA that is also used by ferrichrome.21 Interestingly, of 16 amino acid residues of FhuA that bind CGP 4832, 5 residues recognize those side chains of CGP 4832 in which it differs from unmodified rifamycin. Nine residues that bind CGP 4832 also bind ferrichrome. Two additional amino acid residues specifically bind the unique CGP 4832 side chains, whereas the other residues bind to sites which CGP 4832 shares with rifamycin.The latter residues seem to confer a higher sensitivity to rifamycin to E. coli carrying FhuA than to E. coli lacking FhuA. In contrast to ferrichrome and albomycin, CGP 4832 in the crystal does not cause the large structural change in the periplasmically oriented pocket of FhuA.This does not seem to be caused by restriction of FhuA movements since binding of CGP 4832 to FhuA in solution does not result in intrinsic FhuA tryptophan fluorescence quenching, which is observed when FhuA binds ferrichrome.This finding has an impact on the concept of how FhuA interacts with TonB since, as discussed above, the large structural transition is thought to facilitate interaction of FhuA with TonB.Transport of CGP 4932 depends on TonB; therefore, interaction of TonB with FhuA can also occur in the absence of the structural change. This conclusion is supported by results obtained with a mutant, FhuA∆5–160, in which the entire cork including the TonB box has been deleted by genetic engineering.22 At high concentrations, ferrichrome passively diffuses through FhuA∆5–160, but at low concentrations, ferrichrome is actively transported by FhuA∆5–160. The deletion mutant is highly sensitive to albomycin and CGP 4832; the sensitivity is conferred by FhuA∆5–160. Apparently TonB not only interacts via the TonB box with FhuA, but binds to additional sites of FhuA in a function-relevant manner.
Crystal structure of FhuA with bound rifamycin CGP 4832 In 1987, a group from Ciba-Geigy reported on a semisynthetic rifamycin derivative, CGP 4832, with an activity against many gram-negative bacteria 200-fold higher than that of unmodified rifamycin.20 It was then shown with mutants that rifamycin CGP 4832 is transported by FhuA across the outer membrane of E. coli and that the TonB activity is required. Mutants in the fhuBCD genes, which encode
A number of sulfonamides conjugated to the Fe3+siderophores ferricrocin, a ferrichrome derivative, and ferrioxamine B have been synthesized in the first attempt to examine the efficacy of synthetic antibiotics that enter bacterial cells via Fe3+-siderophore transport systems.23 A sulfanilamido-nicotinic acid ferricrocinyl ester and a sulfanilamido-nicotinic acid ferrioxamine B conjugate display antibiotic activity toward S. aureus, but not toward E. coli; in the latter case, the conjugates could be antagonized by the
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FE3+-HYDROXAMATE-SULFONAMIDE CONJUGATES
Active antibiotic transport Fe3+-siderophores, which points toward competition for the transport systems. However, the activity of the conjugates was too low for their use as therapeutic antibiotics.
FE3+-HYDROXAMATE-Β-LACTAM CONJUGATES Iron-transport-mediated drug delivery has also been investigated by chemical synthesis of the trihydroxamate (N5acetyl-N5-hydroxy-L-ornithine)3 linked to β-lactams. 24,25 However, these compounds, instead of inhibiting growth, promoted growth of strains of E. coli, Shigella flexneri, and Salmonella under iron-limiting conditions in that they served as iron siderophores and not as antibiotics.The β-lactam-hypersensitive E. coli X580 and the Salmonella X514 strain exhibited an increased sensitivity to the β-lactam conjugates as compared to the unmodified β-lactams. Pseudomonas, Staphylococcus aureus, and Streptococcus pneumoniae were resistant. FE3+-CATECHOLATE-CEPHALOSPORIN CONJUGATES Catechol-substituted cephalosporins represent the largest group of antibiotics that have been linked to Fe3+siderophores.27–34 Their MIC values are frequently below 1 µg/ml, particularly against gram-negative bacteria, including P. aeruginosa. Their antimicrobial activities may exceed the activity of the unsubstituted cephalosporins more than 100-fold.The highly active cephalosporin derivatives contain a catechol group, as does the E. coli enterobactin Fe3+ siderophore. Removal of one or both vicinal hydroxyl groups, which bind iron, strongly reduces the activities of the catechol cephalosporins.Their high activity is related to their active transport into the periplasm. For example, resistant mutants of five sensitive E. coli strains are 500-fold less sensitive to the cephalosporin derivative E-0702 than the wild-type strains. The resistant strains are mutated in the tonB gene.27 Since TonB is involved in iron transport, it was determined whether the cephalosporin derivatives bind iron. Specific iron chelation was demonstrated spectroscopically.27 Moreover, the activity of the antibiotics depends on the iron supply of the cells and is high under iron-limiting conditions and low under iron-replete conditions. Lack or substitution of the iron-chelating hydroxyl groups reduces the activity of catechol cephalosporins to that of unsubstituted cephalosporins. Other studies have demonstrated the involvement of the Fe3+-catechol receptor proteins Fiu and Cir26 in the transport of the catechol cephalosporins into E. coli. Iron limitation increases the susceptibility of E. coli strains to E-0702 (Fig. 3) 4000-fold over that of a transportnegative tonB mutant and 2000-fold over that of a transportnegative cir fiu double mutant. The minimal inhibitory concentration for these mutants lacking the Fur iron repressor is even more reduced (8000-fold and 4000 fold, respectively). The cephalosporin derivatives bind to penicillin binding protein 3 with an affinity (I50 0.03–0.13 µg/ml) similar to that of the unsubstituted cephalosporins; this clearly demonstrates that the high activity of catechol-substituted cephalosporins is caused by their active transport. Determination of the rate of iron-free E-0702 hydrolysis in
Fig. 3 Examples of cephalosporin catecholates, E-0702,27 catechol-cephalosporins,28 GR69153,30 L-658,310,31 and KP-736,34 which are actively transported into the periplasm of E. Coli by the Cir and Fiu proteins and the Ton energizing system.
the periplasm by the TEM β-lactamase as a measure of the E-0702 entry into the periplasm has clearly revealed a dependence on Fiu and Cir and suggests that iron-free E-0702 is in fact transported.29 Since the cephalosporins only have to enter the periplasm, where their target site is located, active transport across the outer membrane is sufficient to enhance their antibiotic activity greatly. The efficacy of the catecholate cephalosporin L-658, 310 alone and in combination with gentamicin against P. aeruginosa has been determined in mice.32,33 The median effective dose of L-658,310 is 30.4 mg/kg, that of gentamicin 63.3 mg/kg, and that of the combined antibiotics 2 mg/kg. The challenge contained 32 LD50 doses, and the antibiotics were administered subcutaneously 0 and 6 h after infection. 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 363–369
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Braun The number of survivors was measured after 7 days observation. FERRIMYCIN Ferrimycins are among the first sideromycins discovered. The structure of ferrimycin A1 is shown in Figure 4.Although it inhibits incorporation of amino acids into proteins of Staphylococcus aureus SG511, it does not inhibit, but rather enhances polyphenylalanine synthesis in an in vitro S-30 polyU-directed protein synthesis system of S. aureus. The action of ferrimycins is antagonized by ferroxamine B, which competes for ferrimycin uptake.35 SALMYCINS Salmycins have been isolated from Streptomyces violaceus 37290 (DSM 8286) and are active against staphylococci and streptococci (MIC 10 µg/ml).36 Salmycins consist of an Fe3+siderophore of the ferrioxamine group and an antibiotically active aminodisaccharide, which in salmycin B consists of a 2-ketoglucose linked to the 2-position of a 6-methylaminoheptopyranase (Fig. 4).The salmycin structures are similar to those of danomycin and A-22765, whose complete structures have not been determined. It is assumed that the aminodisaccharide is released from the carrier by cleavage of the ester bond. Like danomycin and A-22765, salmycins inhibit protein synthesis, and how this is achieved by a disaccharide is an interesting, open question. RESISTANCE TO IRON-CARRIER ANTIBIOTICS In the laboratory, resistant bacteria emerge on each nutrient agar plate seeded with sensitive bacteria and antibiotics that are carried into the bacteria by active Fe3+-siderophore transport systems – the higher the number of genes involved in a
particular transport system, the higher the frequency of resistance. However, when two transport systems are used by an antibiotic, for example Cir and Fiu for catechol cephalosporins, the frequency of resistant mutants is low. Although the high resistance frequency seems to prevent development of such antibiotics as antibacterial drugs, the in vivo situation might be quite different. In the few unpublished attempts to evaluate the in vivo efficacy of iron-carrier antibiotics known to the author, resistance in mice was not a problem (see also references 32 and 33). In cases where an iron-transport system is important for the proliferation of the pathogenic bacteria, loss of the iron transport system is detrimental. Even when several iron transport systems exist and only one is inactivated by the antibiotic, the inactivated one may be the transport system that is essential for the bacteria to survive and multiply at the site of infection in the human host. Under these circumstances, it does not matter whether the number of bacteria are reduced by the antibiotic or by the loss of the iron supply since under both conditions the immune defense system gains time to cope with the infection. OUTLOOK The emergence of resistant bacteria is an increasing problem; therefore, the possibility of developing antibiotics which are carried into bacterial cells by iron-transport systems should not be ignored. Active transport reduces the minimal inhibitory concentration by more than 100-fold and thus lowers the risk of antibiotic toxicity. Bacteria are also killed faster because the antibiotic attains intracellular inhibitory concentration sooner. The crystal structures of the FhuA transport protein loaded with the antibiotic albomycin or rifamycin CGP 4832 provides the first insights into the high degree of tolerance to distinct structures that are accepted by the transport protein and strongly supports the proposal for the semisynthesis of antibiotics hooked onto Fe3+-siderophore carriers to facilitate their entry into pathogens. Acknowledgements
The author would like to thank Michael Braun for the preparation of Figures 1 and 2 and Karen A. Brune for critically reading the manuscript. The author’s work was supported by the Deutsche Forschungsgemeinschaft (SFB 323, Graduiertenkolleg Mikrobiologie, Br330/14–2) and the Fonds der Chemischen Industrie. Received 26 October 1999; Revised 8 November 1999; Accepted 8 November 1999 Correspondence to: Volkmar Braun, Mikrobiologie/Membranphysiologie, Universität Tübingen,Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Tel: +49 7071 2972096; Fax: +49 7071 29 5843; E-mail [email protected]
Fig. 4 Examples of antibiotic hydroxamates, ferrimycin A (top) and salmycin (bottom), which are structurally related to ferrioxamine B,5,35, in contrast to albomycin (Fig. 1), which belongs to the antibiotic hydroxamates of the ferrichrome type.
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