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1 Active transport of antibiotics across the outer membrane of gram-negative bacteria and its implications in the development of new antibiotics. 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] PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 2 Abstract The outer membrane of gram-negative bacteria forms a permeability barrier that usually reduces the access of antibiotics to intracellular targets and renders gram-negative bacteria less susceptible to antibiotics than gram-positive bacteria, which lack an outer membrane. However, gram-negative bacteria become highly susceptible to antibiotics if the antibiotics are actively transported across the outer membrane. Some antibiotics can 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 of 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 than 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 of 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 bound by a linker to a transported carrier. A number of Fe3+–siderophore carriers of the hydroxamate and catecholate type linked to antibiotics have been isolated from microbes and synthesized; their superior efficacy has been demonstrated in vitro and in mice. It is proposed that natural sideromycins and synthetic sideromycins should be developped to obtain new antibiotics in light of the increasing problem of resistant pathogens. PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 3 Membranes prevent access to the target sites of antibiotics Slide 1: Antibiotics have to reach their target sites which are located in the periplasm, the cytoplasmic membrane, or the cytoplasm. Most of the antibiotics reach their target by diffusion which is hampered by the membranes that surround bacteria depending on the physical properties of the antibiotics, such as size, hydrophilicity / hydrophobicity. I will concentrate today on the outer membrane of gram-negative bacteria which is the reason why certain antibiotics which are active against gram-positive bacteria, which lack an outer membrane, are not active against gram-negarive bacteria. The obstacle of the outer membrane, which is very pronounced for the curing of bacterial infections with antibiotics, can be turned into an advantage if active transport systems present in the outer membane are used to get antibiotics into cells. Substrate translocation across the outer membrane Slide 2: Gram-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. The proteins are grouped into three functional classes with respect to the uptake of substrates. Slide 3: 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. The second class of proteins also forms pores, which are similar to those of the porins, 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. 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. PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 4 The third class of outer membrane proteins is involved in the uptake of ferric siderophores and of vitamin B12. The molecular mass of the 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. 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. Slide 4: Whenever ferric siderophores meet a cell, the siderophores bind to transport proteins; this binding greatly increases the efficiency of iron uptake. E. coli K-12 expresses 7 such iron siderophore transport systems. The siderophores can be the products of bacteria or fungi. The many transport systems for just a single metal reflects the variety of siderophores in the surrounding of the bacteria but also the absolute need to be able to take up iron which is an essential element in the active center of most of the redox enzymes. 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 of the cytoplasmic membrane. Energy is provided by ATP, and the transporters contain ATP-binding sites and are therefore collectively named ABC (ATP-binding cassette) transporters. Gram-positive bacteria also contain binding proteins that are anchored to the outer surface of the cytoplasmic membrane, recognize ferric siderophores, and transmits them to ABC transporters in the cytoplasmic membrane. In the following I will discuss the Fhu transport system in some detail because it is the best understood active outer membrane transport system and forms the paradigm for the understanding of the other transport systems. PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 5 The crystal structure of FhuA provides the basis for the understanding of FhuA as a transporter Slide 5: FhuA serves as transporter for ferrichrome, a Fe3+–hydroxamate synthesized by the fungus Ustilago sphaerogena, which is released into the growth medium and can be used by E. coli and many other gram-negative and gram-positive bacteria as an iron source. FhuA also transports the antibiotic albomycin, a structural analogue of ferrichrome and the antibiotic rifamycin CGP 4832, 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). Active transport consumes energy. However, there is no energy source in the outer membrane. Rather energy is provided by the proton motive force of the cytoplasmic membrane. The question is how energy is transferred from the cytoplasmic membrane into the outer membrane. This intermembrane energy transfer is achieved by the Ton complex composed of the proteins TonB, ExbB, and ExbD. The TonB protein directly contacts FhuA and there is indirect evidence that energization of TonB changes the conformation of TonB and in this form interacts with FhuA such that FhuA is converted into an active transporter. As we will see, the confomational change in FhuA presumably releases ferrichrome from the binding site at FhuA and opens a channel in FhuA through which ferrichrome is translocated into the periplasm. Slide 6: In 1998 the crystal structures of FhuA and of FhuA loaded with ferrichrome were published at a resolution of 2.7 Å by two groups. FhuA consists of 22 antiparallel βstrands that form a β-barrel, 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. Slide 7: Binding of ferrichrome causes movement of the cork by 1.7 Å towards ferrichrome and a large structural transition in the periplasmically exposed portion, where a PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 6 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 through the energy-transferring device of the Ton system. FhuA is 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 resistance 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, as ß-lactam antibiotics do inhibit there murein biosynthesis, or a steep concentration gradient is formed into the cytoplasm, thereby enhancing the diffusion rate, or the antibiotic may even be actively transported across the cytoplasmic membrane. FhuA–albomycin: the first crystal structure of an antibiotic protein transporter Albomycin is a broad-spectrum antibiotic with an excellent inhibitory activity toward gram-positive and gram-negative bacteria. The minimal inhibitory concentration for E. coli K12 is 100-fold lower than that of ampicillin. Albomycin belongs to the class of sideromycins which contain Fe3+. The albomycin-producing strain Streptomyces specWS116 synthesizes three derivatives that differ in the pyrimidine side chains. Slide 8: 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. The moiety of albomycin that is analogous to ferrichrome serves as the carrier of the antibiotically active thioribosyl pyrimidine group.Albomycin, like PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 7 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. 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. 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 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. The thioribosyl-pyrimidine group makes it 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 into 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. In addition, the extra binding sites on FhuA could result in a stronger binding of FhuA and a lower transport rate of albomycin. Slide 9: The crystal structure reveals that the Fe3+–hydroxamate portion of albomycin occupies the same site on FhuA and is bound by the same amino acid chains as ferrichrome. Slide 10: 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 structure of the FhuA albomycin co-crystal has also revealed the hitherto unknown conformation of albomycin and the conformation in the transport-competent form. Slide 11: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 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 8 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 linked 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. 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. 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 the proteins required for active transport of ferrichrome across the cytoplasmic membrane display unaltered CGP 4832 sensitivity. 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. Slide 12: 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. Therefore, it was particularly attractive to determine the crystal structure of FhuA loaded with CGP 4832. Slide 13: Analysis of the X-ray diffraction data revealed that CGP 4832 largely occupies the site in FhuA that is also used by ferrichrome. 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 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 9 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. Slide 14: The X-ray structure of CGP 4832 bound to FhuA also revaled the conformation of CGP 4832 and showed that it is very different to ferrichrome and albomycin. It is pure chance that the side chains in which CGP 4832 differs from rifamycin are oriented such that they fit into the binding site of FhuA. The cavity of FhuA at the cell surface through which the substrates diffuse to the binding site is large enough to accomodate different and rather large structures. In contrast to CGP 4832 which is actively transported only to the periplasm, albomycin is actively transported also into the cytoplasm. Apparently, the transport proteins that catalyze transport across the cytoplasmic membrane tolerate, like FhuA, rather large additions to ferrichrome. This has actually been demonstrated for the FhuD protein that was crystallized with bound gallichrome, a structural analog of ferrichrome in which iron is replaced by gallium. Slide 15: The crystal structure of the periplasmic FhuD protein reveals a high tolerance to substitutions at ferrichrome since gallichrome is not deeply bound in a pocket but exposed at the surface of the molecule. This is in contrast to the crystal structures of transferrin and lactoferrin which contain the iron bound in a deep pocket that is closed when iron is bound and open when it is relased. Opening and closing of the two FhuD domains is prevented by the rigid helix that fixes the two domains in a single position. Slide 16: Surface exposure has the consequence that the side chain of albomycin is not seen in the co-crystal structure of FhuD since the side chain is flexible. For the design of synthetic antibiotics the data on FhuA and FhuD are very important since they demonstrate the high tolerance of the transport proteins for additions to the true substrates. Antibiotically active portions can be added without impairment of active transport. PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 10 Other sideromycins of the hydroxamate type Slide 17: There are two other sideromycins of which the structures are known. These are ferrimycin A and salmycin. Salmycin inhibits protein biosynthesis through the disaccharide, th emode of action is not known. Both antibotics are probably taken up by active transport by the ferrioxamine B transport system because ferrioxamine B antagonizes their action which presumably occurs by competitive inhibition of transport and not at the target sites. I could extend this review to synthetic Fe3+–catecholate–cephalosporin conjugates which are actively taken up into the periplasm, the location of their activity, by outer membrane transport proteins of the FhuA type which transport linear iron catecholate complexes. 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 is contained in the E. coli enterobactin Fe3+ siderophore. Removal of one or both of the vicinal hydroxyl groups, which bind iron, strongly reduces the activities of the catecholate 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. Since TonB is involved in iron transport, it was determined whether the cephalosporin derivatives bind iron. Specific iron chelation was demonstrated spectroscopically. 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 catecholate cephalosporins to that of the 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) 4 000-fold over that of a transport-negative tonB mutant and 2 000-fold over that of a transport-negative cir fiu double mutant. The minimal inhibitory concentration for these mutants lacking the Fur iron repressor is even more reduced (8 000-fold and 4 000 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 the catechol- PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 11 substituted cephalosporins is caused by their active transport. Determination of the rate of iron-free E-0702 hydrolysis in the periplasm by the TEM β-lactamase as a measure of the E0702 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 gentamycin against P. aeruginosa has been determined in mice.32,33 The medium effective dose of L-658,310 is 30.4 mg/kg, that of gentamycin 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. The number of survivors was measured after 7 days observation. 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 the cephalosporin catecholates, 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 . In cases where an irontransport 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. PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com 12 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 more than 100-fold and thus lowers the risk of antibiotic toxicity. The bacteria are also killed faster because the intracellular inhibitory concentration of the antibiotic is attained 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. More information and material: http://www.um.es/molecula/catedraBFM PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com