Download Active transport of antibiotics across the outer membrane of gram

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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]
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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.
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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.
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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.
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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
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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
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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
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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
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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.
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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-
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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.
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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
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