SIDEROPHORES

INTRODUCTION

• Iron is essntial micronutrient.

• Exist in two oxidation states, so works as iron transporter.

• Enzymes like aconitase, peroxidase, catalase and nitrogenase complex etc contain iron as co factor.

• May have structural role in microbial cells. Magnetite (Fe3O4) have been found in some bacteria.

• Bacterioferritin has been found in Azotobacter vinelandii and E. coli.

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REQUIREMENT OF IRON • Iron is not relatively abundant in nature because of low

solubility at physiological pH.

• In aqueous medium at neutral pH iron exist as insoluble polymer [Fe(OH)3].

• In animal tissues , iron is complexed to various proteins i.e. transferrin, haemoglobin, lectoferrin and ferritin.

• A low mol. wt. iron chelating agent is produced by microorganisms and excreted in to immediate environments. These agents are termed “SIDEROPHORES”

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THE ROLE OF SIDEROPHORES

• The function of siderophore is to solubilize iron as may be available in external environment and transport it in to the cell.

• Have high affinity for ferric iron.

• Production may reduce to 0.1% in presence of iron.

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• Blue green algal blooms may be due to the production of siderophores which bind to all available iron, thus suppressing the growth of other algae.

• Also been found in cheese making bacteria.

• Fungus Pilobolus utilizes ferrichrome and related siderophores produced by other moulds such as Ustilago.

• Arthrobacter terregens responds to siderophores of hydroxamate type only.

• M. paratuberculosis and certain strains of M. avium shows specific growth requirements for Mycobactin, produced by other mycobacterial sp.

• Rhodotorulic acid produced by yeast is utilized by Arthrobacter flavescens and B. megaterium.

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• Salmonella typhimurium – enterochelin and E.coli – aerobactin confers to increase in virulence.

• Specificity of siderophore-iron complex prevent its utilization by other microbes.

• Some siderophores can additionaly have role in conidial germination e.g. Aspergillus nidulans, Neurospora crassa and Penicillium chrysogenum.

• Desferri siderophores

• Desferrioxamine B methanesulfonate by Streptomyces sp. is administered in treatment of iron overload disorders.

• In treatment of infections of L. monocytogenes.

• Enterochelin can chelate metals other than iron.

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UPTAKE AND RELEASE OF IRON FROM SIDEROPHORE COMPLEX • E. coli – enterochelin

• Hexadentate complex with ferric iron. Iron is transported in cell via specific membrane receptor proteins.

• Mechanism of iron release within cell is still unknown. • Hydrolytic destruction of enterochelin

• Reduction of Fe3+ by NAD(P)H- linked reductase.

E. coli can also utilize ferrichrome (fungal), here iron-ferrichrome complex also binds to specific membrane receptors. (not carried to cytoplasm)

In addition, E. coli is also capable of utilizing rhodotorulic acid and citrate in iron uptake.

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• Iron is released when complex is still in c.m.

• Desferri ferrichrome thus created is acetylated and excreted from cell, it cant participate in further iron uptake until deacetylation.

• In U. sphaerogena don’t seem to involve acetylation or other modification of the ligand.

• E. coli also produce “aerobactin”, originally isolated from K. pneumoniae. It is encoded on plasmid and is transmissible between various enterobacteria. H

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• Mycobacteria produces mycobactin within the cell walls. It may be up to 10% dry wt.

• It also produces extracellular siderophore exochelin.

• Fast growing sp. produces water soluble exochelin transport iron by active process independently of mycobactins.

• Slow growing (M.Tb) produce chloroform soluble exochelins which operate by facilitated diffusion mechanism.

• Mechanism of transport by these is unknown.

• It appears that when large amount of iron is made available, firstly small amount is acquired for the immediate growth requirements of the cell and secondly larger amounts which are stored in mycobactin.

• Mycobacteria also utilize citrate for iron transport which appears to be facilitated diffusion mechanism.

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Iron acquisition in Gram Negative Bacteria

• Transport of bound chelates across the outer membrane depends upon TonB–ExbB–ExbD, a cytoplasmic membrane-localized complex.

• TonB-dependent receptors achieve maximal transport efficiency at very low concentrations of ligand.

• Uptake of small (< 1000 Da) microbially produced ferric iron chelators called siderophores and

• Utilization of host iron-binding proteins.

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• The active transport of iron against its concentration gradient requires both the proton motive force (pmf) and ATP hydrolysis.

• Outer Memb -------- Peripl Space -------- Cytoplasmic Memb.

PMF & energy transduction complex including the CM proteins TonB, ExbB and ExbD

ATP-dependent mechanism, conserved among the periplasmic permease subfamily of CM-localized traffic ATPases.

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Ligand–receptor interaction

• Recognition of ferric–enterobactin by its receptor FepA in the OM of Escherichia coli is largely dependent upon charge interactions. (catecholate siderophores – hydrophobic interactions)

• Basic residues in the ligand-binding region of E. coli FepA sequence is conserved among the enterobactin receptors of several Gram-negative bacteria. (contact the negatively charged ferric–enterobactin complex directly)

• Binding of ligand to the ferrichrome–iron receptor [FhuA (receptor for ferrichrome) and FepA of E. coli] results in structural alterations in receptor. All these occurs independently of TonB. (earliest event)

• Receptors may also contain overlapping ligand binding sites.

• The ferrichrome–iron complex is uncharged at neutral pH.

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Fig: - TonB-dependent translocation of ferric iron chelates across the outer membrane (OM).

1. Interaction

2. TonB-independent conf changes, indicate ligand occupancy

3. Physical Contact

4. TonB transduces pmf-derived energy, reducing affinity of receptor for ligand

5. TonB dissociates & recycled

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OM receptors as signal transducers:

• Binding of S-F complex to receptors is reported to be inducing genes responsible for siderophore uptake.

• E.g. ferrichrome binding induces conformational changes in an N-terminal region of FhuA that is exposed to the periplasm.

• Occupancy of the siderophore receptor (free or ligand bound) is then sensed by proteins such as TonB that extend into the periplasm. H

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Fig: - Transport and regulation of siderophores

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• TonB–ExbB–ExbD complex had shown conserved mechanism to transduce energy to high-affinity transporters within the OM (functional link between pmf and active transport)

• TonB box: found on iron chelates (also on vit B12)– mediates TonB and TonB dependent receptors interactions (energy coupling).

• Ligand-induced conformational changes are transmitted from the external surface of the cell through FhuA to the periplasm.

• TonB interacts preferentially with those receptors to which ligand is bound. (receptors can compete even for that e.g. FhuA and BtuB)

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Fig: - TonB-dependent uptake of vitamin B12

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TonB: -

• Localized within the CM by uncleaved signal anchor (N- terminus embeded within CM)

• Proline rich region extends from CM to make contact with OM and high affinity receptor (by β-α-β motif near the C-terminus).

• Cycle between different states (require PMF).

• to recycle TonB back to an energized state within the CM, requires ExbB and ExbD.

• Proposed to detach from the CM transiently in order to provide pmf-derived energy (may circulate more readily through the periplasm but cant leave one without binding other).

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Siderophore receptors: -

• Siderophore receptors adopt a beta-barrel conformation (rich in antiparallel β-sheets ).

• Contain amphipathic residue stretches to span the OM. Some are also organized in to extra membranous loops (H-phobic).

• Some are responsible for ligand binding at the cell surface while others transmit ligand binding dependent signals to the periplasm.

• Contain surface loops that shields underlying channel. FepA and FhuA and perhaps all TonB-dependent receptors act as gated porins.

• Opening and closing of these channels is mediated by the conformational changes of receptors on ligand binding.

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Fig: - The structure of the (prototype) TonB-dependent transporter (TBDT) FhuA.

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Fig: - The FecAapo, FecACit and FecAFeCit crystal structures.

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structures

• Two main structural groups

• Hydroxamates- produced by fungi and bacteria

• Phenolates- found in bacteria

• May contain novel aminoacids.

• Structures varies sp to sp

• Ferric siderophore complexes are often highly colored compounds while the desferri forms are usually colourless in solution.

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HYDROXAMATES

• Contain 3 secondary hydroxamate groups, C(=O)N-(OH)R

• Each siderophore forms a hexadentate octahedral complex with Fe3+.

• Show strong absorption between 425 and 500 nm when bound to iron.

• TYPES: -

• Ferrichrome – cyclic hexapeptides containing tripeptide of glycine, alanin and serine & tripeptide of N-acyl N-hydroxyornithine.

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a. Ferrichrome from U.sp.

b. Ferrioxamine B from St.p.

c. Fusarinine C from Fu.r.

d. Mycobactin from M.s.

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• First siderophore (ferrichrome) was isolated from U. sphaerogena (smut fungus). It can also bind with Al, Ga, Cr, Vn, Cu but not as stable as with Fe.

• Fusarium roseum produce malonichrome (a ferrichrome) where acetyl groups of ferrichrome are replaced by malonyl groups.

• Streptomyces produces albomycin (a ferrichrome) and has antibiotic potential.

• Fusarinines (by Fusarium and Penicillium spp) contain hydroxamate group joined by ester groups rather than peptide bonds.

• A extracellular cyclic triester Fusarinine C is produced by A. nidulans and Penicillium chrysogenum.

• Mycobactins contains 2 hydroxamate groups, the third pair is provided by an oxygen atom on the aromatic residue and nitrogen on oxazoline ring.

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e. Aerobactin from K.p.

f. Enterochelin from E.c.

g. Albomycin δ2 from S.g.

h. Rhodotorulic acid from R.p.

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• Citrate hydroxamates like aerobactin from K.p., Arthrobactin from Arthrobacter sp. & Schizokinen from B.m. also contain two hydroxamate groups. The third chelating residue being provided by citrate moiety.

• Rhodotorulic acid, produced by various yeasts (Rhodotorula pilimanae) is dipeptide.

• Derivatives includes Dimerumic acid produced by Fusarium dimerum & coprogen produced by Neurospora and Penicillium sp.

• Ferribactin is produced by Pseudomonas fluorescens.

• In addition it also produces pyoverdine with iron binding fluorescent component.

• Pseudobactin is linear hexapeptide attached to quinoline derivatives (Pseudomonas B10).

• Gonobactin and nocobactin are produced by Neisseria gonorrhoeae & N. maningitidis.

• Nocobactin has priority for mycobactin like siderophore from Nocardia spp.

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i. Ferrimycin from St. spp

j. Siderochelin A fron Nocardia sp.

k. Sepedonin from Sepedonium chrysogenum

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PHENOLATES/CATACHOLATES

• Its Prototype is Enterochelin (by E.c., S.t. & K.p.) and is the cyclic triester of 2, 3-dihydroxybenzoylserine.

• Each catecholate grp provides two O-atoms for chelation with iron forming hexadentate octahedral complex.

• Dihydroxybenzoate and 2,3-dihydroxybenzoylserine are also found in above organisms.

• Ferric enterochelin- soluble in ethyl acetate, acetone, dioxane, dimethylsulfoxide and methanol but very sparingly in water.

• Gallium and scandium complexes of enterochelin have antibiotic activity.

• Threonine conjugate analogues to enterochelin are produced by E.c. and K.p. while glycine conjugate is produced by B.s.

• Arobactin (by A.t.) and parabactin (by P.d.) are linear catacholate siderophores.

• Pseudomonas syringae phaseolicola produces mixed catecholate-hydroxamate siderophore.

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Siderophores with antibiotic activity

• Sideromycins- streptomyces.

• e.g. Albomycin and ferrimycin

• Both contain extra chemical group attached to the basic siderophore structure to gain access to the cell where they exert antibiotic action by inhibiting protein synthesis.

• IRON UPTAKE INTERFERENCE- • Desferritriacetylfusarinine C by A. deflectus, inhibits the

growth of bacteria only.

• Pseudomonas alcaligens produce cyclic hydroxamate inhibiting Gram +ve and –ve bacteria as well as Trichomonas vaginalis.

• ANALOGUES-

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USES OF SIDEROPHORES IRON IN BODY

• 3-4 gm iron in human body.

• Ferritin in liver, spleen & other organs- non toxic storage up to 4500 Fe atoms per molecule.

• Transferrin with 2 iron binding sites (30% saturated)– transport of iron in blood around body. Accept iron from intestinal mucosa and in liver.

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• A siderophore from Streptomyces pilosus, desferrioxamine B, is marketed as the mesylate salt under the trade name Desferal.

• Desferal is widely used by physicians.

• Desferrioxamine B is administered parenterally, filtered through kidney/ taken up by liver.

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• HYPERCHROMATOSIS: - progressive increase in body iron stores cousing iron deposits in the cells of organs such as liver, heart or pancreas.

• CHELATION THERAPY: - Treating iron overload - administering chelator forming complex with iron and excreted in urine or feces.

• OTHER CLINICAL APPLICATIONS: -

• Deferrization of patients suffering from transfusion-induced siderosis.

• For removal of excess iron resulting from the supportive therapy for thalassemia.

• AGRICULTURAL INTERESTS: -

• Pseudobactin or pyoverdine type siderophores - improved plant growth - direct effect on the plant or through control of noxious organisms in the soil.

• Rhizobial Nitrogenase - iron-intensive enzyme complex - may require an intact siderophore system for expression of this exclusively prokaryotic catalyst

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