Rebecca J. Abergel

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2

Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that C-glucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence.

Anthrax pathogen evades the mammalian immune system through stealth siderophore production

Systemic anthrax, caused by inhalation or ingestion of Bacillus anthracis spores, is characterized by rapid microbial growth stages that require iron. Tightly bound and highly regulated in a mammalian host, iron is scarce during an infection. To scavenge iron from its environment, B. anthracis synthesizes by independent pathways two small molecules, the siderophores bacillibactin (BB) and petrobactin (PB). Despite the great efficiency of BB at chelating iron, PB may be the only siderophore necessary to ensure full virulence of the pathogen. In the present work, we show that BB is specifically bound by siderocalin, a recently discovered innate immune protein that is part of an antibacterial iron-depletion defense. In contrast, neither PB nor its ferric complex is bound by siderocalin. Although BB incorporates the common 2,3-dihydroxybenzoyl iron-chelating subunit, PB is novel in that it incorporates the very unusual 3,4-dihydroxybenzoyl chelating subunit. This structural variation results in a large change in the shape of both the iron complex and the free siderophore that precludes siderocalin binding, a stealthy evasion of the immune system. Our results indicate that the blockade of bacterial siderophore-mediated iron acquisition by siderocalin is not restricted to enteric pathogenic organisms and may be a general defense mechanism against several different bacterial species. Significantly, to evade this innate immune response, B. anthracis produces PB, which plays a key role in virulence of the organism. This analysis argues for antianthrax strategies targeting siderophore synthesis and uptake.

The siderocalin/enterobactin interaction: a link between mammalian immunity and bacterial iron transport.

The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [Fe (III)(Ent)] (3-). This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an antibacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidic endosomes and [Fe (III)(Ent)] (3-) is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[Fe (III)(Ent)] (3-) and Scn-Y106F:[Fe (III)(Ent)] (3-) complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe (III)(Ent)] (3-). Fluorescence, UV-vis, and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogues of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.

Petrobactin-mediated iron transport in pathogenic bacteria: coordination chemistry of an unusual 3,4-catecholate/citrate siderophore.

The hexadentate 3,4-catecholate/citrate siderophore petrobactin is produced by the mammalian pathogens Bacillus anthracis and Bacillus cereus apparently to evade the immune protein siderocalin and mediate their iron acquisition during infection; it is essential for the pathogenicity of these organisms. Solution thermodynamic and kinetic measurements show that incorporation of 3,4-catecholate subunits into the siderophore structure does not enhance its affinity for iron as compared to hydroxamate units, but it promotes fast iron removal from human diferric transferrin. The function of the photoreactive ferric citrate moiety remains unexplored in pathogenic Bacilli; however, the soil organism Bacillus subtilis can utilize both ferric petrobactin and its photoproduct as iron sources and therefore carries a specific uptake system for this exogenous siderophore.

Siderophore-mediated iron acquisition systems in Bacillus cereus: identification of receptors for anthrax virulence-associated petrobactin

During growth under iron limitation, Bacillus cereus and Bacillus anthracis, two human pathogens from the Bacillus cereus group of Gram-positive bacteria, secrete two siderophores, bacillibactin (BB) and petrobactin (PB), for iron acquisition via membrane-associated substrate-binding proteins (SBPs) and other ABC transporter components. Since PB is associated with virulence traits in B. anthracis, the PB-mediated iron uptake system presents a potential target for antimicrobial therapies; its characterization in B. cereus is described here. Separate transporters for BB, PB, and several xenosiderophores are suggested by (55)Fe-siderophore uptake studies. The PB precursor, 3,4-dihydroxybenzoic acid (3,4-DHB), and the photoproduct of FePB (FePB(nu)) also mediate iron delivery into iron-deprived cells. Putative SBPs were recombinantly expressed, and their ligand specificity and binding affinity were assessed using fluorescence spectroscopy. The noncovalent complexes of the SBPs with their respective siderophores were characterized using ESI-MS. The differences between solution phase behavior and gas phase measurements are indicative of noncovalent interactions between the siderophores and the binding sites of their respective SBPs. These studies combined with bioinformatics sequence comparison identify SBPs from five putative transporters specific for BB and enterobactin (FeuA), 3,4-DHB and PB (FatB), PB (FpuA), schizokinen (YfiY), and desferrioxamine and ferrichrome (YxeB). The two PB receptors show different substrate ranges: FatB has the highest affinity for ferric 3,4-DHB, iron-free PB, FePB, and FePB(nu), whereas FpuA is specific to only apo- and ferric PB. The biochemical characterization of these SBPs provides the first identification of the transporter candidates that most likely play a role in the B. cereus group pathogenicity.

Biomimetic Actinide Chelators: An Update on the Preclinical Development of the Orally Active Hydroxypyridonate Decorporation Agents 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO)

The threat of a dirty bomb or other major radiological contamination presents a danger of large-scale radiation exposure of the population. Because major components of such contamination are likely to be actinides, actinide decorporation treatments that will reduce radiation exposure must be a priority. Current therapies for the treatment of radionuclide contamination are limited and extensive efforts must be dedicated to the development of therapeutic, orally bioavailable, actinide chelators for emergency medical use. Using a biomimetic approach based on the similar biochemical properties of plutonium(IV) and iron(III), siderophore-inspired multidentate hydroxypyridonate ligands have been designed and are unrivaled in terms of actinide-affinity, selectivity, and efficiency. A perspective on the preclinical development of two hydroxypyridonate actinide decorporation agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), is presented. The chemical syntheses of both candidate compounds have been optimized for scale-up. Baseline preparation and analytical methods suitable for manufacturing large amounts have been established. Both ligands show much higher actinide-removal efficacy than the currently approved agent, diethylenetriaminepentaacetic acid (DTPA), with different selectivity for the tested isotopes of plutonium, americium, uranium and neptunium. No toxicity is observed in cells derived from three different human tissue sources treated in vitro up to ligand concentrations of 1 mM, and both ligands were well tolerated in rats when orally administered daily at high doses (>100 micromol kg d) over 28 d under good laboratory practice guidelines. Both compounds are on an accelerated development pathway towards clinical use.

Microbial Evasion of the Immune System: Structural Modifications of Enterobactin Impair Siderocalin Recognition

The mammalian protein siderocalin binds and inactivates the ferric complex of the bacterial siderophore enterobactin with a Kd value similar to that of the bacterial receptor FepA. However, microorganisms can evade this immune response by structural modifications of the siderophore. The binding of siderophores by siderocalin relies in part on electrostatic interactions and does not depend greatly on what metal is in the complex. It is also sterically limited by the rigid conformation of the protein calyx; methylation of the three catecholate rings of enterobactin hinders siderocalin recognition. The siderocalin binding has been probed for a series of enterobactin analogues in order to investigate in detail the specificity of siderocalin recognition.

The Role of Electrostatics in Siderophore Recognition by the Immunoprotein Siderocalin

Iron is required for virulence of most bacterial pathogens, many of which rely on siderophores, small-molecule chelators, to scavenge iron in mammalian hosts. As an immune response, the human protein Siderocalin binds both apo and ferric siderophores in order to intercept delivery of iron to the bacterium, impeding virulence. The introduction of steric clashes into the siderophore structure is an important mechanism of evading sequestration. However, in the absence of steric incompatibilities, electrostatic interactions determine siderophore strength of binding by Siderocalin. By using a series of isosteric enterobactin analogues, the contribution of electrostatic interactions, including both charge-charge and cation-pi, to the recognition of 2,3-catecholate siderophores has been deconvoluted. The analogues used in the study incorporate a systematic combination of 2,3-catecholamide (CAM) and N-hydroxypyridinonate (1,2-HOPO) binding units on a tris(2-aminoethyl)amine (tren) backbone, [tren(CAM)(m)(1,2-HOPO)(n), where m = 0, 1, 2, or 3 and n = 3 - m]. The shape complementarity of the synthetic analogue series was determined through small-molecule crystallography, and the binding interactions were investigated through a fluorescence-based binding assay. These results were modeled and correlated through ab initio calculations of the electrostatic properties of the binding units. Although all the analogues are accommodated in the binding pocket of Siderocalin, the ferric complexes incorporating decreasing numbers of CAM units are bound with decreasing affinities (K(d) = >600, 43, 0.8, and 0.3 nM for m = 0-3). These results elucidate the role of electrostatics in the mechanism of siderophore recognition by Siderocalin.

Temporal production of the two Bacillus anthracis siderophores, petrobactin and bacillibactin

Bacillus anthracis secretes two siderophores, petrobactin (PB) and bacillibactin (BB). These siderophores were temporally produced during germination and outgrowth of spores (the usual infectious form of B. anthracis) in low-iron medium. The siderophore PB was made first while BB secretion began several hours later. Spore outgrowth early in an infection may require PB, whereas delayed BB production suggests a role for BB in the later stages of the infection. Incubation of cultures (inoculated as vegetative cells) at 37°C, as compared to 2°C, increased PB production and decreased secretion of BB, suggesting that the production of PB and BB responded to the host temperature signal. The dual siderophores of B. anthracis may fulfill independent roles in the life cycle of B. anthracis.

Using the Antenna Effect as a Spectroscopic Tool; Photophysics and Solution Thermodynamics of the Model Luminescent Hydroxypyridonate Complex [EuIII(3,4,3-LI(1,2-HOPO))]-

Although widely used in bioassays, the spectrofluorimetric method described here uses the antenna effect as a tool to probe the thermodynamic parameters of ligands that sensitize lanthanide luminescence. The Eu(3+) coordination chemistry, solution thermodynamic stability, and photophysical properties of the spermine-based hydroxypyridonate octadentate chelator 3,4,3-LI(1,2-HOPO) are reported. The complex [Eu(III)(3,4,3-LI(1,2-HOPO))](-) luminesces with a long lifetime (805 mus) and a quantum yield of 7.0% in aqueous solution, at pH 7.4. These remarkable optical properties were exploited to determine the high (and proton-independent) stability of the complex (log beta(110) = 20.2(2)) and to define the influence of the ligand scaffold on the stability and photophysical properties.