Anthrax pathogen evades the mammalian immune system through stealth siderophore production

Abstract

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.

Fig. 1.

Molecular structures of B. anthracis siderophores (a and b) and the corresponding siderophores from Gram-negative enteric bacteria (c and d). (a) BB. (b) PB. (c) Ent.; (d) Aerobactin. The iron-coordinating oxygen atoms (when deprotonated) are indicated in red. All completely sequester the Fe^III by six-coordinate binding.

Fig. 2.

Crystal structure of the adduct formed by [Fe^III(Ent)]³⁻ specifically bound to siderocalin. The specific binding of ferric Ent to siderocalin is caused by hybrid electrostatic/cation-π interactions between [Fe^III(Ent)]³⁻ and the side chains of residues Arg-81, Lys-125, and Lys-134 colored by atom type.

Fig. 3.

Fluorescence quenching titrations measuring the affinity of siderocalin for BB, PB, and their ferric complexes. Symbols give the fluorescence measurements at 340 nm (pH 7.4) upon addition of ligand to a 100-nM solution of siderocalin; lines give the calculated fits.

Fig. 4.

Comparison of the tris-bidentate complexes [Fe^III(2,3-DHBA)₃]³⁻ and [Fe^III(3,4-DHBA)₃]³⁻ for siderocalin binding. (a) A view of the crystal structure of the tris-bidentate ferric complex of 2,3-DHBA, [Fe^III(2,3-DHBA)₃]³⁻ bound to siderocalin. Steric conflicts between siderocalin and the carboxyl groups of a 3,4-DHBA complex (yellow) are generated in simulated binding. (b) A cutaway showing the penetration of the protein surface by [Fe^III(3,4-DHBA)₃]³⁻ in two of the pockets during simulated binding. (c) Molecular structure of a 2,3-dihydroxybenzoyl subunit bound to iron. The dashed arc represents the wall of the protein calyx. Cocrystallization of siderocalin with [Fe^III(2,3-DHBA)₃]³⁻ gives red crystals, indicative of binding. (d) Molecular structure of a 3,4-dihydroxybenzoyl subunit bound to iron. The dashed arc represents the wall of the protein calyx. Cocrystallization of siderocalin with [Fe^III(3,4-DHBA)₃]³⁻ gives colorless crystals, suggestive of nonbinding.

Fig. 5.

Synthetic analogues of BB and Ent. (a) TRENglyCAM. (b) SERglyCAM. (c) _D-Ent. The iron-coordinating oxygen atoms (when deprotonated) are in red.