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.bacillibactin ͉ Bacillus anthracis ͉ petrobactin ͉ siderocalin

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SummaryMany strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5Ј region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.

Bacillus anthracis Sterne produced a catecholate siderophore named anthrachelin that was based on 3,4-dihydroxybenzoic acid (3,4-DHB, or protocatechuic acid), a catechol moiety previously unreported as a siderophore component. During iron restriction, both anthrachelin and free 3,4-DHB were excreted. Growth at 37 degrees C (as compared with 23 degrees C) decreased excretion of anthrachelin but not its precursor 3,4-DHB, suggesting that anthrachelin assembly is temperature regulated. A plasmidless strain also produced anthrachelin in an iron- and temperature-regulated fashion, indicating that anthrachelin genes are chromosomal. In addition to anthrachelin-mediated iron delivery, B. anthracis also used heme, hemoproteins, iron-transferrin, and certain heterologous siderophores (xenosiderophores) produced by other microorganisms as iron sources. Downregulation of anthrachelin production at the temperature of the mammalian host (which triggers toxin production in this pathogen) may focus the B. anthracis iron acquisition systems to exploit the iron sources prevailing in the infected host.