Discovery and Biosynthesis of Anthrochelin, a Growth‐Promoting Metallophore of the Human PathogenLuteibacter anthropi

Abstract

Various human pathogens have emerged from environmental strains by adapting to higher growth temperatures and the ability to produce virulence factors. A remarkable example of a pathoadapted bacterium is found in the genus Luteibacter, which typically comprises harmless soil microbes, yet Luteibacter anthropi was isolated from the blood of a diseased child. Up until now, nothing has been known about the specialized metabolism of this pathogen. By comparative genome analyses we found that L. anthropi has a markedly higher biosynthetic potential than other bacteria of this genus and uniquely bears an NRPS gene locus tentatively coding for the biosynthesis of a metallophore. By metabolic profiling, stable isotope labeling, and NMR investigation of a gallium complex, we identified a new family of salicylate-derived nonribosomal peptides named anthrochelins A-D. Surprisingly, anthrochelins feature a Cterminal homocysteine tag, which might be introduced during peptide termination. Mutational analyses provided insight into the anthrochelin assembly and revealed the unexpected involvement of a cytochrome P450 monooxygenase in oxazole formation. Notably, this heterocycle plays a key role in the binding of metals, especially copper(II). Bioassays showed that anthrochelin significantly promotes the growth of L. anthropi in the presence of low and high copper concentrations, which occur during infections.

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

“…Notably, in vivo formation of the bufferin-Cu 2+ complex most likely occurs while the unfolded bufferin emerges from the Sec machinery in the periplasm and folds around copper, which explains that our in vitro loading attempts were not successful. The defensive role of bufferins is reminiscent of those of non-ribosomal peptides, anthrochelin and yersiniabactin (43, 44), although the putative multiple metal binding sites of large bufferins suggest that some of them might serve as intracellular metal reservoirs. Future studies on this large, diverse family will likely also unveil roles for bufferins in the homeostasis of other transition metals.…”

“…Most prominently, methanobactins scavenge copper from the milieu to feed it to cuproenzymes, and some of them also protect bacteria from toxic metals such as mercury (44). The defensive role of bufferins is reminiscent of those of the non-ribosomal siderophore peptides anthrochelin and yersiniabactin, which can passivate copper by chelation (2, 45). However, the fate of copper ions bound by bufferins remains unknown.…”

“…Purified His-SUMO-BufA1* was incubated with trypsin (1:100 w/w) at 37°C overnight in 50 mM Tris-HCl (pH 7.6), 10 mM TCEP. Purification of the 19-residue central trypsin fragment containing two modified Cys residues was conducted by HPLC (UltiMate TM 3000 System, Thermo Scientific) on a preparative C18 column (Luna C18 (2), 5 µm, 100 Å, 250 x 10 mm, Phenomenex) at ambient temperature. Separation was performed using a linear gradient (6 min at 5% B, 6-14 min from 5-30 % B, 14-18 min to 80% B, and 18-22 min at 80%B) of mobile phases A (0.1% FA) and B (ACN) at 3 mL/min, and monitored at 280 and 305 nm.…”

“…One strategy is the production of specialised metabolites which play key roles in microbial adaptive processes, including defence against competing species, nutrient acquisition and resistance to stresses (1). Bacterial metallophores are prominent examples that can promote bacterial growth under different conditions (2)(3)(4)(5). Several transition metals, including copper, are essential for living organisms (6,7).…”

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“…Anthrochelin A 6 is a growth-promoting metallophore of the human pathogen Luteibacter anthropi . 5 The biosynthetic pathway to anthrochelin A 6 had been studied using genome analyses and stable isotope labelling. Anthrochelin A 6 contains the unusual C-terminal homocysteine but there is no module for the incorporation of this amino acid.…”