Nitro versus Hydroxamate in Siderophores of Pathogenic Bacteria: Effect of Missing Hydroxylamine Protection in Malleobactin Biosynthesis

Abstract

Siderophores play a major role as virulence factors of pathogenic bacteria. Known as the strongest Fe-binding agents, these structurally diverse compounds are used to scavenge scarcely soluble iron from the human or animal hosts. Strikingly, infamous human pathogenic bacteria like Yersinia pestis, the causative agent of devastating black death epidemics, completely lose their virulence in the absence of a siderophore. Thus, knowledge of a pathogen s siderophore structures and of the corresponding biosynthetic machineries is considered a prerequisite for new therapeutic approaches such as targeting pathogenicity factors and Trojan horse strategies using siderophore–drug conjugates. Over the past two decades, much research has been devoted to elucidating the siderophores of Burkholderia mallei and Burkholderia pseudomallei, the causative agents of the infectious diseases glanders and melioidosis. Infections with these b-proteobacteria are often lethal, even with the best treatment available. Both species have thus been categorized as potential biological warfare agents, and indeed B. mallei has been abused in World War I to kill enemy horses and mules. Although it has long been known that B. pseudomallei and B. mallei produce two types of siderophores, pyochelin (1 and 2’’-epi-1, Figure 1) and malleobactin (2), surprisingly, the structure of the latter has remained obscure. It was only a matter of speculation that malleobactin could be related to the ornibactins (3–5, Scheme 1), hydroxamate siderophores produced by the Burkholderia cepacia complex (Bcc). Here we disclose the unusual structure and absolute configuration of malleobactin, the siderophore of the human pathogenic B. mallei family and reveal the biogenetic origin of an unprecedented aliphatic nitro amino acid. To gain first insights into the structural deviations of the encoded siderophores, we compared the gene loci for ornibactin and malleobactin biosynthesis. Both types of gene clusters share genes for a tetramodular nonribosomal peptide synthetase (NRPS) and accessory enzymes. Furthermore, genes for putative amino acid tailoring enzymes (ornithine monooxygenase, aspartic acid b-hydroxylase, N-formyltransferase), siderophore receptors and transporters are present in both types of gene clusters. However, we noted that the gene loci in bacteria belonging to the B. mallei family harbor an additional gene for a hypothetical protein (mbaM), but lack orthologues of orbK and orbL (Figure 1A, yellow open reading frames (orfs)). The latter genes code for acyltransferases, and although their biochemical function has not yet been studied, one may assume that they are required for loading acyl units onto the N-terminal ornithine residue. We concluded that ornibactins and malleobactin differ in their substitution patterns. Figure 1. Comparison of malleobactin and ornibactin biosynthesis gene clusters (mba and orb, respectively) of Burkholderia spp., and analysis of siderophore production of B. thailandensis. A) Malleobactin biosynthesis gene clusters: Bm : B. mallei ; Bp : B. pseudomallei ; Bt : B. thailandensis. Ornibactin biosynthesis gene clusters: Bc : B. cenocepacia ; Ba : B. ambifaria. Deduced functions of mba biosynthesis genes, see Table S2 in the Supporting Information. pvd genes refer to orthologues from the pyoverdin biosynthesis gene cluster in Pseudomonas spp. B) CAS agar plate assay of B. thailandensis wild type (a), DpchE mutant (b, pch=pyochelin biosynthesis gene cluster), DmbaA mutant (c), DpchEDmbaA mutant (d). C) LC–MS profiles monitoring malleobactin (2, m/z 637) and pyochelin (1 and 2’’-epi-1, m/z 325) production of mutant strains (as in (B)).