Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration.

The causes of nigrostriatal cell death in idiopathic Parkinson's disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C10H11NO2S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3-4 µM. It was less toxic for other neurons (10-20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the "basal slowing response" assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease.

QSDB-a graphical Quorum Sensing Database.

The human microbiome is largely shaped by the chemical interactions of its microbial members, which includes cross-talk via shared signals or quenching of the signalling of other species. Quorum sensing is a process that allows microbes to coordinate their behaviour in dependence of their population density and to adjust gene expression accordingly. We present the Quorum Sensing Database (QSDB), a comprehensive database of all published sensing and quenching relations between organisms and signalling molecules of the human microbiome, as well as an interactive web interface that allows browsing the database, provides graphical depictions of sensing mechanisms as Systems Biology Graphical Notation diagrams and links to other databases. Database URL: QSDB (Quorum Sensing DataBase) is freely available via an interactive web interface and as a downloadable csv file at http://qsdb.org.

A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity.

Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood-feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito-microbiome studies and highlights an understudied source of bioactive small molecules.

Engineering siderophores.

Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate siderophores by various biochemical approaches based on the example of Shewanella algae. The marine gamma-proteobacterium S. algae produces three different cyclic hydroxamate siderophores as metabolites via a single biosynthetic gene cluster and one of them is an important key player in interspecies competition blocking swarming of Vibrio alginolyticus. AvbD is the key metabolic enzyme assembling the precursors into three different core structures and hence an interesting target for metabolic and biochemical engineering. Synthetic natural and unnatural precursors can be converted in vitro with purified AvbD to generate siderophores with various ring sizes ranging from analytical to milligram scale. These engineered siderophores can be applied, for example, as swarming inhibitors against V. alginolyticus. Here, we describe the synthesis of the natural and unnatural siderophore precursors HS[X]A and provide our detailed protocols for protein expression of AvbD, conversion of HS[X]A with the enzyme to produce ring-size engineered siderophores and secondly for a biosynthetic feeding strategy that allows to extract engineered siderophores in the milligram scale.

Inhibitors of Bacterial Swarming Behavior.

Bacteria can migrate in groups of flagella-driven cells over semisolid surfaces. This coordinated form of motility is called swarming behavior. Swarming is associated with enhanced virulence and antibiotic resistance of various human pathogens and may be considered as favorable adaptation to the diverse challenges that microbes face in rapidly changing environments. Consequently, the differentiation of motile swarmer cells is tightly regulated and involves multi-layered signaling networks. Controlling swarming behavior is of major interest for the development of novel anti-infective strategies. In addition, compounds that block swarming represent important tools for more detailed insights into the molecular mechanisms of the coordination of bacterial population behavior. Over the past decades, there has been major progress in the discovery of small-molecule modulators and mechanisms that allow selective inhibition of swarming behavior. Herein, an overview of the achievements in the field and future directions and challenges will be presented.

Dissecting the Mechanism of Oligomerization and Macrocyclization Reactions of NRPS-Independent Siderophore Synthetases.

Macrocyclic and linear hydroxamate siderophores produced by NRPS-independent siderophore (NIS; NRPS=nonribosomal peptide synthetase) synthetases are important in the bacterial competition for iron, as virulence factors, and as drugs for medical use in humans. Despite their importance, the mechanistic details of NIS synthetases have so far remained obscure. Using synthetic substrate analogues as tools allowed for an interrogation of the mechanism of the two closely related NIS synthetases AvbD and DesD. While AvbD produces macrocyclic homo- and heterodimers as native products, DesD is responsible for the synthesis of trimeric desferrioxamines. These enzymes comprise two adjacent binding sites with different substrate selectivities, which direct oligomerization and macrocyclization steps. Exploiting this difference, synthetic substrates were used to invert the native affinities for the sites resulting in switching from trimerization to dimerization reactions for DesD. Based on this work, a comprehensive model explaining the mechanistic details of the reactions and the differences between trimerizing and dimerizing enzymes was developed. Finally, a DesD mutant demonstrated the tuneability of the enzyme's substrate selectivity by only minor changes in the protein sequence. This finding confirms the affinity-directed mechanism responsible for the iterativity of oligomerization and macrocyclization steps.

One Enzyme To Build Them All: Ring-Size Engineered Siderophores Inhibit the Swarming Motility of Vibrio.

Bacteria compete for ferric iron by producing siderophores, and some microbes engage in piracy by scavenging siderophores of their competitors. The macrocyclic hydroxamate siderophore avaroferrin of Shewanella algae inhibits swarming of Vibrio alginolyticus by evading this piracy. Avaroferrin, as well as related putrebactin and bisucaberin, are produced by the IucC-like synthetases AvbD, PubC, and BibCC. Here, we have established that they are capable of synthesizing not only their native product but also other siderophores. Exploiting this relaxed substrate specificity by synthetic precursors generated 15 different ring-size engineered macrocycles ranging from 18- to 28-membered rings, indicating unprecedented biosynthetic flexibility of the enzymes. Two of the novel siderophores could be obtained in larger quantities by precursor-directed biosynthesis in S. algae. Both inhibited swarming motility of Vibrio and, similar to avaroferrin, the most active one exhibited a heterodimeric architecture. Our results demonstrate the impact of minor structural changes on biological activity, which may trigger the evolution of siderophore diversity.

From pirates and killers: does metabolite diversity drive bacterial competition?

Bacteria engage in numerous collaborative and competitive interactions, which are often mediated by small molecule metabolites. Bacterial competition involves for example the production of compounds that effectively kill or inhibit growth of their neighbours but also the secretion of siderophores that allow securing the essential and fiercely embattled resource of ferric iron. Yet, the enormous diversity of metabolites produced has remained puzzling in many cases. We here present examples of both types of competition from our recent work. These include the human pathogen Pseudomonas aeruginosa producing HQNO derived 4-quinolone N-oxides varying in chain length and saturation as antibiotics against Staphylococcus aureus and two marine bacteria, Shewanella algae and Vibrio alginolyticus competing for iron acquisition via homodimeric and heterodimeric cyclic hydroxamate siderophores. In each case, bacteria not only produce one but a whole set of closely related metabolites encoded by a single biosynthetic gene cluster. Our recent work has demonstrated that individual metabolites can have significantly different biological activities and we speculate on the reasons for maintaining this metabolite diversity from the perspective of interspecies competition.

One Enzyme, Three Metabolites: Shewanella algae Controls Siderophore Production via the Cellular Substrate Pool.

Shewanella algae B516 produces avaroferrin, an asymmetric hydroxamate siderophore, which has been shown to inhibit swarming motility of Vibrio alginolyticus. We aimed to elucidate the biosynthesis of this siderophore and to investigate how S. algae coordinates the production of avaroferrin and its two symmetric counterparts. We reconstituted the reaction in vitro with the main enzyme AvbD and the putative biosynthetic precursors, and demonstrate that multispecificity of this enzyme results in the production of all three cyclic hydroxamate siderophores that were previously isolated as natural products from S. algae. Surprisingly, purified AvbD exhibited a clear preference for the larger cadaverine-derived substrate. In live cells, however, siderophore ratios are maximized toward avaroferrin production, and we demonstrate that these siderophore ratios are the result of a regulation on substrate pool level, which may allow rapid evolutionary adaptation to environmental changes. Our results thereby give insights into a unique evolutionary strategy toward metabolite diversity.