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.

Biosynthetic flexibility of Pseudomonas aeruginosa leads to hydroxylated 2-alkylquinolones with proinflammatory host response.

The human pathogen Pseudomonas aeruginosa produces various 4(1H)-quinolones with diverse functions. Among these, 2-nonyl-4(1H)-quinolone (NQ) and its N-oxide (NQNO) belong to the main metabolites. Their biosynthesis involves substrates from the fatty acid metabolism and we hypothesized that oxidized fatty acids could be responsible for a so far undetected class of metabolites. We developed a divergent synthesis strategy for 2'-hydroxy (2'-OH) and 2'-oxo- substituted quinolones and N-oxides and demonstrated for the first time that 2'-OH-NQ and 2'-OH-NQNO but not the corresponding 2'-oxo compounds are naturally produced by PAO1 and PA14 strains of P. aeruginosa. The main metabolite 2'-OH-NQ is produced even in concentrations comparable to NQ. Exogenous availability of ß-hydroxydecanoic acid can further increase the production of 2'-OH-NQ. In contrast to NQ, 2'-OH-NQ potently induced the cytokine IL-8 in a human cell line at 100 nм, suggesting a potential role in host immune modulation.

Beyond iron: metal-binding activity of the Pseudomonas quinolone signal-motif.

The Pseudomonas quinolone signal (PQS) is an important quorum sensing signal controlling virulence of the human pathogen Pseudomonas aeruginosa. PQS also exhibits multiple additional biological functions for P. aeruginosa, including the trapping of ferric iron. The PQS-motif has proven as privileged structure with great potential which is why we here explored the synthesis of two different types of crosslinked dimeric PQS-motifs as potential iron chelators. These compounds indeed chelated ferric iron and produced colorful and fluorescent complexes also with other metal ions. Inspired by these results, we revisited the metal ion binding capabilities of the natural product PQS and were able to detect further metal complexes beyond ferric iron and confirm the complex stoichiometry by mass spectrometry.

A ligand selection strategy to customize small molecule probes for activity-based protein profiling (LS-ABPP).

Activity-based probes (ABPs) are the key components of activity-based protein profiling (ABPP). However, designing a probe that shows target-specific as well as site-selective binding can be a challenging and time-consuming task, often requiring complex synthetic procedures to provide a selection of probes from which to choose the ideal one. In this chapter, we present a ligand selection (LS) approach that allows us to rapidly diversify probe molecules in order to meet the steric and electronic demands of the binding site of any target enzyme. The central element of this method is a trifunctional LS probe synthesized from tyrosine in five steps, consisting of a highly reactive pentafluorophenyl (PFP) ester in addition to an electrophilic chloroacetamide warhead, and a bioorthogonal alkyne reporter group. By reacting a variety of primary amine ligands with the PFP ester, a probe library is created and screened for optimal binding characteristics to the target enzyme. With the optimized probe in hand, a compound library is subsequently screened by competitive profiling to identify potential enzyme inhibitors. Conveniently, this protocol is highly adaptable to a large variety of target proteins, representing a valuable tool for enzyme characterization and the discovery of enzyme inhibitors. Here, we apply this method exemplarily to the cysteine protease 3CLpro of the coronavirus SARS-CoV-2.

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.

Inhibiting quinolone biosynthesis of Burkholderia.

2-Alkylquinolones are important signalling molecules of Burkholderia species. We developed a substrate-based chemical probe against the central quinolone biosynthesis enzyme HmqD and applied it in competitive profiling experiments to discover the first known HmqD inhibitors. The most potent inhibitors quantitatively blocked quinolone production in Burkholderia cultures with single-digit micromolar efficacy.

A Metabolite of Pseudomonas Triggers Prophage-Selective Lysogenic to Lytic Conversion in Staphylococcus aureus.

Bacteriophages have major impact on their microbial hosts and shape entire microbial communities. The majority of these phages are latent and reside as prophages integrated in the genomes of their microbial hosts. A variety of intricate regulatory systems determine the switch from a lysogenic to lytic life style, but so far strategies are lacking to selectively control prophage induction by small molecules. Here we show that Pseudomonas aeruginosa deploys a trigger factor to hijack the lysogenic to lytic switch of a polylysogenic Staphylococcus aureus strain causing the selective production of only one of its prophages. Fractionating extracts of P. aeruginosa identified the phenazine pyocyanin as a highly potent prophage inducer of S. aureus that, in contrast to mitomycin C, displayed prophage selectivity. Mutagenesis and biochemical investigations confirm the existence of a noncanonical mechanism beyond SOS-response that is controlled by the intracellular oxidation level and is prophage-selective. Our results demonstrate that human pathogens can produce metabolites triggering lysogenic to lytic conversion in a prophage-selective manner. We anticipate our discovery to be the starting point of unveiling metabolite-mediated microbe-prophage interactions and laying the foundations for a selective small molecule controlled manipulation of prophage activity. These could be for example applied to control microbial communities by their built-in destruction mechanism in a novel form of phage therapy or for the construction of small molecule-inducible switches in synthetic biology.

A Ligand Selection Strategy Identifies Chemical Probes Targeting the Proteases of SARS-CoV-2.

Activity-based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS-CoV-2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases.

Unveiling the Hidden Theatre of Microbes.

The chemical interactions of microbes shape and structure their communities and have important effects on the health and diseases of multicellular hosts. This special collection on Microbial Biosynthesis and Interactions sheds light on some of these interactions and takes a closer look at the molecules involved in them.

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.

Profiling structural diversity and activity of 2-alkyl-4(1H)-quinolone N-oxides of Pseudomonas and Burkholderia.

We synthesized all major saturated and unsaturated 2-alkyl-4(1H)-quinolone N-oxides of Pseudomonas and Burkholderia, quantified their native production levels and characterized their antibiotic activities against competing Staphylococcus aureus. We demonstrate that quinolone core methylation and position of unsaturation in the alkyl-chain dictate antibiotic potency which supports the proposed mechanism of action.

Competitive Metabolite Profiling of Natural Products Reveals Subunit Specific Inhibitors of the 20S Proteasome.

We have developed a syringolin-based chemical probe and explored its utility for the profiling of metabolite extracts as potent inhibitors of the 20S proteasome. Activity-guided fractionation by competitive labeling allowed us to isolate and identify glidobactin A and C as well as luminmycin A from a Burkholderiales strain. The natural products exhibited unique subunit specificities for the proteolytic subunits of human and mouse constitutive and immunoproteasome in the lower nanomolar range. In particular, glidobactin C displayed an unprecedented ß2/ß5 coinhibition profile with single-digit nanomolar potency in combination with sufficiently high cell permeability. These properties render glidobactin C a promising live cell proteasome inhibitor with potent activity against human breast cancer cell lines and comparably low immunotoxicity.

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.

Competitive profiling for enzyme inhibitors using chemical probes.

Enzyme inhibitors are central tools for chemical biology. In this chapter we will discuss the application of chemical probes for competitive profiling of inhibitors of the quinolone biosynthesis enzyme PqsD of Pseudomonas aeruginosa. The human pathogen P. aeruginosa produces a large diversity of 2-alkyl-4(1H)-quinolones and their derivatives as metabolites with major roles in quorum sensing, virulence, and interspecies competition. PqsD is a central enzyme in the biosynthesis of all of these quinolones and hence an interesting target for inhibitor discovery. Activity-based probes with an electrophilic warhead bind covalently to active site nucleophiles like cysteine or serine. An α-chloroacetamide probe with terminal alkyne tag allowed to selectively label the active site cysteine of PqsD and was demonstrated to be a useful tool for inhibitor discovery using competition experiments. Potent inhibitors bind to the active site and thereby prevent labeling of the enzyme by the probe. Labeling intensity is quantified on polyacrylamide gels by the fluorescence of a reporter tag appended by bioorthogonal click chemistry. The competitive inhibitor profiling strategy has many advantages over traditional screening approaches and is applicable in vitro as well as in live cells. Here we describe the synthesis of an activity-based probe and provide our detailed protocols for target enzyme labeling as well as its application for the screening for potent enzyme inhibitors of PqsD by a competitive profiling strategy.

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.

The Multifaceted Inhibitory Effects of an Alkylquinolone on the Diatom Phaeodactylum tricornutum.

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far-reaching ecological consequences. Recently, 2-alkyl-4-quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom-bacteria interactions. The effects of several quinolones on three diatom species have been investigated. The growth of all three was inhibited, with half-maximal inhibitory concentrations reaching the sub-micromolar range. By using multiple techniques, dual inhibition mechanisms were uncovered for 2-heptyl-4-quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily through inhibition of the cytochrome b6 f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria through organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments.

A thiochromenone antibiotic derived from the Pseudomonas quinolone signal selectively targets the Gram-negative pathogen Moraxella catarrhalis.

The Pseudomonas quinolone signal (PQS) is an important quorum sensing signal of the pathogen Pseudomonas aeruginosa. We discovered an additional activity of PQS as a narrow spectrum antibiotic. Exploiting the privileged structure of PQS by the synthesis of heteroatom-substituted analogues led to a class of 2-alkyl-3-hydroxythiochromen-4-ones with highly potent antibiotic activity against the nasopharyngeal pathogen Moraxella catarrhalis. Synthetic optimization resulted in minimum inhibitory concentrations in the nanomolar range even for clinical isolates of M. catarrhalis. Surprisingly, the growth of other human pathogens and commensals, including closely related Moraxella species, was not inhibited, indicating exceptional species selectivity. Mechanistic studies revealed that the antibiotic was bactericidal and likely inhibits a target in the primary energy metabolism causing rapid depletion of the cellular ATP pool.

Close the ring to break the cycle: tandem quinolone-alkyne-cyclisation gives access to tricyclic pyrrolo[1,2-a]quinolin-5-ones with potent anti-protozoal activity.

Expanding the chemical space of quinolones led to a tandem quinolone-alkyne-cyclisation reaction allowing chemoselective control of the synthesis of tricyclic pyrrolo[1,2-a]quinolin-5-ones. Importantly, we discovered anti-protozoal activity against Plasmodium and Toxoplasma with specific potency of one of the compounds against the liver stage of the malaria parasite in the nanomolar range.

Pseudo-Biomineralization: Complex Mineral Structures Shaped by Microbes.

Biomineralization is an active, biologically governed process of mineral formation, established early on in the history of life. The appearance of biomineralizing organisms heavily influenced the course of evolution, leading to the development of the large diversity of the extant taxa. Yet, we are still only beginning to grasp the intricate, genetically regulated mechanisms involved. Since prokaryotic organisms were the first to emerge from the primordial environments, we investigated bacteria-mineral interactions using titration and gas diffusion systems adapted to emulate conditions, which may have facilitated the development of biomineralization initially. By screening the minerals and bacteria from titration experiments with scanning electron microscopy, we discovered a broad spectrum of behavioral strategies employed by bacteria confronted with calcification, which fell into three main categories: (1) evasion of mineralization by the formation of the biofilm, (2) random embedding into the mineral, and (3) control over the mineral shape during its formation. The latter phenomenon we termed pseudo-biomineralization. Our experiments indicate that pseudo-biomineralization is an active process obligatorily reliant on the external calcifying conditions and allowing considerable degree of control over mineral shape, thus producing structures reminiscent of true biominerals. Here, we describe this notion for the first time, thus providing vital insight into the genesis of a transitional stage to calcium carbonate-based biomineralization systems.