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Bacterial isoprenoids are necessary for many biological processes, including maintaining membrane integrity, facilitating intercellular communication, and preventing oxidative damage. All bacterial isoprenoids are biosynthesized from two five carbon structural isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are cell impermeant. Herein, we demonstrate exogenous delivery of IPP and DMAPP into Bacillus subtilis by utilizing a self-immolative ester (SIE)-caging approach. We initially evaluated native B. subtilis esterase activity, which revealed a preference for short straight chain esters. We then examined the viability of the SIE-caging approach in B. subtilis and demonstrate that the released caging groups are well tolerated and the released IPP and DMAPP are bioavailable, such that isoprenoid biosynthesis can be rescued in the presence of pathway inhibitors. We further show that IPP and DMAPP are both toxic and inhibit growth of B. subtilis at the same concentration. Lastly, we establish the optimal ratio of IPP to DMAPP (5 : 1) for B. subtilis growth and find that, surprisingly, DMAPP alone is insufficient to rescue isoprenoid biosynthesis under high concentrations of fosmidomycin. These findings showcase the potential of the SIE-caging approach in B. subtilis and promise to both aid in novel isoprenoid discovery and to inform metabolic engineering efforts in bacteria., (© 2024 Wiley-VCH GmbH.)

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

Artificial protein hydrogels are an emerging class of biomaterials with numerous prospective applications in tissue engineering and regenerative medicine. These materials are likely to be immunogenic due to their frequent incorporation of novel amino acid sequence domains, which often serve a functional role within the material itself. We engineered injectable "self" and "nonself" artificial protein hydrogels, which were predicted to have divergent immune outcomes in vivo on the basis of their primary amino acid sequence. Following implantation in mouse, the nonself gels raised significantly higher antigel antibody titers than the corresponding self gels. Prophylactic administration of a fusion antibody targeting the nonself hydrogel epitopes to DEC-205, an endocytic receptor involved in T reg induction, fully suppressed the elevated antibody titer against the nonself gels. These results suggest that the clinical immune response to artificial protein biomaterials, including those that contain highly antigenic sequence domains, can be tuned through the induction of antigen-specific tolerance.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the β-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP., (© 2022 Wiley-VCH GmbH.)

We report boronate-caged guanidine-lipid 1 that activates liposomes for cellular delivery only upon uncaging of this compound by reactive oxygen species (ROS) to produce cationic lipid products. These liposomes are designed to mimic the exceptional cell delivery properties of cell-penetrating peptides (CPPs), while the inclusion of the boronate cage is designed to enhance selectivity such that cell entry will only be activated in the presence of ROS. Boronate uncaging by hydrogen peroxide was verified by mass spectrometry and zeta potential (ZP) measurements. A microplate-based fluorescence assay was developed to study the ROS-mediated vesicle interactions between 1-liposomes and anionic membranes, which were further elucidated via dynamic light scattering (DLS) analysis. Cellular delivery studies utilizing fluorescence microscopy demonstrated significant enhancements in cellular delivery only when 1-liposomes were incubated with hydrogen peroxide. Our results showcase that lipid 1 exhibits strong potential as an ROS-responsive liposomal platform for targeted drug delivery applications., (© 2022 Wiley-VCH GmbH.)

Liposomes are effective therapeutic delivery nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of therapeutics. Two primary areas in which improvement is needed for liposomal drug delivery is to enhance the ability to infiltrate cells and to facilitate derivatization of the liposome surface. Herein, we report a liposome platform incorporating a cyclic disulfide lipid (CDL) for the dual purpose of enhancing cell entry and functionalizing the liposome membrane through thiol-disulfide exchange. In order to accomplish this, CDL-1 and CDL-2, composed of lipoic acid (LA) or asparagusic acid (AA) appended to a lipid scaffold, were designed and synthesized. A fluorescence-based microplate immobilization assay was implemented to show that these compounds enable convenient membrane decoration through reaction with thiol-functionalized small molecules. Additionally, fluorescence microscopy experiments indicated dramatic enhancements in cellular delivery when CDLs were incorporated within liposomes. These results demonstrate that multifunctional CDLs serve as an exciting liposome system for surface decoration and enhanced cellular delivery., (© 2022 Wiley-VCH GmbH.)

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells., (© 2021 Wiley-VCH GmbH.)

Untargeted metabolomics indicates that the number of unidentified small-molecule metabolites may exceed the number of protein-coding genes for many organisms, including humans, by orders of magnitude. Uncovering the underlying metabolic networks is essential for elucidating the physiological and ecological significance of these biogenic small molecules. Here we develop a click-chemistry-based enrichment strategy, DIMEN (deep interrogation of metabolism via enrichment), that we apply to investigate metabolism of the ascarosides, a family of signaling molecules in the model organism C. elegans . Using a single alkyne-modified metabolite and a solid-phase azide resin that installs a diagnostic moiety for MS/MS-based identification, DIMEN uncovered several hundred novel compounds originating from diverse biosynthetic transformations that reveal unexpected intersection with amino acid, carbohydrate, and energy metabolism. Many of the newly discovered transformations could not be identified or detected by conventional LC-MS analyses without enrichment, demonstrating the utility of DIMEN for deeply probing biochemical networks that generate extensive yet uncharacterized structure space.

Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5-diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses. Here we show that DKP formation en route to the fungal virulence factor gliotoxin requires a seemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP. In vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxin and all other gli pathway metabolites. Point mutation of conserved active sites in the C and T domains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified NRPSs of other fungal DKPs terminate with similar CT domain couplets, suggesting a conserved strategy for DKP biosynthesis by fungal NRPSs., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial plant pathogens cause significant crop damage worldwide. They invade plant cells by producing a variety of virulence factors, including small-molecule toxins and phytohormone mimics. Virulence of the model pathogen Pseudomonas syringae pv. tomato DC3000 ( Pto ) is regulated in part by the sigma factor HrpL. Our study of the HrpL regulon identified an uncharacterized, three-gene operon in Pto that is controlled by HrpL and related to the Erwinia hrp -associated systemic virulence ( hsv ) operon. Here, we demonstrate that the hsv operon contributes to the virulence of Pto on Arabidopsis thaliana and suppresses bacteria-induced immune responses. We show that the hsv -encoded enzymes in Pto synthesize a small molecule, phevamine A. This molecule consists of l-phenylalanine, l-valine, and a modified spermidine, and is different from known small molecules produced by phytopathogens. We show that phevamine A suppresses a potentiation effect of spermidine and l-arginine on the reactive oxygen species burst generated upon recognition of bacterial flagellin. The hsv operon is found in the genomes of divergent bacterial genera, including ∼37% of P. syringae genomes, suggesting that phevamine A is a widely distributed virulence factor in phytopathogens. Our work identifies a small-molecule virulence factor and reveals a mechanism by which bacterial pathogens overcome plant defense. This work highlights the power of omics approaches in identifying important small molecules in bacteria-host interactions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

In the nematodes Caenorhabditis elegans and Pristionchus pacificus, a modular library of small molecules control behavior, lifespan, and development. However, little is known about the final steps of their biosynthesis, in which diverse building blocks from primary metabolism are attached to glycosides of the dideoxysugar ascarylose, the ascarosides. We combine metabolomic analysis of natural isolates of P. pacificus with genome-wide association mapping to identify a putative carboxylesterase, Ppa-uar-1, that is required for attachment of a pyrimidine-derived moiety in the biosynthesis of ubas#1, a major dauer pheromone component. Comparative metabolomic analysis of wild-type and Ppa-uar-1 mutants showed that Ppa-uar-1 is required specifically for the biosynthesis of ubas#1 and related metabolites. Heterologous expression of Ppa-UAR-1 in C. elegans yielded a non-endogenous ascaroside, whose structure confirmed that Ppa-uar-1 is involved in modification of a specific position in ascarosides. Our study demonstrates the utility of natural variation-based approaches for uncovering biosynthetic pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Microbial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties. Although the first naturally occurring isocyanide (xanthocillin) was isolated from the fungus Penicillium notatum over half a century ago, the biosynthetic origins of fungal isocyanides remain unknown. Here we report the identification of a family of isocyanide synthases (ICSs) from the opportunistic human pathogen Aspergillus fumigatus Comparative metabolomics of overexpression or knockout mutants of ICS candidate genes led to the discovery of a fungal biosynthetic gene cluster (BGC) that produces xanthocillin ( xan ). Detailed analysis of xanthocillin biosynthesis in A. fumigatus revealed several previously undescribed compounds produced by the xan BGC, including two novel members of the melanocin family of compounds. We found both the xan BGC and a second ICS-containing cluster, named the copper-responsive metabolite ( crm ) BGC, to be transcriptionally responsive to external copper levels and further demonstrated that production of metabolites from the xan BGC is increased during copper starvation. The crm BGC includes a novel type of fungus-specific ICS-nonribosomal peptide synthase (NRPS) hybrid enzyme, CrmA. This family of ICS-NRPS hybrid enzymes is highly enriched in fungal pathogens of humans, insects, and plants. Phylogenetic assessment of all ICSs spanning the tree of life shows not only high prevalence throughout the fungal kingdom but also distribution in species not previously known to harbor BGCs, indicating an untapped resource of fungal secondary metabolism. IMPORTANCE Fungal ICSs are an untapped resource in fungal natural product research. Their isocyanide products have been implicated in plant and insect pathogenesis due to their ability to coordinate transition metals and disable host metalloenzymes. The discovery of a novel isocyanide-producing family of hybrid ICS-NRPS enzymes enriched in medically and agriculturally important fungal pathogens may reveal mechanisms underlying pathogenicity and afford opportunities to discover additional families of isocyanides. Furthermore, the identification of noncanonical ICS BGCs will enable refinement of BGC prediction algorithms to expand on the secondary metabolic potential of fungal and bacterial species. The identification of genes related to ICS BGCs in fungal species not previously known for secondary metabolite-producing capabilities (e.g., Saccharomyces spp.) contributes to our understanding of the evolution of BGC in fungi., (Copyright © 2018 Lim et al.)

Small-molecule signaling is one major mode of communication within the polymicrobial consortium of soil and rhizosphere. While microbial secondary metabolite (SM) production and responses of individual species have been studied extensively, little is known about potentially conserved roles of SM signals in multilayered symbiotic or antagonistic relationships. Here, we characterize the SM-mediated interaction between the plant-pathogenic bacterium Ralstonia solanacearum and the two plant-pathogenic fungi Fusarium fujikuroi and Botrytis cinerea We show that cellular differentiation and SM biosynthesis in F. fujikuroi are induced by the bacterially produced lipopeptide ralsolamycin (synonym ralstonin A). In particular, fungal bikaverin production is induced and preferentially accumulates in fungal survival spores (chlamydospores) only when exposed to supernatants of ralsolamycin-producing strains of R. solanacearum Although inactivation of bikaverin biosynthesis moderately increases chlamydospore invasion by R. solanacearum , we show that other metabolites such as beauvericin are also induced by ralsolamycin and contribute to suppression of R. solanacearum growth in vitro Based on our findings that bikaverin antagonizes R. solanacearum and that ralsolamycin induces bikaverin biosynthesis in F. fujikuroi , we asked whether other bikaverin-producing fungi show similar responses to ralsolamycin. Examining a strain of B. cinerea that horizontally acquired the bikaverin gene cluster from Fusarium , we found that ralsolamycin induced bikaverin biosynthesis in this fungus. Our results suggest that conservation of microbial SM responses across distantly related fungi may arise from horizontal transfer of protective gene clusters that are activated by conserved regulatory cues, e.g., a bacterial lipopeptide, providing consistent fitness advantages in dynamic polymicrobial networks. IMPORTANCE Bacteria and fungi are ubiquitous neighbors in many environments, including the rhizosphere. Many of these organisms are notorious as economically devastating plant pathogens, but little is known about how they communicate chemically with each other. Here, we uncover a conserved antagonistic communication between the widespread bacterial wilt pathogen Ralstonia solanacearum and plant-pathogenic fungi from disparate genera, Fusarium and Botrytis Exposure of Fusarium fujikuroi to the bacterial lipopeptide ralsolamycin resulted in production of the antibacterial metabolite bikaverin specifically in fungal tissues invaded by Ralstonia Remarkably, ralsolamycin induction of bikaverin was conserved in a Botrytis cinerea isolate carrying a horizontally transferred bikaverin gene cluster. These results indicate that horizontally transferred gene clusters may carry regulatory prompts that contribute to conserved fitness functions in polymicrobial environments., (Copyright © 2018 Spraker et al.)

Bacterial-fungal interactions are presumed to be mediated chiefly by small-molecule signals; however, little is known about the signaling networks that regulate antagonistic relationships between pathogens. Here, we show that the ralstonins, lipopeptides produced by the plant pathogenic bacteria Ralstonia solanacearum, interfere with germination of the plant-pathogenic fungus Aspergillus flavus by down-regulating expression of a cryptic biosynthetic gene cluster (BGC), named imq. Comparative metabolomic analysis of overexpression strains of the transcription factor ImqK revealed imq-dependent production of a family of tripeptide-derived alkaloids, the imizoquins. These alkaloids are produced via a nonribosomal peptide synthetase- (NRPS-)derived tripeptide and contain an unprecedented tricyclic imidazo[2,1-a]isoquinoline ring system. We show that the imizoquins serve a protective role against oxidative stress that is essential for normal A. flavus germination. Supplementation of purified imizoquins restored wildtype germination to a ΔimqK A. flavus strain and protected the fungus from ROS damage. Whereas the bacterial ralstonins retarded A. flavus germination and suppressed expression of the imq cluster, the fungal imizoquins in turn suppressed growth of R. solanacearum. We suggest such reciprocal small-molecule-mediated antagonism is a common feature in microbial encounters affecting pathogenicity and survival of the involved species.

Targeted cancer therapies that use genetics are successful, but principles for selectively targeting tumor metabolism that is also dependent on the environment remain unknown. We now show that differences in rate-controlling enzymes during the Warburg effect (WE), the most prominent hallmark of cancer cell metabolism, can be used to predict a response to targeting glucose metabolism. We establish a natural product, koningic acid (KA), to be a selective inhibitor of GAPDH, an enzyme we characterize to have differential control properties over metabolism during the WE. With machine learning and integrated pharmacogenomics and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond the status of individual genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method that tests for the direct physical interaction of a candidate receptor protein and a peptide ligand. This protocol describes the generation of the ligand probe, transient expression of a receptor protein, enrichment of membrane-bound receptors, photo-crosslinking and click chemistry-mediated reporter addition, and detection of the receptor-ligand complex. Copper-based click chemistry confers several advantages, including the versatility to use almost any azide-containing reporter molecule for detection or visualization of the complex and addition of the reporter molecule after receptor-ligand binding which reduces the need for bulky ligand modifications that could interfere with the interaction.

As microbial genome sequencing becomes more widespread, the capacity of microorganisms to produce an immense number of metabolites has come into better view. Utilizing a metabolite/gene cluster correlation platform, the biosynthetic origins of a new family of natural products, the rimosamides, were discovered. The rimosamides were identified in Streptomyces rimosus and associated with their NRPS/PKS-type gene cluster based upon their high frequency of co-occurrence across 179 strains of actinobacteria. This also led to the discovery of the related detoxin gene cluster. The core of each of these families of natural products contains a depsipeptide bond at the point of bifurcation in their unusual branched structures, the origins of which are definitively assigned to nonlinear biosynthetic pathways via heterologous expression in Streptomyces lividans. The rimosamides were found to antagonize the antibiotic activity of blasticidin S against Bacillus cereus., Competing Interests: The authors declare no competing financial interest.

Plants and animals detect the presence of potential pathogens through the perception of conserved microbial patterns by cell surface receptors. Certain solanaceous plants, including tomato, potato and pepper, detect flgII-28, a region of bacterial flagellin that is distinct from that perceived by the well-characterized FLAGELLIN-SENSING 2 receptor. Here we identify and characterize the receptor responsible for this recognition in tomato, called FLAGELLIN-SENSING 3. This receptor binds flgII-28 and enhances immune responses leading to a reduction in bacterial colonization of leaf tissues. Further characterization of FLS3 and its signalling pathway could provide new insights into the plant immune system and transfer of the receptor to other crop plants offers the potential of enhancing resistance to bacterial pathogens that have evolved to evade FLS2-mediated immunity.

Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multimodular polyketide synthases and nonribosomal peptide synthetases; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several new isoquinoline alkaloids known as the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi., Competing Interests: The authors declare no competing financial interests.

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing life-threatening infections in immunocompromised patients. Adaptation to different habitats and also virulence of the fungus depends on signal perception and transduction by modules such as the cyclic AMP-dependent protein kinase A (PKA) pathway. Here, by transcriptome analysis, 632 differentially regulated genes of this important signaling cascade were identified, including 23 putative transcriptional regulators. The highest upregulated transcription factor gene was located in a previously unknown secondary metabolite gene cluster, which we named fmp, encoding an incomplete non-ribosomal peptide synthetase, FmpE. Overexpression of the regulatory gene fmpR using the Tet(On) system led to the specific expression of the other six genes of the fmp cluster. Metabolic profiling of wild type and fmpR overexpressing strain by HPLC-DAD and HPLC-HRESI-MS and structure elucidation by NMR led to identification of 5-benzyl-1H-pyrrole-2-carboxylic acid, which we named fumipyrrole. Fumipyrrole was not described as natural product yet. Chemical synthesis of fumipyrrole confirmed its structure. Interestingly, deletion of fmpR or fmpE led to reduced growth and sporulation of the mutant strains. Although fmp cluster genes were transcribed in infected mouse lungs, deletion of fmpR resulted in wild-type virulence in a murine infection model., (© 2015 John Wiley & Sons Ltd.)

Thiocillins from Bacillus cereus ATCC 14579 are members of the well-known thiazolyl peptide class of natural product antibiotics, the biosynthesis of which has recently been shown to proceed via post-translational modification of ribosomally encoded precursor peptides. It has long been hypothesized that the final step of thiazolyl peptide biosynthesis involves a formal [4 + 2] cycloaddition between two dehydroalanines, a unique transformation that had eluded enzymatic characterization. Here we demonstrate that TclM, a single enzyme from the thiocillin biosynthetic pathway, catalyzes this transformation. To facilitate characterization of this new class of enzyme, we have developed a combined chemical and biological route to the complex peptide substrate, relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue leader peptide by means of native chemical ligation (NCL). This strategy, combined with active enzyme, provides a new chemoenzymatic route to this promising class of antibiotics.

Iron plays a critical role in survival and virulence of the opportunistic pathogen Aspergillus fumigatus. Two transcription factors, the GATA-factor SreA and the bZip-factor HapX oppositely monitor iron homeostasis with HapX activating iron acquisition pathways (e.g., siderophores) and shutting down iron consumptive pathways (and SreA) during iron starvation conditions whereas SreA negatively regulates HapX and corresponding pathways during iron sufficiency. Recently the non-ribosomal peptide, hexadehydroastechrome (HAS; a tryptophan-derived iron (III)-complex), has been found important in A. fumigatus virulence. We found that HAS overproduction caused an iron starvation phenotype, from alteration of siderophore pools to regulation of iron homeostasis gene expression including sreA. Moreover, we uncovered an iron dependent secondary metabolism network where both SreA and HapX oppositely regulate multiple other secondary metabolites including HAS. This circuitry links iron-acquisition and consumption pathways with secondary metabolism-thus placing HAS as part of a metabolic feedback circuitry designed to balance iron pools in the fungus and presenting iron availability as one environmental trigger of secondary metabolism.

Small molecules (SMs) play central roles as virulence factors of pathogenic fungi and bacteria; however, genomic analyses suggest that the majority of microbial SMs have remained uncharacterized. Based on microarray analysis followed by comparative metabolomics of overexpression/knockout mutants, we identified a tryptophan-derived iron(III)-complex, hexadehydro-astechrome (HAS), as the major product of the cryptic has nonribosomal peptide synthetase (NRPS) gene cluster in the human pathogen Aspergillus fumigatus. Activation of the has cluster created a highly virulent A. fumigatus strain that increased mortality of infected mice. Comparative metabolomics of different mutant strains allowed to propose a pathway for HAS biosynthesis and further revealed cross-talk with another NRPS pathway producing the anticancer fumitremorgins.