The histone deacetylase Hda1 affects oxidative and osmotic stress response as well as mycoparasitic activity and secondary metabolite biosynthesis in Trichoderma atroviride.

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride, regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens.

The use of immunoaffinity purification approaches coupled with LC-MS/MS offers a powerful strategy to identify protein complexes in filamentous fungi.

Fungi are a diverse group of organisms that can be both beneficial and harmful to mankind. They have advantages such as producing food processing enzymes and antibiotics, but they can also be pathogens and produce mycotoxins that contaminate food. Over the past two decades, there have been significant advancements in methods for studying fungal molecular biology. These advancements have led to important discoveries in fungal development, physiology, pathogenicity, biotechnology, and natural product research. Protein complexes and protein-protein interactions (PPIs) play crucial roles in fungal biology. Various methods, including yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), are used to investigate PPIs. However, affinity-based PPI methods like co-immunoprecipitation (Co-IP) are highly preferred because they represent the natural conditions of PPIs. In recent years, the integration of liquid chromatography coupled with mass spectrometry (LC-MS/MS) has been used to analyse Co-IPs, leading to the discovery of important protein complexes in filamentous fungi. In this review, we discuss the tandem affinity purification (TAP) method and single affinity purification methods such as GFP, HA, FLAG, and MYC tag purifications. These techniques are used to identify PPIs and protein complexes in filamentous fungi. Additionally, we compare the efficiency, time requirements, and material usage of Sepharose™ and magnetic-based purification systems. Overall, the advancements in fungal molecular biology techniques have provided valuable insights into the complex interactions and functions of proteins in fungi. The methods discussed in this review offer powerful tools for studying fungal biology and will contribute to further discoveries in this field.

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells.

Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.

Online Biomass Monitoring Enables Characterization of the Growth Pattern of Aspergillus fumigatus in Liquid Shake Conditions.

Numerous filamentous fungal species are extensively studied due to their role as model organisms, workhorses in biotechnology, or as pathogens for plants, animals, and humans. Growth studies are mainly carried out on solid media. However, studies concerning gene expression, biochemistry, or metabolism are carried out usually in liquid shake conditions, which do not correspond to the growth pattern on solid media. The reason for this practice is the problem of on-line growth monitoring of filamentous fungal species, which usually form pellets in liquid shake cultures. Here, we compared the time-consuming and tedious process of dry-weight determination of the mold Aspergillus fumigatus with online monitoring of biomass in liquid shake culture by the parallelizable CGQ ("cell growth quantifier"), which implements dynamic biomass determination by backscattered light measurement. The results revealed a strong correlation of CGQ-mediated growth monitoring and classical biomass measurement of A. fumigatus grown over a time course. Moreover, CGQ-mediated growth monitoring displayed the difference in growth of A. fumigatus in response to the limitation of iron or nitrogen as well as the growth defects of previously reported mutant strains (ΔhapX, ΔsrbA). Furthermore, the frequently used wild-type strain Af293 showed largely decreased and delayed growth in liquid shake cultures compared to other strains (AfS77, A1160p+, AfS35). Taken together, the CGQ allows for robust, automated biomass monitoring of A. fumigatus during liquid shake conditions, which largely facilitates the characterization of the growth pattern of filamentous fungal species.

The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis.

Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.

Regulation of sensory perception and motor abilities by brain-specific action of chromatin remodeling factor CHD1.

The ATP-dependent chromatin remodeling factor CHD1 (chromodomain-helicase-DNA binding protein 1) is involved in both the de novo assembly and the remodeling of chromatin. Recently, we discovered a crucial role of CHD1 in the incorporation of the histone variant H3.3 in the fly brain illustrated by widespread transcriptional upregulation and shortened lifespan in Chd1-mutant animals. Because many genes linked to sensory perception were dysregulated in Chd1-mutant heads, we studied the role of CHD1 in these processes. Here we show that Chd1-mutant flies have severe defects in their response behavior to olfactory and gustatory but not visual stimuli. Further analyses suggested that poor performance in gustatory response assays was caused by reduced motivation for foraging and feeding rather than defects in taste perception. Moreover, we show that shortened lifespan of Chd1-mutant flies is accompanied by indications of premature functional aging as suggested by defects in negative geotaxis and exploratory walking assays. The latter phenotype was rescued by neuronal re-expression of Chd1, while the olfactory defects were not. Interestingly, we found evidence for indirect regulation of the non-neuronal expression of odorant binding proteins (Obp) by neuronal expression of Chd1. Together, these results emphasize the crucial role of CHD1 activity controlling diverse neuronal processes thereby affecting healthy lifespan.

CHD1 controls H3.3 incorporation in adult brain chromatin to maintain metabolic homeostasis and normal lifespan.

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.

Fungal Lysine Deacetylases in Virulence, Resistance, and Production of Small Bioactive Compounds.

The growing number of immunocompromised patients begs for efficient therapy strategies against invasive fungal infections. As conventional antifungal treatment is increasingly hampered by resistance to commonly used antifungals, development of novel therapy regimens is required. On the other hand, numerous fungal species are industrially exploited as cell factories of enzymes and chemicals or as producers of medically relevant pharmaceuticals. Consequently, there is immense interest in tapping the almost inexhaustible fungal portfolio of natural products for potential medical and industrial applications. Both the pathogenicity and production of those small metabolites are significantly dependent on the acetylation status of distinct regulatory proteins. Thus, classical lysine deacetylases (KDACs) are crucial virulence determinants and important regulators of natural products of fungi. In this review, we present an overview of the members of classical KDACs and their complexes in filamentous fungi. Further, we discuss the impact of the genetic manipulation of KDACs on the pathogenicity and production of bioactive molecules. Special consideration is given to inhibitors of these enzymes and their role as potential new antifungals and emerging tools for the discovery of novel pharmaceutical drugs and antibiotics in fungal producer strains.

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers.

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

RcLS2F - A Novel Fungal Class 1 KDAC Co-repressor Complex in Aspergillus nidulans.

The fungal class 1 lysine deacetylase (KDAC) RpdA is a promising target for prevention and treatment of invasive fungal infection. RpdA is essential for survival of the most common air-borne mold pathogen Aspergillus fumigatus and the model organism Aspergillus nidulans. In A. nidulans, RpdA depletion induced production of previously unknown small bioactive substances. As known from yeasts and mammals, class 1 KDACs act as components of multimeric protein complexes, which previously was indicated also for A. nidulans. Composition of these complexes, however, remained obscure. In this study, we used tandem affinity purification to characterize different RpdA complexes and their composition in A. nidulans. In addition to known class 1 KDAC interactors, we identified a novel RpdA complex, which was termed RcLS2F. It contains ScrC, previously described as suppressor of the transcription factor CrzA, as well as the uncharacterized protein FscA. We show that recruitment of FscA depends on ScrC and we provide clear evidence that ΔcrzA suppression by ScrC depletion is due to a lack of transcriptional repression caused by loss of the novel RcLS2F complex. Moreover, RcLS2F is essential for sexual development and engaged in an autoregulatory feed-back loop.

The Lysine Deacetylase RpdA Is Essential for Virulence in Aspergillus fumigatus.

Current suboptimal treatment options of invasive fungal infections and emerging resistance of the corresponding pathogens urge the need for alternative therapy strategies and require the identification of novel antifungal targets. Aspergillus fumigatus is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel in vivo conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of A. fumigatus in a murine model for pulmonary aspergillosis. The xylP promoter used has previously been shown to allow xylose-induced gene expression in different molds. Here, we demonstrate for the first time that this promoter also allows in vivo tuning of A. fumigatus gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an A. fumigatus strain expressing rpdA under control of the xylP promoter, rpdA xylP , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, rpdA xylP displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused rpdA induction in vivo. These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the xylP expression system is a powerful new tool for in vivo gene silencing in A. fumigatus.

The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Type I and II PRMTs regulate catabolic as well as detoxifying processes in Aspergillus nidulans.

In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs). In Aspergillus nidulans, three conserved type I and II PRMTs are present that catalyze asymmetric or symmetric dimethylation of arginines. We generated a double type I mutant (ΔrmtA/rmtB) and a combined type I and type II mutant (ΔrmtB/rmtC) to perform genome-wide comparison of their effects on gene expression, but also to monitor putative overlapping activities and reciprocal regulations of type I and type II PRMTs in Aspergillus. Our study demonstrates, that rmtA and rmtC as type I and type II representatives act together as repressors of proteins that are secreted into the extracellular region as the majority of up-regulated genes are mainly involved in catabolic pathways that constitute the secretome of Aspergillus. In addition to a strong up-regulation of secretory genes we found a significant enrichment of down-regulated genes involved in processes related to oxidation-reduction, transmembrane transport and secondary metabolite biosynthesis. Strikingly, nearly 50% of down-regulated genes in both double mutants correspond to redox reaction/oxidoreductase processes, a remarkable finding in light of our recently observed oxidative stress phenotypes of ΔrmtA and ΔrmtC. Finally, analysis of nuclear and cytoplasmic extracts for mono-methylated proteins revealed the presence of both, common and specific substrates of RmtA and RmtC. Thus, our data indicate that type I and II PRMTs in Aspergillus seem to co-regulate the same biological processes but also specifically affect other pathways in a non-redundant fashion.

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

A Class 1 Histone Deacetylase as Major Regulator of Secondary Metabolite Production in Aspergillus nidulans.

An outstanding feature of filamentous fungi is their ability to produce a wide variety of small bioactive molecules that contribute to their survival, fitness, and pathogenicity. The vast collection of these so-called secondary metabolites (SMs) includes molecules that play a role in virulence, protect fungi from environmental damage, act as toxins or antibiotics that harm host tissues, or hinder microbial competitors for food sources. Many of these compounds are used in medical treatment; however, biosynthetic genes for the production of these natural products are arranged in compact clusters that are commonly silent under growth conditions routinely used in laboratories. Consequently, a wide arsenal of yet unknown fungal metabolites is waiting to be discovered. Here, we describe the effects of deletion of hosA, one of four classical histone deacetylase (HDAC) genes in Aspergillus nidulans; we show that HosA acts as a major regulator of SMs in Aspergillus with converse regulatory effects depending on the metabolite gene cluster examined. Co-inhibition of all classical enzymes by the pan HDAC inhibitor trichostatin A and the analysis of HDAC double mutants indicate that HosA is able to override known regulatory effects of other HDACs such as the class 2 type enzyme HdaA. Chromatin immunoprecipitation analysis revealed a direct correlation between hosA deletion, the acetylation status of H4 and the regulation of SM cluster genes, whereas H3 hyper-acetylation could not be detected in all the upregulated SM clusters examined. Our data suggest that HosA has inductive effects on SM production in addition to its classical role as a repressor via deacetylation of histones. Moreover, a genome wide transcriptome analysis revealed that in addition to SMs, expression of several other important protein categories such as enzymes of the carbohydrate metabolism or proteins involved in disease, virulence, and defense are significantly affected by the deletion of HosA.

A Class 1 Histone Deacetylase with Potential as an Antifungal Target.

Histone deacetylases (HDACs) remove acetyl moieties from lysine residues at histone tails and nuclear regulatory proteins and thus significantly impact chromatin remodeling and transcriptional regulation in eukaryotes. In recent years, HDACs of filamentous fungi were found to be decisive regulators of genes involved in pathogenicity and the production of important fungal metabolites such as antibiotics and toxins. Here we present proof that one of these enzymes, the class 1 type HDAC RpdA, is of vital importance for the opportunistic human pathogen Aspergillus fumigatus Recombinant expression of inactivated RpdA shows that loss of catalytic activity is responsible for the lethal phenotype of Aspergillus RpdA null mutants. Furthermore, we demonstrate that a fungus-specific C-terminal region of only a few acidic amino acids is required for both the nuclear localization and catalytic activity of the enzyme in the model organism Aspergillus nidulans Since strains with single or multiple deletions of other classical HDACs revealed no or only moderate growth deficiencies, it is highly probable that the significant delay of germination and the growth defects observed in strains growing under the HDAC inhibitor trichostatin A are caused primarily by inhibition of catalytic RpdA activity. Indeed, even at low nanomolar concentrations of the inhibitor, the catalytic activity of purified RpdA is considerably diminished. Considering these results, RpdA with its fungus-specific motif represents a promising target for novel HDAC inhibitors that, in addition to their increasing impact as anticancer drugs, might gain in importance as antifungals against life-threatening invasive infections, apart from or in combination with classical antifungal therapy regimes. IMPORTANCE: This paper reports on the fungal histone deacetylase RpdA and its importance for the viability of the fungal pathogen Aspergillus fumigatus and other filamentous fungi, a finding that is without precedent in other eukaryotic pathogens. Our data clearly indicate that loss of RpdA activity, as well as depletion of the enzyme in the nucleus, results in lethality of the corresponding Aspergillus mutants. Interestingly, both catalytic activity and proper cellular localization depend on the presence of an acidic motif within the C terminus of RpdA-type enzymes of filamentous fungi that is missing from the homologous proteins of yeasts and higher eukaryotes. The pivotal role, together with the fungus-specific features, turns RpdA into a promising antifungal target of histone deacetylase inhibitors, a class of molecules that is successfully used for the treatment of certain types of cancer. Indeed, some of these inhibitors significantly delay the germination and growth of different filamentous fungi via inhibition of RpdA. Upcoming analyses of clinically approved and novel inhibitors will elucidate their therapeutic potential as new agents for the therapy of invasive fungal infections-an interesting aspect in light of the rising resistance of fungal pathogens to conventional therapies.

Novel insights into the functional role of three protein arginine methyltransferases in Aspergillus nidulans.

Protein arginine methylation has been implicated in different cellular processes including transcriptional regulation by the modification of histone proteins. Here we demonstrate significant in vitro activities and multifaceted specificities of Aspergillus protein arginine methyltransferases (PRMTs) and we provide evidence for a role of protein methylation in mechanisms of oxidative stress response. We have isolated all three Aspergillus PRMTs from fungal extracts and could assign significant histone specificity to RmtA and RmtC. In addition, both enzymes were able to methylate several non-histone proteins in chromatographic fractions. For endogenous RmtB a remarkable change in its substrate specificity compared to the recombinant enzyme form could be obtained. Phenotypic analysis of mutant strains revealed that growth of DeltarmtA and DeltarmtC strains was significantly reduced under conditions of oxidative stress. Moreover, mycelia of DeltarmtC mutants showed a significant retardation of growth under elevated temperatures.

Design, synthesis and biological evaluation of carboxy analogues of arginine methyltransferase inhibitor 1 (AMI-1).

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI-1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis-amidic moiety; 3) the introduction of a N-containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates. Molecular modeling studies for a deep binding-mode analysis of test compounds were also performed. The bis-carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI-1) and practically inactive against the lysine methyltransferase SET7/9.