Essential gene identification and drug target prioritization in Aspergillus fumigatus.
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ABSTRACT:
SUBMITTER: Hu W
PROVIDER: S-EPMC1817658 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UnlabelledFungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in both Cryptococcus and ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity.ImportanceFungal infections are very common in humans but may be neglected due to misdiagnosis and inattention. Cryptococcus neoformans is a yeast that infects mainly immunocompromised people, causing high mortality rates in developing countries. The fungus infects the lungs, crosses the blood-brain barrier, and invades the cerebrospinal fluid, causing fatal meningitis. C. neoformans infections are treated with amphotericin B, flucytosine, and azoles, all developed decades ago. However, problems with antifungal agents highlight the urgent need for more-effective drugs to treat C. neoformans and other invasive fungal infections. These issues include the negative side effects of amphotericin B, the spontaneous resistance of C. neoformans to azoles, and the inefficacy of the echinocandin antifungals. In this study, we report the identification of C. neoformans essential genes as targets for the development of novel antifungals. Because of the level of evolutionary divergence between C. neoformans and the ascomycetes, a subset of these genes is likely essential in all fungi. Genes identified in this study represent an excellent starting point for the future development of new antifungals by pharmaceutical companies.
| S-EPMC4453551 | biostudies-literature
Project description:Fungal diseases are a serious health burden worldwide with drug resistance compromising efficacy of the limited arsenal of antifungals available. New drugs with novel mechanisms of action are desperately needed to overcome current challenges. The screening of the Aspergillus fumigatus genome identified 35 phosphatases, four of which were previously reported as essential for viability. In addition, we validated another three essential phosphatases. Phosphatases control critical events in fungi from cell wall integrity to cell cycle, thus they are attractive targets for drug development. We used VSpipe v1.0, a virtual screening pipeline, to evaluate the druggability of the seven essential phosphatases and identify starting points for drug discovery. Targeted virtual screening and evaluation of the ligand efficiency plots created by VSpipe, enabled us to define the most favourable chemical space for drug development and suggested different modes of inhibition for each phosphatase. Interestingly, the identified ligand binding sites match with functional sites (active site and protein interaction sites) reported for other yeast and human homologues. Thus, the VSpipe virtual screening approach identified both druggable and functional sites in these essential phosphatases for further experimental validation and antifungal drug development.
| S-EPMC6769645 | biostudies-literature
Project description:Drugs bind to their target proteins, which interact with downstream effectors and ultimately perturb the transcriptome of a cancer cell. These perturbations reveal information about their source, i.e., drugs' targets. Here, we investigate whether these perturbations and protein interaction networks can uncover drug targets and key pathways. We performed the first systematic analysis of over 500 drugs from the Connectivity Map. First, we show that the gene expression of drug targets is usually not significantly affected by the drug perturbation. Hence, expression changes after drug treatment on their own are not sufficient to identify drug targets. However, ranking of candidate drug targets by network topological measures prioritizes the targets. We introduce a novel measure, local radiality, which combines perturbed genes and functional interaction network information. The new measure outperforms other methods in target prioritization and proposes cancer-specific pathways from drugs to affected genes for the first time. Local radiality identifies more diverse targets with fewer neighbors and possibly less side effects.
| S-EPMC4663505 | biostudies-literature
Project description:Aspergillus fumigatus (Afu) causes allergic and invasive forms of diseases in humans. In order to identify genes relevant for pathogenesis, a total of 235 cDNA clones were randomly selected and sequenced from cDNA library of Afu. One of the partially sequenced cDNA clones was homologous to polyubiquitin. Sequencing of the complete cDNA clone showed an open reading frame of 912 bases. Comparison with genomic sequence of Afu using BlastN program, revealed that polyubiquitin gene comprises of 992 bases and contains one intron of 80 bases. The recombinant expression of fusion protein showed an approximately molecular weight of 43-kDa on SDS-PAGE. The translation product of the cDNA sequence showed four tandem repeats of 76 amino acid residues in a single polyubiquitin protein and showed 100% identity with polyubiquitin protein sequences of S. cerevisiae, N. crassa, C. albicans, S. pombe, and M. grisae. Polyubiquitin gene is known to play important role in a variety of cellular processes and recently have been implicated in fungal pathogenesis. Identification of polyubiquitin gene of Afu has opened up scope to study its role in understanding Aspergillus biology and pathogenesis.
| S-EPMC3454157 | biostudies-other
Project description:The beta(1-3)glucanosyltransferase GEL family of Aspergillus fumigatus contains 7 genes, among which only 3 are expressed during mycelial growth. The role of the GEL4 gene was investigated in this study. Like the other Gelps, it encodes a glycosylphosphatidylinositol (GPI)-anchored protein. In contrast to the other beta(1-3)glucanosyltransferases analyzed to date, it is essential for this fungal species.
| S-EPMC2918925 | biostudies-literature
Project description:Aspergillus fumigatus is an opportunistic human pathogen responsible for deadly, invasive infections in immunocompromised patients. The A. fumigatus cell wall is a complex network of polysaccharides among them galactofuran, which is absent in humans. UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactofuranose (UDP-Galf) to UDP-galactopyranose (UDP-Galp) and is an important virulence factor. UGM is a flavin-dependent enzyme that requires the reduced flavin for activity; flavin reduction is achieved by reaction with NADPH. The aim of this work was to discover inhibitors of UGM by targeting the NADPH binding site using an ADP-TAMRA probe in a high-throughput screening assay. The flavonoids (2S)-hesperetin and (2S)-naringenin were validated as competitive inhibitors of UGM against NADPH with Ki values of 6 µM and 74 µM, respectively. To gain insight into the active chemical substituents involved in the inhibition of UGM, several derivatives of these inhibitors were studied. The results show that the hydroxyl groups of (2S)-hesperetin are important for inhibition, in particular the phenyl-chroman moiety. Congo red susceptibility assay and growth temperature effects showed that these compounds affected cell wall biosynthesis in A. fumigatus. This work is the first report of inhibition studies on UGM from eukaryotic human pathogens.
| S-EPMC5589893 | biostudies-literature
Project description:The fungal pathogen Aspergillus fumigatus causes serious illness and often death when it invades tissues, especially in immunocompromised individuals. The azole class of drugs is the most commonly prescribed treatment for many fungal infections and acts on the ergosterol biosynthesis pathway. One common mechanism of acquired azole drug resistance in fungi is the prevention of drug accumulation to toxic levels in the cell. While drug efflux is a well-known resistance strategy, reduced azole import would be another strategy to maintain low intracellular azole levels. Recently, azole uptake in Candida albicans and other yeasts was analyzed using [(3)H]fluconazole. Defective drug import was suggested to be a potential mechanism of drug resistance in several pathogenic fungi, including Cryptococcus neoformans, Candida krusei, and Saccharomyces cerevisiae. We have adapted and developed an assay to measure azole accumulation in A. fumigatus using radioactively labeled azole drugs, based on previous work done with C. albicans. We used this assay to study the differences in azole uptake in A. fumigatus isolates under a variety of drug treatment conditions, with different morphologies and with a select mutant strain with deficiencies in the sterol uptake and biosynthesis pathway. We conclude that azole drugs are specifically selected and imported into the fungal cell by a pH- and ATP-independent facilitated diffusion mechanism, not by passive diffusion. This method of drug transport is likely to be conserved across most fungal species.
| S-EPMC4432157 | biostudies-literature
Project description:Treatment of filamentous fungal infections relies on a limited repertoire of antifungal agents. Compounds possessing novel modes of action are urgently required. N-myristoylation is a ubiquitous modification of eukaryotic proteins. The enzyme N-myristoyltransferase (NMT) has been considered a potential therapeutic target in protozoa and yeasts. Here, we show that the filamentous fungal pathogen Aspergillus fumigatus possesses an active NMT enzyme that is essential for survival. Surprisingly, partial repression of the gene revealed downstream effects of N-myristoylation on cell wall morphology. Screening a library of inhibitors led to the discovery of a pyrazole sulphonamide compound that inhibits the enzyme and is fungicidal under partially repressive nmt conditions. Together with a crystallographic complex showing the inhibitor binding in the peptide substrate pocket, we provide evidence of NMT being a potential drug target in A. fumigatus.
| S-EPMC4477619 | biostudies-literature
Project description:Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol ?-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1 In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis.IMPORTANCE Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.
| S-EPMC6414702 | biostudies-literature
Project description:Sexual reproduction of the human pathogen Aspergillus fumigatus (teleomorph: Neosartorya fumigata) was assumed to be absent or cryptic until recently, when fertile crosses among geographically restricted environmental isolates were described. Here, we provide evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated, clinical isolates of A. fumigatus, and this evidence demonstrates the generality and reproducibility of this long-time-undisclosed phase in the life cycle of this heterothallic fungus. Successful mating requires the presence of both mating-type idiomorphs MAT1-1 and MAT1-2, as does expression of genes encoding factors presumably involved in this process. Moreover, analysis of an A. fumigatus mutant deleted for the nsdD gene suggests a role of this conserved regulator of cleistothecium development in hyphal fusion and hence heterokaryon formation.
| S-EPMC2863953 | biostudies-literature