Genome Of Aspergillus Fumigatus Research Articles

Elevated mutation rates in multi-azole resistant Aspergillus fumigatus drive rapid evolution of antifungal resistance

The environmental use of azole fungicides has led to selective sweeps across multiple loci in the Aspergillus fumigatus genome causing the rapid global expansion of a genetically distinct cluster of resistant genotypes. Isolates within this cluster are also more likely to be resistant to agricultural antifungals with unrelated modes of action. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to next generation antifungals because of variants in the DNA mismatch repair system. A variant in msh6-G233A is found almost exclusively within azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR34/L98H. Naturally occurring isolates with this msh6 variant display up to 5-times higher rate of mutation, leading to an increased likelihood of evolving resistance to other antifungals. Furthermore, unlike hypermutator strains, the G233A variant conveys no measurable fitness cost and has become globally distributed. Our findings further suggest that resistance to next-generation antifungals is more likely to emerge within organisms that are already multi-azole resistant due to close linkage between TR34/L98H and msh6-G233A, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings.

The New GPI-Anchored Protein, SwgA, Is Involved in Nitrogen Metabolism in the Pathogenic Filamentous Fungus Aspergillus fumigatus.

GPI-anchored proteins display very diverse biological (biochemical and immunological) functions. An in silico analysis has revealed that the genome of Aspergillus fumigatus contains 86 genes coding for putative GPI-anchored proteins (GPI-APs). Past research has demonstrated the involvement of GPI-APs in cell wall remodeling, virulence, and adhesion. We analyzed a new GPI-anchored protein called SwgA. We showed that this protein is mainly present in the Clavati of Aspergillus and is absent from yeasts and other molds. The protein, localized in the membrane of A. fumigatus, is involved in germination, growth, and morphogenesis, and is associated with nitrogen metabolism and thermosensitivity. swgA is controlled by the nitrogen regulator AreA. This current study indicates that GPI-APs have more general functions in fungal metabolism than cell wall biosynthesis.

Characterization of the mbsA Gene Encoding a Putative APSES Transcription Factor in Aspergillus fumigatus.

The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ΔmbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ΔmbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ΔmbsA conidium. Comparative transcriptomic analyses revealed that the ΔmbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ΔmbsA mutant produced higher amount of GT, ΔmbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.

Computational approach for identification, characterization, three-dimensional structure modelling and machine learning-based thermostability prediction of xylanases from the genome of Aspergillus fumigatus

Identification of thermostable and alkaline xylanases from different fungal and bacterial species have gained an interest for the researchers because of its biotechnological relevance in many industries, such as pulp, paper, and bioethanol. In this study, we have identified and characterized xylanases from the genome of the thermophilic fungus of Aspergillus fumigatus by in silico analysis. Genome data mining revealed that the A fumigatus genome has six xylanase genes that belong to GH10, GH11, GH43 glycoside hydrolase families. In general, most of the bacterial and fungal GH11 xylanases are alkaline, and GH10 xylanases are acidic; however, we found that one identified xylanase from A fumigatus that belongs to the GH10 family is alkaline while the rest are acidic. Moreover, physicochemical properties also stated that most of the xylanases identified have lower molecular weight except one that belongs to the GH43 family. Structure prediction by homology modelling gave optimized structures of the xylanases. It suggests that GH10 family structure models adapt (β∕α) 8 barrel type, GH11 homology models adapt β-jelly type, and the GH43 family has a fivefold β-propeller type structure. Molecular docking of identified xylanases with xylan revealed that GH11 xylanases have strong interaction (-9.6 kcal/mol) with xylan than the GH10 (-8.5 and -9.3 kcal/mol) and GH43 (-8.8 kcal/mol). We used the machine learning approach based TAXyl server to predict the thermostability of the xylanases. It revealed that two GH10 xylanases and one GH11 xylanase are thermo-active up to 75ᵒC. We have explored the physiochemical properties responsible for maintaining thermostability for bacterial and fungal GH10 and GH11 xylanases by comparing crystal structures. All the analyzed parameters specified that GH10 xylanases from both the fungi and bacteria are more thermostable due to higher hydrogen bonds, salt bridges, and helical content.

Comparative analysis of two recombinant LPMOs from Aspergillus fumigatus and their effects on sugarcane bagasse saccharification

Lytic polysaccharide monooxygenases (LPMOs) have been introduced into industrial cocktails used for biomass saccharification due to their capacity to boost enzymatic conversion of recalcitrant cellulose. The genome of the thermotolerant ascomycete Aspergillus fumigatus encodes 7 genes for LPMOs that belong to auxiliary activity family 9 (AA9). Here, we cloned, successfully expressed and performed biochemical evaluation of two CBM-less A. fumigatus LPMOs (AfAA9A and AfAA9B). A high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis demonstrated that AfAA9A and AfAA9B are able to oxide cellulose at C1 and C1/C4 positions, respectively. Synergic effects of LPMOs (separately and in combination) with cellulases were investigated. Supplementation of Celluclast 1.5 L with a low concentration of AfAA9B improved in 20 % the saccharification of sugarcane bagasse pretreated by steam explosion (SEB), while AfAA9A did not improvethe saccharification. Analysis of the hydrolyzed biomass by confocal laser scanning microscopy (CLSM) showed the LPMOs are promoting lignin oxidation in the lignocellulosic material. This study complements the available results concerning the utilization of LPMOs in the enzymatic saccharification of lignocellulosic biomass.

Identification and mutational analyses of KpsF gene containing PxIxIT motif in Aspergillus fumigatus

Invasive Aspergillosis (IA) has increased significantly in recent years in which about 90% is caused by Aspergillus fumigatus, and the mortality rate is on the rise. Its treatment has become a common problem clinically. Calcineurin (CaN) plays an important role in the morphogenesis and toxicity of Aspergillus fumigatus, making it an attractive antifungal target. Although the Calcineurin inhibitors FK506 and cyclosporine A have successfully applied in modern transplantation medicine, the systemic toxic and side effects of the two drugs have greatly limited their clinical antifungal applications. Previous studies have shown that CaN of the Aspergillus fumigatus is localized at the top and division of hyphae, and interactions of CaN with cbpA are closely related to the PxIxIT motif. The functioning of CaN is closely related to the PxIxIT motif. Calcineurin substrates or target proteins such as Crz1, SIm1, SIm2, Hph1, and CbpA all contain similar PxIxIT motifs, and similar motifs are present in the Aspergillus fumigatus KpsF gene sequence. We can find KpsF gene in the Aspergillus fumigatus genome through bioinformatics methods, which contains a similar motif in its sequence. Therefore, we can speculate that this motif may interact with Calcineurin, further illustrate the specific mechanism of pathogenesis of CaN in Aspergillus fumigatus, which will help us to have a deeper understanding of the pathogenesis of invasive aspergillosis and provide new ideas for us. By completing the construction of the Aspergillus fumigatus KpsF gene-deficient strain and over-exp strain, we found that the KpsF gene over-exp strain of Aspergillus fumigatus is significantly different from that of the control strain in morphology, and is different from the cbpA in calcium ion regulation. In the low concentration of Ca2+, KpsF may be involved in the negative feedback regulation of Calcineurin, and this negative feedback regulation is inhibited under the condition of high concentration Ca2+.

GPI Anchored Proteins in Aspergillus fumigatus and Cell Wall Morphogenesis.

Glycosylphosphatidylinositol (GPI) anchored proteins are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. GPI anchored proteins are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, and immune response. In this review, we investigated for the first time an exhaustive list of all the GPI anchored proteins present in the Aspergillus fumigatus genome. An A. fumigatus mutant library of all the genes that encode in silico identified GPI anchored proteins has been constructed and the phenotypic analysis of all these mutants has been characterized including their growth, conidial viability or morphology, adhesion and the ability to form biofilms. We showed the presence of different fungal categories of GPI anchored proteins in the A. fumigatus genome associated to their role in cell wall remodeling, adhesion, and biofilm formation.

RgsA Attenuates the PKA Signaling, Stress Response, and Virulence in the Human Opportunistic Pathogen Aspergillus fumigatus.

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA, brlA, and wetA are elevated in the ΔrgsA mutant. Moreover, ΔrgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by ΔrgsA. In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA, and the ΔrgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the ΔrgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by ΔrgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation—likely by attenuating PKA signaling.

Identification of Functional and Druggable Sites in Aspergillus fumigatus Essential Phosphatases by Virtual Screening.

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.

A sphingolipid synthesis-related protein OrmA in Aspergillus fumigatus is responsible for azole susceptibility and virulence.

Previous studies identified that the budding yeast Saccharomyces cerevisiae have two sphingolipid synthesis-related proteins, Orm1p and Orm2p, that negatively regulate the activities of SPT, which is a key rate-limiting enzyme in sphingolipid synthesis. However, little is known about whether sphingolipids in the cell membrane, which are closely related to ergosterols, could affect the efficacy of azole drugs, which target to the ergosterol biosynthesis. In this study, through genome-wide homologue search analysis, we found that the Aspergillus fumigatus genome only contains one Orm homologue, referred to as OrmA for which the protein expression could be induced by azole antifungals in a dose-dependent manner. Deletion of ormA caused hypersensitivity to azoles, and adding the sphingolipid synthesis inhibitor myriocin rescued the azole susceptibility induced by lack of ormA. In contrast, overexpression of OrmA resulted in azole resistance, indicating that OrmA is a positive azole-response regulator. Further mechanism analysis verified that OrmA is related to drug susceptibility by affecting endoplasmic reticulum stress responses in an unfolded protein response pathway-HacA-dependent manner. Lack of ormA led to an abnormal profile of sphingolipid ceramide components accompanied by hypersensitivity to low temperatures. Furthermore, deletion of OrmA significantly reduced virulence in an immunosuppressed mouse model. The findings in this study collectively suggest that the sphingolipid metabolism pathway in A. fumigatus plays a critical role in azole susceptibility and fungal virulence.

Conservation and Recombinational Contexts in Apoptosis Pathway in Aspergillus Fumigatus

Introduction: Aspergillus fumigatus is a highly pathogenic fungus for immunocompromised patients and invasive aspergillosis exhibits mortality rates around 50-80%. Thus, the apoptosis pathway in this fungus may be an important target for the development of new drugs. Materials and Methods: From a data bank, we analyzed five different proteins (Bax, Bir, Nma111, Pca1, and Rad9) belonging to the apoptosis pathway in the genome of Aspergillus fumigatus. Phylogenetic trees, splits graphs, recombination analyses, and assessment of selection strength from codon to codon were developed to understand the evolution and the function of these proteins in the fungus. Results: Our results suggest a conservation of these proteins in the Aspergillaceae family, as well as a low rate of gene recombination with a putative preservation of the function. Conclusion: We thus propose that the apoptosis pathway in Aspergillus fumigatus is started with the Rad9 protein followed by the activation of Bax and Pca1 proteins. Also, Nma111 cleaves the Bir protein resulting in the consequent activation of the Bax protein. Further studies are necessary to better understand the apoptosis process in Aspergillus fumigatus and to confirm the proteins belonging to the pathway.

Structural and functional insights of β-glucosidases identified from the genome of Aspergillus fumigatus

Thermostable glucose tolerant β-glucosidase from Aspergillus species has attracted worldwide interest for their potentiality in industrial applications and bioethanol production. A strain of Aspergillus fumigatus (AfNITDGPKA3) identified by our laboratory from straw retting ground showed higher cellulase activity, specifically the β-glucosidase activity, compared to other contemporary strains. Though A. fumigatus has been known for high cellulase activity, detailed identification and characterization of the cellulase genes from their genome is yet to be done. In this work we have been analyzed the cellulase genes from the genome sequence database of Aspergillus fumigatus (Af293). Genome analysis suggests two cellobiohydrolase, eleven endoglucanase and seventeen β-glucosidase genes present. β-Glucosidase genes belong to either Glycohydro1 (GH1 or Bgl1) or Glycohydro3 (GH3 or Bgl3) family. The sequence similarity suggests that Bgl1 and Bgl3 of A. fumagatus are phylogenetically close to those of A. fisheri and A. oryzae. The modelled structure of the Bgl1 predicts the (β/α)8 barrel type structure with deep and narrow active site, whereas, Bgl3 shows the (α/β)8 barrel and (α/β)6 sandwich structure with shallow and open active site. Docking results suggest that amino acids Glu544, Glu466, Trp408,Trp567,Tyr44,Tyr222,Tyr770,Asp844,Asp537,Asn212,Asn217 of Bgl3 and Asp224,Asn242,Glu440, Glu445, Tyr367, Tyr365,Thr994,Trp435,Trp446 of Bgl1 are involved in the hydrolysis. Binding affinity analyses suggest that Bgl3 and Bgl1 enzymes are more active on the substrates like 4-methylumbelliferyl glycoside (MUG) and p-nitrophenyl-β-D-1, 4-glucopyranoside (pNPG) than on cellobiose. Further docking with glucose suggests that Bgl1 is more glucose tolerant than Bgl3. Analysis of the Aspergillus fumigatus genome may help to identify a β-glucosidase enzyme with better property and the structural information may help to develop an engineered recombinant enzyme.

Conserved cysteine residues determine substrate specificity in a novel As(III) S-adenosylmethionine methyltransferase from Aspergillus fumigatus.

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S-adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono- (MAs(III)), di- (DMAs(III)) and tri- (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C-terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).

GH16 and GH81 family β-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis.

The fungal cell wall is a rigid structure because of fibrillar and branched β-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on β-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-β-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo β-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.

A putative mitochondrial calcium uniporter in A. fumigatus contributes to mitochondrial Ca2+ homeostasis and stress responses

Ca2+ uptake into mitochondria plays a central role in cell physiology by stimulating ATP production, shaping cytosolic Ca2+ transients and regulating cell survival or death. Although this system has been studied extensively in mammalian cells, the physiological implications of Ca2+ uptake into mitochondria in fungal cells are still unknown. In this study, a bi-directional best-hit BLASTP search revealed that the genome of Aspergillus fumigatus encodes a homolog of a putative mitochondrial Ca2+ uniporter (MCU) and a mitochondrial carrier protein AGC1/MICU1 homolog. Both putative homologs are mitochondrially localized and required for the response to azole and oxidative stress such that the loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress, suggesting a role in environmental stress adaptation. Overexpressing mcuA restores the azole-resistance phenotype of the ΔagcA strain to wild-type levels, but not vice versa, indicating McuA plays a dominant role during these stress responses. Using a mitochondrially targeted version of the calcium-sensitive photoprotein aequorin, we found that only mcuA deletion leads to dysfunctional [Ca2+]mt and [Ca2+]c homeostasis, suggesting that McuA, but not AgcA, contributes to Ca2+ uptake into mitochondria. Further point-mutation experiments combined with extracellular Ca2+ chelator treatment verified that two predicted Ca2+-binding sites in McuA are required for Ca2+ uptake into mitochondria and stress responses through the regulation of [Ca2+]c homeostasis.

Divergent Protein Kinase A isoforms co‐ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus

The genome of Aspergillus fumigatus encodes two isoforms of the catalytic subunit of the cAMP-dependent Protein Kinase (PKA). Although deletion of the class I isoform, pkaC1, leads to an attenuation of virulence, the function of the class II subunit, PkaC2, was previously uninvestigated. In this report, we demonstrate that both isoforms act in concert to support various physiologic processes that promote the virulence of this pathogen. Whereas pkaC1 and pkaC2 single-deletion mutants display wild-type conidial germination, a double-deletion mutant is delayed in germination in response to environmental nutrients. Furthermore, PkaC1 and PkaC2 interact to positively regulate flux through the carbohydrate catabolic pathway and, consequently, the ΔpkaC1ΔpkaC2 mutant is unable to grow on low glucose concentrations. Importantly, the reduced germinative capacity and inability to utilize glucose observed for the ΔpkaC1ΔpkaC2 strain correlated with an inability of the mutant to establish infection in a murine model. Conversely, overexpression of pkaC2 both promotes the in vitro growth on glucose, and restores the fungal burden and mortality associated with the ΔpkaC1 to that of the wild-type organism. Taken together, these data demonstrate the functional capacity of pkaC2 and emphasize the importance of PKA-mediated metabolic control in the pathogenic potential of A. fumigatus.

Calculation of the ratio of the mononucleosome mapping number to the dinucleosome mapping number for each nucleotide position in the Aspergillus fumigatus genome

correspondence: hiromi Nishida Agricultural Bioinformatics Research Unit, graduate school of Agricultural and Life sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Tel +81 3 5841 1297 Fax +81 3 5841 1136 email hnishida@iu.a.u-tokyo.ac.jp Abstract: The ratio of the mononucleosome mapping site-number to the dinucleosome mapping site-number calculated using nucleosome mapping data of Aspergillus fumigatus was employed as a marker for the sensitivity of nucleosomes to micrococcal nuclease (MNase). This ratio was obtained for four different regions, namely, active gene bodies, active gene promoters, inactive gene bodies, and inactive gene promoters and compared. It was found that the nucleosomes located in the active gene promoters were more sensitive to MNase than those in the active gene bodies but less sensitive than those in the inactive gene bodies and promoters. In addition, compared to the gene bodies, gene promoters have a greater variety of nucleosomes that are sensitive to MNase, regardless of the transcriptional activity of these genes. This study showed that in addition to a mononucleosome map, a dinucleosome map is useful for estimating the sensitivity of nucleosomes to MNase.

The GC and window-averaged DNA curvature profile of secondary metabolite gene cluster in Aspergillus fumigatus genome

An immense variety of complex secondary metabolites is produced by filamentous fungi including Aspergillus fumigatus, a main inducer of invasive aspergillosis. The identification of fungal secondary metabolite gene cluster is essential for the characterization of fungal secondary metabolism in terms of genetics and biochemistry through recombinant technologies such as gene disruption and cloning. Most of the prediction methods for secondary metabolite gene cluster severely depend on homology searches. However, homology-based approach has intrinsic limitation to unknown or novel gene cluster. We analyzed the GC and window-averaged DNA curvature profile of 26 secondary metabolite gene clusters in the A. fumigatus genome to find out potential conserved features of secondary metabolite gene cluster. Fifteen secondary metabolite gene clusters showed a conserved pattern in window-averaged DNA curvature profile, that is, the DNA regions including secondary metabolic signature genes such as polyketide synthase, nonribosomal peptide synthase, and/or dimethylallyl tryptophan synthase consisted of window-averaged DNA curvature values lower than 0.18 and these DNA regions were at least 20 kb. Forty percent of secondary metabolite gene clusters with this conserved pattern were related to severe regulation by a transcription factor, LaeA. Our result could be used for identification of other fungal secondary metabolite gene clusters, especially for secondary metabolite gene cluster that is severely regulated by LaeA or other proteins with similar function to LaeA.

GDP-mannose pyrophosphorylase is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus

GDP-mannose pyrophosphorylase (GMPP) catalyses the synthesis of GDP-mannose, which is the precursor for the mannose residues in glycoconjugates, using mannose 1-phosphate and GTP as substrates. Repression of GMPP in yeast leads to phenotypes including cell lysis, defective cell wall, and failure of polarized growth and cell separation. Although several GMPPs have been isolated and characterized in filamentous fungi, the physiological consequences of their actions are not clear. In this study, Afsrb1, which is a homologue of yeast SRB1/PSA1/VIG9, was identified in the Aspergillus fumigatus genome. The Afsrb1 gene was expressed in Escherichia coli, and recombinant AfSrb1 was functionally confirmed as a GMPP. By the replacement of the native Afsrb1 promoter with an inducible Aspergillus nidulans alcA promoter, the conditional inactivation mutant strain YJ-gmpp was constructed. The presence of 3 % glucose completely blocked transcription of P(alcA)-Afsrb1, and was lethal to strain YJ-gmpp. Repression of Afsrb1 expression in strain YJ-gmpp led to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance, and branching site selection. Also, rapid germination and reduced conidiation were documented. However, in contrast to yeast, strain YJ-gmpp retained the ability to direct polarity establishment and septation. Our results showed that the Afsrb1 gene is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

Proteome analysis for pathogenicity and new diagnostic markers forAspergillus fumigatus

With the completion of the Aspergillus fumigatus genome it is now possible to study protein regulation on a global scale. One of the most suitable protein separation techniques is based on 2D-gel electrophoresis, which allows the separation of proteins based on their charge and size in a gel matrix. In addition, gel-free proteomics techniques based on liquid-chromatography coupled with mass spectrometry have gained importance. With the application of proteomic tools a comprehensive overview about the proteins of A. fumigatus present or induced during environmental changes and stress conditions can be obtained. For A. fumigatus, several proteomic studies have already been published including the response of the fungus to oxidative stress that induced the up-regulation of many proteins including catalases and thioredoxin peroxidase. Since many of the identified proteins/genes were apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified, designated Afyap1 and further characterized. In addition, some of the gene products expressed under stress conditions are also known fungal antigens, such as the thioredoxin peroxidase AspF3. Thus, besides pathogenicity studies, proteomics also delivers the tools to screen for new antigens which could improve the diagnosis of diseases caused by A. fumigatus.