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Neil A Brown,
Patrícia Alves de Castro,
Bárbara de Castro Pimentel Figueiredo,
Marcela Savoldi,
Marcos Silveira Buckeridge,
Mário Lúcio Lopes,
Silene Cristina de Lima Paullilo,
Eduardo P Borges,
Henrique V Amorim,
Maria Helena S Goldman,
Diego Bonatto, Iran Malavazi,
Gustavo Henrique Goldman
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ABSTRACT: Brazil played a pioneering role in the global establishment of the sugarcane-bioethanol industry. The bioethanol fermentation process currently used in Brazil is unique due to the acid wash and recycling of yeast cells. Two, industrially adopted, wild yeast strains, CAT-1 and PE-2, have become the most widely used in Brazil. How these strains respond to the unique fermentation process is poorly understood. The improved performance of CAT-1 and PE-2 is hypothesised to be related to enhanced stress tolerance. This study presents a genome wide analysis of the CAT-1 and PE-2 transcriptomes during a small-scale fermentation process that mimicked the industrial conditions. The common and unique transcriptional responses of the two strains to the Brazilian fermentation process were identified. Environmental stress response genes were up regulated post fermenter feeding, demonstrating the impact of the prior acid wash and high glucose environment. Cell wall and oxidative stress tolerance were subsequently demonstrated to be enhanced for the industrial strains. Conversely, numerous genes involved in protein synthesis were down regulated at the end of fermentation revealing the later impact of ethanol induced stress. Subsequently, the industrial strains demonstrated a greater tolerance of ethanol and the disruption of endoplasmic reticulum homeostasis. This increased ethanol tolerance was finally correlated with an increased unfolded protein response and increased HAC1 splicing. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
FEMS Yeast Research 01/2013; · 2.40 Impact Factor
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ABSTRACT: BACKGROUND: Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. METHODS: We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. RESULTS: We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. CONCLUSIONS: In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.
BMC Complementary and Alternative Medicine 10/2012; 12(1):194. · 2.24 Impact Factor
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ABSTRACT: Here, we characterize the Aspergillus fumigatus homologue ncsA Neuronal Calcium Sensor. We showed that ncsA is not an essential gene and ∆ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ∆ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm and its cellular localization is not affected
by the cellular response to either calcium chloride or EGTA. The ∆ncsA mutant strain is more sensitive to voriconazole, itraconazole, and amphotericin. Polar growth in the ΔncsA mutant was also considerably more affected by lovastatin than in the wild type strain. The Spitzenkörper can be visualized
in both strains and although the vacuolar system does not seem to be very different, there is an increase in the staining
intensity on the germling surface of the ∆ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the ΔncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding
wild type strain.
Molecular and General Genetics 04/2012; 280(6):483-495. · 2.63 Impact Factor
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ABSTRACT: Aspergillus fumigatus is a ubiquitous, filamentous fungal saprophyte and is the causative agent of the vast majority of aspergillosis in that invasive aspergillosis is the life-threatening form of infection by this fungus. The study of gene function using null mutants in this organism can be achieved through DNA-mediated transformation with an engineered deletion cassette containing about 2 kb of the 5'- and 3'-flanking region of the target gene and a selectable marker. Here, we describe the use of a PCR-based strategy and "in vivo" recombination in Saccharomyces cerevisiae to produce gene deletion cassettes for A. fumigatus, using an auxotrophic marker for gene replacement. This protocol produces highly effective deletion cassettes and permits the rapid disruption of genes identified in the recently available A. fumigatus genome.
Methods in molecular biology (Clifton, N.J.) 01/2012; 845:99-118.
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Christopher A Desjardins,
Mia D Champion,
Jason W Holder,
Anna Muszewska,
Jonathan Goldberg,
Alexandre M Bailão,
Marcelo Macedo Brigido,
Márcia Eliana da Silva Ferreira,
Ana Maria Garcia,
Marcin Grynberg, [......],
Maria Sueli Felipe,
Gustavo H Goldman,
Brian J Haas,
Juan G McEwen,
Gustavo Nino-Vega,
Rosana Puccia,
Gioconda San-Blas,
Celia Maria de Almeida Soares,
Bruce W Birren,
Christina A Cuomo
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ABSTRACT: Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.
PLoS Genetics 10/2011; 7(10):e1002345. · 8.69 Impact Factor
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ABSTRACT: ABSTRACT:
Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB).
Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB.
Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol.
Biotechnology for Biofuels 01/2011; 4:40. · 6.09 Impact Factor
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ABSTRACT: Farnesol (FOH) is a nonsterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. Here, we show that Aspergillus nidulans AifA encoding the apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I. Additionally, we demonstrated that ndeA-B and ndiA encode external and internal alternative NADH dehydrogenases, respectively, that have a function in FOH resistance. When exposed to FOH, the ΔaifA and ΔndeA strains have increased ROS production while ΔndeB, ΔndeA ΔndeB, and ΔndiA mutant strains showed the same ROS accumulation than in the absence of FOH. We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH.
Fungal Genetics and Biology 12/2010; 47(12):1055-69. · 3.74 Impact Factor
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ABSTRACT: Previously, we demonstrated that the Aspergillus nidulans calC2 mutation in protein kinase C pkcA was able to confer tolerance to farnesol (FOH), an isoprenoid that has been shown to inhibit proliferation and induce apoptosis. Here, we investigate in more detail the role played by A. nidulans pkcA in FOH tolerance. We demonstrate that pkcA overexpression during FOH exposure causes increased cell death. FOH is also able to activate several markers of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Our results suggest an intense cross-talk between PkcA and the UPR during FOH-induced cell death. Furthermore, the overexpression of pkcA increases both mRNA accumulation and metacaspases activity, and there is a genetic interaction between PkcA and the caspase-like protein CasA. Mutant analyses imply that MAP kinases are involved in the signal transduction in response to the effects caused by FOH.
Molecular Microbiology 12/2010; 78(5):1259-79. · 5.01 Impact Factor
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ABSTRACT: Abstract
Background
Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and ΔAfcrzA mutant strains.
Results
We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the ΔcrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 μM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl<sub>2 </sub>25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.
Conclusion
We have performed a transcriptional profiling analysis of the A. fumigatus ΔAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity. Concomitantly with A. fumigatus AfrcnA molecular analysis, we decided to exploit the conserved features of A. nidulans calcineurin system and investigated the A. nidulans AnRcnA homologue. A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.
BMC Microbiology. 01/2010;
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ABSTRACT: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and DeltaAfcrzA mutant strains.
We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the DeltacrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 microM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.
We have performed a transcriptional profiling analysis of the A. fumigatus DeltaAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity. Concomitantly with A. fumigatus AfrcnA molecular analysis, we decided to exploit the conserved features of A. nidulans calcineurin system and investigated the A. nidulans AnRcnA homologue. A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.
BMC Microbiology 01/2010; 10:12. · 3.04 Impact Factor
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ABSTRACT: Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges.
The results shown here are for an adapted subset of a Saccharomyces cerevisiae gene expression data set with low temporal resolution and poor statistics. The method reveals common transcription factors with a high confidence level and allows the construction of subnetworks with high biological relevance that reveals characteristic features of the processes driving the organism adaptations to specific environmental conditions.
Our method allows a reliable and sophisticated analysis of microarray data even under severe constraints. The utilization of systems biology improves the biologists ability to elucidate the mechanisms underlying cellular processes and to formulate new hypotheses.
BMC Bioinformatics 09/2009; 10:270. · 2.75 Impact Factor
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ABSTRACT: Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory subunit B. A mutant of Aspergillus fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and DeltacalA mutant strains. Our results showed that the mitochondrial copy number is reduced in the DeltacalA mutant strain, and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified four genes that encode transcription factors that have increased mRNA expression in the DeltacalA mutant. Deletion mutants for these transcription factors had reduced susceptibility to itraconazole, caspofungin, and sodium dodecyl sulfate (SDS).
Fungal Genetics and Biology 07/2009; 46(10):791-802. · 3.74 Impact Factor
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ABSTRACT: Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here, we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage.
Fungal Genetics and Biology 06/2009; 46(5):410-7. · 3.74 Impact Factor
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ABSTRACT: Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here, we describe the homologues of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage.
Fungal Genetics and Biology 03/2009; · 3.74 Impact Factor
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ABSTRACT: The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.
Plant physiology 01/2009; 149(3):1211-30. · 6.53 Impact Factor
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ABSTRACT: We show indirect evidences for the possible involvement of NIT2-like binding motifs in transcription modulation of the PbGP43 gene, which codes for an important antigen from the human fungal pathogen Paracoccidioides brasiliensis. This investigation was motivated by the finding of 23 NIT2-like sites within the proximal -2047 nucleotides of the PbGP43 5' intergenic region from the Pb339 isolate. They compose four clusters, two of them identical. We found four NIT2-containing probes that were positive in electrophoretic mobility shift assays and further analyzed them. PbGP43 could be modulated by nitrogen primary sources in Pb339, Pb3 and Pb18 isolates, as observed by reverse transcription (RT) real time-PCR. Gene reporter assays conducted in Aspergillus nidulans suggested that the minimal fragment responsible for nitrogen modulation lies within -480 bp of the PbGP43 gene. This is the first report on PbGP43 transcription modulation in response to nitrogen primary sources, which might help understand its regulation during infection.
Fungal Genetics and Biology 11/2008; 46(1):85-93. · 3.74 Impact Factor
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ABSTRACT: Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The DeltaaifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 microM FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 microM FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.
Molecular Microbiology 09/2008; 70(1):44-59. · 5.01 Impact Factor
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ABSTRACT: Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P(i)) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P(i) at normal or high external P(i) concentrations) and a high-affinity (activated in response to P(i) starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB(PHO80). We show that the DeltaphoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and DeltaphoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the DeltaphoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1mM P(i). Epifluorescence microscopy revealed accumulation of poly-phosphate in DeltaphoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, DeltaphoB(PHO80) and DeltaphoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis.
Fungal Genetics and Biology 08/2008; 45(7):1135-46. · 3.74 Impact Factor
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Natalie D Fedorova,
Nora Khaldi,
Vinita S Joardar,
Rama Maiti,
Paolo Amedeo,
Michael J Anderson,
Jonathan Crabtree,
Joana C Silva,
Jonathan H Badger,
Ahmed Albarraq, [......],
J Craig Venter,
Owen R White,
Brett R Whitty,
Phil Youngman,
Kenneth H Wolfe,
Gustavo H Goldman,
Jennifer R Wortman,
Bo Jiang,
David W Denning,
William C Nierman
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ABSTRACT: We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".
PLoS Genetics 05/2008; 4(4):e1000046. · 8.69 Impact Factor
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ABSTRACT: Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the DeltaddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The DeltaddbA mutation can genetically interact with uvsB (ATR), atmA(ATM), nkuA (KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne's syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP::DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.
Molecular and General Genetics 04/2008; 279(3):239-53. · 2.63 Impact Factor
