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1.  SCI1, the first member of the tissue-specific inhibitors of CDK (TIC) class, is probably connected to the auxin signaling pathway 
Plant Signaling & Behavior  2012;7(1):53-58.
The recent finding of a tissue-specific cell cycle regulator (SCI1) that inhibits cell proliferation/differentiation in the upper pistil points to an unanticipated way of controlling plant morphogenesis. The similarity between the SCI1 RNAi-silenced plants and some auxin-related phenotypes suggested that SCI1 could be involved in the auxin signaling pathway. To address this hypothesis, we analyzed the expression of three auxin-related genes in transgenic plants in which SCI1 was silenced and overexpressed. The results showed that the expression levels of the auxin-related genes largely correlated with the SCI1 expression level. Additionally, we analyzed the Arabidopsis SCI1 upstream regulatory region and found putative cis-acting elements also present in the AtCYCB1;1 AtYUC1, AtYUC2 and AtYUC4 URRs, suggesting a cell cycle- and auxin-related transcriptional regulation. Based on our previous and the current studies, we propose SCI1 as a signal transducer engaging auxin signaling and cell division/differentiation.
doi:10.4161/psb.7.1.18525
PMCID: PMC3357369  PMID: 22301969
auxin signaling; CDK inhibitors; cell cycle; SCI1; stigma/style; TIC class
3.  Protein Kinase C Overexpression Suppresses Calcineurin-Associated Defects in Aspergillus nidulans and Is Involved in Mitochondrial Function 
PLoS ONE  2014;9(8):e104792.
In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca2+ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca2+ and Pkc-mediated pathways has mainly been described in Saccharomyces cerevisiae and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus Aspergillus nidulans. Overexpression of pkcA partially rescues the phenotypes caused by a cnaA deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of pkcA in a cnaA deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an A. nidulans and identifies a novel interaction of both pathways in the regulation of cellular respiration.
doi:10.1371/journal.pone.0104792
PMCID: PMC4143261  PMID: 25153325
4.  The Involvement of the Mid1/Cch1/Yvc1 Calcium Channels in Aspergillus fumigatus Virulence 
PLoS ONE  2014;9(8):e103957.
Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Calcium homeostasis and signaling is essential for numerous biological processes and also influences A. fumigatus pathogenicity. The presented study characterized the function of the A. fumigatus homologues of three Saccharomyces cerevisiae calcium channels, voltage-gated Cch1, stretch-activated Mid1 and vacuolar Yvc1. The A. fumigatus calcium channels cchA, midA and yvcA were regulated at transcriptional level by increased calcium levels. The YvcA::GFP fusion protein localized to the vacuoles. Both ΔcchA and ΔmidA mutant strains showed reduced radial growth rate in nutrient-poor minimal media. Interestingly, this growth defect in the ΔcchA strain was rescued by the exogenous addition of CaCl2. The ΔcchA, ΔmidA, and ΔcchA ΔmidA strains were also sensitive to the oxidative stress inducer, paraquat. Restriction of external Ca2+ through the addition of the Ca2+-chelator EGTA impacted upon the growth of the ΔcchA and ΔmidA strains. All the A. fumigatus ΔcchA, ΔmidA, and ΔyvcA strains demonstrated attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Infection with the parental strain resulted in a 100% mortality rate at 15 days post-infection, while the mortality rate of the ΔcchA, ΔmidA, and ΔyvcA strains after 15 days post-infection was only 25%. Collectively, this investigation strongly indicates that CchA, MidA, and YvcA play a role in A. fumigatus calcium homeostasis and virulence.
doi:10.1371/journal.pone.0103957
PMCID: PMC4118995  PMID: 25083783
5.  Functional characterization of a xylose transporter in Aspergillus nidulans 
Background
The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization.
Results
In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene.
Conclusions
This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.
doi:10.1186/1754-6834-7-46
PMCID: PMC4021826  PMID: 24690493
Aspergillus nidulans; xylose transporters; Saccharomyces cerevisiae; second generation bioethanol
6.  Identification of Glucose Transporters in Aspergillus nidulans 
PLoS ONE  2013;8(11):e81412.
To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.
doi:10.1371/journal.pone.0081412
PMCID: PMC3839997  PMID: 24282591
7.  The Aspergillus nidulans ATM Kinase Regulates Mitochondrial Function, Glucose Uptake and the Carbon Starvation Response 
G3: Genes|Genomes|Genetics  2013;4(1):49-62.
Mitochondria supply cellular energy and also perform a role in the adaptation to metabolic stress. In mammals, the ataxia-telangiectasia mutated (ATM) kinase acts as a redox sensor controlling mitochondrial function. Subsequently, transcriptomic and genetic studies were utilized to elucidate the role played by a fungal ATM homolog during carbon starvation. In Aspergillus nidulans, AtmA was shown to control mitochondrial function and glucose uptake. Carbon starvation responses that are regulated by target of rapamycin (TOR) were shown to be AtmA-dependent, including autophagy and hydrolytic enzyme secretion. AtmA also regulated a p53-like transcription factor, XprG, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Thus, AtmA possibly represents a direct or indirect link between mitochondrial stress, metabolism, and growth through the influence of TOR and XprG function. The coordination of cell growth and division with nutrient availability is crucial for all microorganisms to successfully proliferate in a heterogeneous environment. Mitochondria supply cellular energy but also perform a role in the adaptation to metabolic stress and the cross-talk between prosurvival and prodeath pathways. The present study of Aspergillus nidulans demonstrated that AtmA also controlled mitochondrial mass, function, and oxidative phosphorylation, which directly or indirectly influenced glucose uptake. Carbon starvation responses, including autophagy, shifting metabolism to the glyoxylate cycle, and the secretion of carbon scavenging enzymes were AtmA-dependent. Transcriptomic profiling of the carbon starvation response demonstrated how TOR signaling and the retrograde response, which signals mitochondrial dysfunction, were directly or indirectly influenced by AtmA. The AtmA kinase was also shown to influence a p53-like transcription factor, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Therefore, in response to metabolic stress, AtmA appears to perform a role in the regulation of TOR signaling, involving the retrograde and SnfA pathways. Thus, AtmA may represent a link between mitochondrial function and cell cycle or growth, possibly through the influence of the TOR and XprG function.
doi:10.1534/g3.113.008607
PMCID: PMC3887539  PMID: 24192833
ATM kinase; glucose starvation; cell death; autophagy
8.  Genetic Bypass of Aspergillus nidulans crzA Function in Calcium Homeostasis 
G3: Genes|Genomes|Genetics  2013;3(7):1129-1141.
After dephosphorylation by the phosphatase calcineurin, the fungal transcription factor CrzA enters the nucleus and activates the transcription of genes responsible for calcium homeostasis and many other calcium-regulated activities. A lack of CrzA confers calcium-sensitivity to the filamentous fungus Aspergillus nidulans. To further understand calcium signaling in filamentous fungi and to identify genes that interact genetically with CrzA, we selected for mutations that were able to suppress crzAΔ calcium intolerance and identified three genes. Through genetic mapping, gene sequencing, and mutant rescue, we were able to identify these as cnaB (encoding the calcineurin regulatory subunit), folA (encoding an enzyme involved in folic acid biosynthesis, dihydroneopterin aldolase), and scrC (suppression of crzA-, encoding a hypothetical protein). By using a calcium indicator, Fluo-3, we were able to determine that the wild-type and the suppressor strains were either able to regulate intracellular calcium levels or were able to take up and or store calcium correctly. The increased expression of calcium transporters, pmcA and/or pmcB, in suppressor mutants possibly enabled tolerance to high levels of calcium. Our results suggest that a cnaB suppressor mutation confers calcium tolerance to crzAΔ strains through restoration of calcium homeostasis. These results stress that in A. nidulans there are calcineurin-dependent and CrzA-independent pathways. In addition, it is possible that CrzA is able to contribute to the modulation of folic acid biosynthesis.
doi:10.1534/g3.113.005983
PMCID: PMC3704241  PMID: 23665873
Aspergillus nidulan; extragenic suppression; calcineurin; CrzA; folate biosynthesis
9.  The Genome of Anopheles darlingi, the main neotropical malaria vector 
Marinotti, Osvaldo | Cerqueira, Gustavo C. | de Almeida, Luiz Gonzaga Paula | Ferro, Maria Inês Tiraboschi | Loreto, Elgion Lucio da Silva | Zaha, Arnaldo | Teixeira, Santuza M. R. | Wespiser, Adam R. | Almeida e Silva, Alexandre | Schlindwein, Aline Daiane | Pacheco, Ana Carolina Landim | da Silva, Artur Luiz da Costa | Graveley, Brenton R. | Walenz, Brian P. | Lima, Bruna de Araujo | Ribeiro, Carlos Alexandre Gomes | Nunes-Silva, Carlos Gustavo | de Carvalho, Carlos Roberto | Soares, Célia Maria de Almeida | de Menezes, Claudia Beatriz Afonso | Matiolli, Cleverson | Caffrey, Daniel | Araújo, Demetrius Antonio M. | de Oliveira, Diana Magalhães | Golenbock, Douglas | Grisard, Edmundo Carlos | Fantinatti-Garboggini, Fabiana | de Carvalho, Fabíola Marques | Barcellos, Fernando Gomes | Prosdocimi, Francisco | May, Gemma | de Azevedo Junior, Gilson Martins | Guimarães, Giselle Moura | Goldman, Gustavo Henrique | Padilha, Itácio Q. M. | Batista, Jacqueline da Silva | Ferro, Jesus Aparecido | Ribeiro, José M. C. | Fietto, Juliana Lopes Rangel | Dabbas, Karina Maia | Cerdeira, Louise | Agnez-Lima, Lucymara Fassarella | Brocchi, Marcelo | de Carvalho, Marcos Oliveira | Teixeira, Marcus de Melo | Diniz Maia, Maria de Mascena | Goldman, Maria Helena S. | Cruz Schneider, Maria Paula | Felipe, Maria Sueli Soares | Hungria, Mariangela | Nicolás, Marisa Fabiana | Pereira, Maristela | Montes, Martín Alejandro | Cantão, Maurício E. | Vincentz, Michel | Rafael, Miriam Silva | Silverman, Neal | Stoco, Patrícia Hermes | Souza, Rangel Celso | Vicentini, Renato | Gazzinelli, Ricardo Tostes | Neves, Rogério de Oliveira | Silva, Rosane | Astolfi-Filho, Spartaco | Maciel, Talles Eduardo Ferreira | Ürményi, Turán P. | Tadei, Wanderli Pedro | Camargo, Erney Plessmann | de Vasconcelos, Ana Tereza Ribeiro
Nucleic Acids Research  2013;41(15):7387-7400.
Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.
doi:10.1093/nar/gkt484
PMCID: PMC3753621  PMID: 23761445
10.  Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family 
PLoS ONE  2013;8(4):e60209.
Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.
doi:10.1371/journal.pone.0060209
PMCID: PMC3616161  PMID: 23560078
11.  Functional Characterization of Aspergillus nidulans ypkA, a Homologue of the Mammalian Kinase SGK 
PLoS ONE  2013;8(3):e57630.
The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.
doi:10.1371/journal.pone.0057630
PMCID: PMC3589345  PMID: 23472095
13.  Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis 
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.
doi:10.1186/1472-6882-12-194
PMCID: PMC3598864  PMID: 23092287
14.  Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus 
Eukaryotic Cell  2012;11(4):518-531.
Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.
doi:10.1128/EC.00016-12
PMCID: PMC3318302  PMID: 22345349
15.  Characterization and optimization of ArtinM lectin expression in Escherichia coli 
BMC Biotechnology  2012;12:44.
Background
ArtinM is a d-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system.
Results
The ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized d-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure.
Conclusions
Overall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.
doi:10.1186/1472-6750-12-44
PMCID: PMC3431236  PMID: 22857259
16.  Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection 
PLoS ONE  2012;7(5):e37591.
Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5′-CACAGCCAC-3′ and 5′-CCCTGCCCC-3′ sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis.
doi:10.1371/journal.pone.0037591
PMCID: PMC3359301  PMID: 22649543
17.  Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse 
Background
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).
Results
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.
Conclusions
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.
doi:10.1186/1754-6834-4-40
PMCID: PMC3219568  PMID: 22008461
18.  Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae▿† 
Eukaryotic Cell  2011;10(3):398-411.
Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G (Nuc1p) is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation 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.
doi:10.1128/EC.00256-10
PMCID: PMC3067468  PMID: 21193549
19.  The Aspergillus nidulans nucAEndoG Homologue Is Not Involved in Cell Death ▿ 
Eukaryotic Cell  2011;10(2):276-283.
Upon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if the Aspergillus nidulans homologue of the EndoG gene, named nucAEndoG, is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response in A. nidulans.
doi:10.1128/EC.00224-10
PMCID: PMC3067401  PMID: 21131437
20.  Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA 
BMC Microbiology  2010;10:12.
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 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.
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.
doi:10.1186/1471-2180-10-12
PMCID: PMC2818617  PMID: 20078882
21.  Transcriptome Analysis of Aspergillus nidulans Exposed to Camptothecin-Induced DNA Damage†  
Eukaryotic Cell  2006;5(10):1688-1704.
We have used an Aspergillus nidulans macroarray carrying sequences of 2,787 genes from this fungus to monitor gene expression of both wild-type and uvsBATR (the homologue of the ATR gene) deletion mutant strains in a time course exposure to camptothecin (CPT). The results revealed a total of 1,512 and 1,700 genes in the wild-type and uvsBATR deletion mutant strains that displayed a statistically significant difference at at least one experimental time point. We characterized six genes that have increased mRNA expression in the presence of CPT in the wild-type strain relative to the uvsBATR mutant strain: fhdA (encoding a forkhead-associated domain protein), tprA (encoding a hypothetical protein that contains a tetratrico peptide repeat), mshA (encoding a MutS homologue involved in mismatch repair), phbA (encoding a prohibitin homologue), uvsCRAD51 (the homologue of the RAD51 gene), and cshA (encoding a homologue of the excision repair protein ERCC-6 [Cockayne's syndrome protein]). The induced transcript levels of these genes in the presence of CPT require uvsBATR. These genes were deleted, and surprisingly, only the ΔuvsC mutant strain was sensitive to CPT; however, the others displayed sensitivity to a range of DNA-damaging and oxidative stress agents. These results indicate that the selected genes when inactivated display very complex and heterogeneous sensitivity behavior during growth in the presence of agents that directly or indirectly cause DNA damage. Moreover, with the exception of UvsC, deletion of each of these genes partially suppressed the sensitivity of the ΔuvsB strain to menadione and paraquat. Our results provide the first insight into the overall complexity of the response to DNA damage in filamentous fungi and suggest that multiple pathways may act in parallel to mediate DNA repair.
doi:10.1128/EC.00167-06
PMCID: PMC1595335  PMID: 17030995
22.  Fungal Metabolic Model for Tyrosinemia Type 3: Molecular Characterization of a Gene Encoding a 4-Hydroxy-Phenyl Pyruvate Dioxygenase from Aspergillus nidulans 
Eukaryotic Cell  2006;5(8):1441-1445.
Mutations in the human HPD gene (encoding 4-hydroxyphenylpyruvic acid dioxygenase) cause hereditary tyrosinemia type 3 (HT3). We deleted the Aspergillus nidulans homologue (hpdA). We showed that the mutant strain is not able to grow in the presence of phenylalanine and that it accumulates increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid, mimicking the human HT3 phenotype.
doi:10.1128/EC.00160-06
PMCID: PMC1539140  PMID: 16896227
23.  The akuBKU80 Mutant Deficient for Nonhomologous End Joining Is a Powerful Tool for Analyzing Pathogenicity in Aspergillus fumigatus 
Eukaryotic Cell  2006;5(1):207-211.
To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuBKU80). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuBKU80 mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuBKU80 had no influence on pathogenicity in a low-dose murine infection model.
doi:10.1128/EC.5.1.207-211.2006
PMCID: PMC1360264  PMID: 16400184
24.  Transcriptome Analysis of Paracoccidioides brasiliensis Cells Undergoing Mycelium-to-Yeast Transition 
Eukaryotic Cell  2005;4(12):2115-2128.
Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.
doi:10.1128/EC.4.12.2115-2128.2005
PMCID: PMC1317488  PMID: 16339729
25.  Aspergillus nidulans uvsBATR and scaANBS1 Genes Show Genetic Interactions during Recovery from Replication Stress and DNA Damage 
Eukaryotic Cell  2005;4(7):1239-1252.
The ATM/ATR kinases and the Mre11 (Mre11-Rad50-Nbs1) protein complex are central players in the cellular DNA damage response. Here we characterize possible interactions between Aspergillus nidulans uvsBATR and the Mre11 complex (scaANBS1). We demonstrate that there is an epistatic relationship between uvsBATR, the homolog of the ATR/MEC1 gene, and scaANBS1, the homolog of the NBS1/XRS2 gene, for both repair and checkpoint functions and that correct ScaANBS1 expression during recovery from replication stress depends on uvsBATR. In addition, we also show that the formation of UvsC foci during recovery from replication stress is dependent on both uvsBATR and scaANBS1 function. Furthermore, ScaANBS1 is also dependent on uvsBATR for nuclear focus formation upon the induction of DNA double-strand breaks by phleomycin. Our results highlight the extensive genetic interactions between UvsB and the Mre11 complex that are required for S-phase progression and recovery from DNA damage.
doi:10.1128/EC.4.7.1239-1252.2005
PMCID: PMC1168959  PMID: 16002650

Results 1-25 (29)