Aspergillus e Aspergilose

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Purification of mitochondrial RNase P in A. nidulans

Résumé La ribonucléase P (RNase P) est une ribonucléoprotéine omniprésente dans tous les règnes du vivant, elle est responsable de la maturation en 5’ des précurseurs des ARNs de transfert (ARNts) et quelques autres petits ARNs. L’enzyme est composée d'une sous unité catalytique d'ARN (ARN-P) et d'une ou de plusieurs protéines selon les espèces. Chez les eucaryotes, l’activité de la RNase P cytoplasmique est distincte de celles des organelles (mitochondrie et chloroplaste). Chez la plupart des espèces, les ARN-P sont constituées de plusieurs éléments structuraux secondaires critiques conservés au cours de l’évolution. En revanche, au niveau de la structure, une réduction forte été observé dans la plupart des mtARN-Ps. Le nombre de protéines composant la RNase P est extrêmement variable : une chez les bactéries, environ quatre chez les archéobactéries, et dix chez la forme cytoplasmique des eucaryotes. Cet aspect est peu connu pour les formes mitochondriales. Dans la plupart des cas, l’identification de la mtRNase P est le résultat de longues procédures de purification comprenant plusieurs étapes dans le but de réduire au minimum le nombre de protéines requises pour l’activité (exemple de la levure et A. nidulans). Cela mène régulièrement à la perte de l’activité et de l’intégrité des complexes ribonucléo-protéiques natifs. Dans ce travail, par l’utilisation de la technique de BN-PAGE, nous avons développé une procédure d’enrichissement de l’activité RNase P mitochondriale native, donnant un rendement raisonnable. Les fractions enrichies capables de cette activité enzymatique ont été analysées par LC/MS/MS et les résultats montrent que l’holoenzyme de la RNase P de chacune des fractions contient un nombre de protéines beaucoup plus grand que ce qui était connue. Nous suggérons une liste de protéines (principalement hypothétiques) qui accompagnent l’activité de la RNase P. IV De plus, la question de la localisation de la mtRNase P de A. nidulans a été étudiée, selon nos résultats, la majorité de la mtRNase P est attachée á la membrane interne de la mitochondrie. Sa solubilisation se fait par l’utilisation de différents types de détergent. Ces derniers permettent l’obtention d’un spectre de complexes de la RNase P de différentes tailles.

β-glucosidases from Aspergillus unguis NII 08123: Molecular characterization, properties and applications

Lignocellulosic biomass is probably the best alternative resource for biofuel production and it is composed mainly of cellulose, hemicelluloses and lignin. Cellulose is the most abundant among the three and conversion of cellulose to glucose is catalyzed by the enzyme cellulase. Cellulases are groups of enzymes act synergistically upon cellulose to produce glucose and comprise of endoglucanase, cellobiohydrolase and β-glucosidase. β -glucosidase assumes great importance due to the fact that it is the rate limiting enzyme. Endoglucanases (EG) produces nicks in the cellulose polymer exposing reducing and non reducing ends, cellobiohydrolases (CBH) acts upon the reducing or non reducing ends to liberate cellobiose units, and β - glucosidases (BGL) cleaves the cellobiose to liberate glucose completing the hydrolysis. . β -glucosidases undergo feedback inhibition by their own product- β glucose, and cellobiose which is their substrate. Few filamentous fungi produce glucose tolerant β - glucosidases which can overcome this inhibition by tolerating the product concentration to a particular threshold. The present study had targeted a filamentous fungus producing glucose tolerant β - glucosidase which was identified by morphological as well as molecular method. The fungus showed 99% similarity to Aspergillus unguis strain which comes under the Aspergillus nidulans group where most of the glucose tolerant β -glucosidase belongs. The culture was designated the strain number NII 08123 and was deposited in the NII culture collection at CSIR-NIIST. β -glucosidase multiplicity is a common occurrence in fungal world and in A.unguis this was demonstrated using zymogram analysis. A total 5 extracellular isoforms were detected in fungus and the expression levels of these five isoforms varied based on the carbon source available in the medium. Three of these 5 isoforms were expressed in higher levels as identified by the increased fluorescence (due to larger amounts of MUG breakdown by enzyme action) and was speculated to contribute significantly to the total _- β glucosidase activity. These isoforms were named as BGL 1, BGL3 and BGL 5. Among the three, BGL5 was demonstrated to be the glucose tolerant β -glucosidase and this was a low molecular weight protein. Major fraction was a high molecular weight protein but with lesser tolerance to glucose. BGL 3 was between the two in both activity and glucose tolerance.121 Glucose tolerant .β -glucosidase was purified and characterized and kinetic analysis showed that the glucose inhibition constant (Ki) of the protein is 800mM and Km and Vmax of the enzyme was found to be 4.854 mM and 2.946 mol min-1mg protein-1respectively. The optimumtemperature was 60°C and pH 6.0. The molecular weight of the purified protein was ~10kDa in both SDS as well as Native PAGE indicating that the glucose tolerant BGL is a monomeric protein.The major β -glucosidase, BGL1 had a pH and temperature optima of 5.0 and 60 °C respectively. The apparent molecular weight of the Native protein is 240kDa. The Vmax and Km was 78.8 mol min-1mg protein-1 and 0.326mM respectively. Degenerate primers were designed for glycosyl hydrolase families 1, 3 and 5 and the BGL genes were amplified from genomic DNA of Aspergillus unguis. The sequence analyses performed on the amplicons results confirmed the presence of all the three genes. Amplicon with a size of ~500bp was sequenced and which matched to a GH1 –BGL from Aspergillus oryzae. GH3 degenerate primers producing amplicons were sequenced and the sequences matched to β - glucosidase of GH3 family from Aspergillus nidulans and Aspergillus acculateus. GH5 degenerate primers also gave amplification and sequencing results indicated the presence of GH5 family BGL gene in the Aspergillus unguis genomic DNA.From the partial gene sequencing results, specific as well as degenerate primers were designed for TAIL PCR. Sequencing results of the 1.0 Kb amplicon matched Aspergillus nidulans β -glucosidase gene which belongs to the GH1 family. The sequence mainly covered the N-Terminal region of the matching peptide. All the three BGL proteins ie. BGL1, BGL3 and BGL5 were purified by chromatography an electro elution from Native PAGE gels and were subjected to MALDI-TOF mass spectrometric analysis. The results showed that BGL1 peptide mass matched to . β -glucosidase-I of Aspergillus flavus which is a 92kDa protein with 69% protein coverage. The glucose tolerant β -glucosidase BGL5 mass matched to the catalytic C-terminal domain of β -glucosidase-F from Emericella nidulans, but the protein coverage was very low compared to the size of the Emericella nidulans protein. While comparing the size of BGL5 from Aspergillus unguis, the protein sequence coverage is more than 80%. BGL F is a glycosyl hydrolase family 3 protein.The properties of BGL5 seem to be very unique, in that it is a GH3 β -glucosidase with a very low molecular weight of ~10kDa and at the same time having catalytic activity and glucose 122 tolerance which is as yet un-described in GH β -glucosidases. The occurrence of a fully functional 10kDA protein with glucose tolerant BGL activity has tremendous implications both from the points of understanding the structure function relationships as well as for applications of BGL enzymes. BGL-3 showed similarity to BGL1 of Aspergillus aculateus which was another GH3 β -glucosidase. It may be noted that though PCR could detect GH1, GH3 and GH5 β-glucosidases in the fungus, the major isoforms BGL1 BGL3 and BGL5 were all GH3 family enzymes. This would imply that β-glucosidases belonging to other families may also co-exist in the fungus and the other minor isoforms detected in zymograms may account for them. In biomass hydrolysis, GT-BGL containing BGL enzyme was supplemented to cellulase and the performances of blends were compared with a cocktail where commercial β- glucosidase was supplemented to the biomass hydrolyzing enzyme preparation. The cocktail supplemented with A unguis BGL preparation yielded 555mg/g sugar in 12h compared to the commercial enzyme preparation which gave only 333mg/g in the same period and the maximum sugar yield of 858 mg/g was attained in 36h by the cocktail containing A. unguis BGL. While the commercial enzyme achieved almost similar sugar yield in 24h, there was rapid drop in sugar concentration after that, indicating probably the conversion of glucose back to di-or oligosaccharides by the transglycosylation activity of the BGl in that preparation. Compared this, the A.unguis enzyme containing preparation supported peak yields for longer duration (upto 48h) which is important for biomass conversion to other products since the hydrolysate has to undergo certain unit operations before it goes into the next stage ie – fermentation in any bioprocesses for production of either fuels or chemicals.. Most importantly the Aspergillus unguis BGL preparation yields approximately 1.6 fold increase in the sugar release compared to the commercial BGL within 12h of time interval and 2.25 fold increase in the sugar release compared to the control ie. Cellulase without BGL supplementation. The current study therefore leads to the identification of a potent new isolate producing glucose tolerant β - glucosidase. The organism identified as Aspergillus unguis comes under the Aspergillus nidulans group where most of the GT-BGL producers belong and the detailed studies showed that the glucose tolerant β -glucosidase was a very low molecular weight protein which probably belongs to the glycosyl hydrolase family 3. Inhibition kinetic studies helped to understand the Ki and it is the second highest among the nidulans group of Aspergilli. This has promoted us for a detailed study regarding the mechanism of glucose tolerance. The proteomic 123 analyses clearly indicate the presence of GH3 catalytic domain in the protein. Since the size of the protein is very low and still its active and showed glucose tolerance it is speculated that this could be an entirely new protein or the modification of the existing β -glucosidase with only the catalytic domain present in it. Hydrolysis experiments also qualify this BGL, a suitable candidate for the enzyme cocktail development for biomass hydrolysis

Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-beta-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-beta-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 degrees C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan beta-1,3 or beta-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in beta-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 degrees C and 81.3 degrees C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 degrees C. the enzymatic assays demonstrated that XegA is more active in its monomeric state. (c) 2012 Elsevier B.V. All rights reserved.

The microtubule plus-end localization of Aspergillus dynein is important for dynein-early-endosome interaction but not for dynein ATPase activation

Cytoplasmic dynein in filamentous fungi accumulates at microtubule plus-ends near the hyphal tip, which is important for minus-end-directed transport of early endosomes. It was hypothesized that dynein is switched on at the plus-end by cargo association. Here, we show in Aspergillus nidulans that kinesin-1-dependent plus-end localization is not a prerequisite for dynein ATPase activation. First, the Walker A and Walker B mutations in the dynein heavy chain AAA1 domain implicated in blocking different steps of the ATPase cycle cause different effects on dynein localization to microtubules, arguing against the suggestion that ATPase is inactive before arriving at the plus-end. Second, dynein from kinA (kinesin 1) mutant cells has normal ATPase activity despite the absence of dynein plus-end accumulation. In kinA hyphae, dynein localizes along microtubules and does not colocalize with abnormally accumulated early endosomes at the hyphal tip. This is in contrast to the colocalization of dynein and early endosomes in the absence of NUDF/LIS1. However, the Walker B mutation allows dynein to colocalize with the hyphal-tip-accumulated early endosomes in the kinA background. We suggest that the normal ability of dyenin to interact with microtubules as an active minus-end-directed motor demands kinesin-1-mediated plus-end accumulation for effective interactions with early endosomes.

Aspergillus has distinct fatty acid synthases for primary and secondary metabolism

Aspergillus nidulans contains two functionally distinct fatty acid synthases (FASs): one required for primary fatty acid metabolism (FAS) and the other required for secondary metabolism (sFAS). FAS mutants require long-chain fatty acids for growth, whereas sFAS mutants grow normally but cannot synthesize sterigmatocystin (ST), a carcinogenic secondary metabolite structurally and biosynthetically related to aflatoxin. sFAS mutants regain the ability to synthesize ST when provided with hexanoic acid, supporting the model that the ST polyketide synthase uses this short-chain fatty acid as a starter unit. The characterization of both the polyketide synthase and FAS may provide novel means for modifying secondary metabolites.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Supercomplexes multifonctionnels chez les mitochondries, et chez E. coli

Les processus mitochondriaux tels que la réplication et la traduction sont effectués par des complexes multiprotéiques. Par contre, le métabolisme et la voie de maturation des ARN mitochondriaux (p. ex précurseurs des ARNt et des ARNr) sont habituellement traités comme une suite de réactions catalysées par des protéines séparées. L’exécution fidèle et optimale de ces processus mitochondriaux, exige un couplage étroit nécessaire pour la canalisation des intermédiaires métaboliques. Or, les évidences en faveur de l'interconnexion postulée de ces processus cellulaires sont peu nombreuses et proviennent en grande partie des interactions protéine-protéine. Contrairement à la perception classique, nos résultats révèlent l’organisation des fonctions cellulaires telles que la transcription, la traduction, le métabolisme et la régulation en supercomplexes multifonctionnels stables, dans les mitochondries des champignons (ex Saccharomyces cerevisiae, Aspergillus nidulans et Neurospora crassa), des animaux (ex Bos taurus), des plantes (B. oleracea et Arabidopsis thaliana) et chez les bactéries (ex E. coli) à partir desquelles les mitochondries descendent. La composition de ces supercomplexes chez les champignons et les animaux est comparable à celle de levure, toutefois, chez les plantes et E. coli ils comportent des différences notables (ex, présence des enzymes spécifiques à la voie de biosynthèse des sucres et les léctines chez B. oleracea). Chez la levure, en accord avec les changements dûs à la répression catabolique du glucose, nos résultats révèlent que les supercomplexes sont dynamiques et que leur composition en protéines dépend des stimulis et de la régulation cellulaire. De plus, nous montrons que l’inactivation de la voie de biosynthèse des lipides de type II (FASII) perturbe l’assemblage et/ou la biogenèse du supercomplexe de la RNase P (responsable de la maturation en 5’ des précurseurs des ARNt), ce qui suggère que de multiples effets pléiotropiques peuvent être de nature structurale entre les protéines. Chez la levure et chez E. coli, nos études de la maturation in vitro des précurseurs des ARNt et de la protéomique révèlent l’association de la RNase P avec les enzymes de la maturation d’ARNt en 3’. En effet, la voie de maturation des pré-ARNt et des ARNr, et la dégradation des ARN mitochondriaux semblent êtres associées avec la machinerie de la traduction au sein d’un même supercomplexe multifonctionnel dans la mitochondrie de la levure. Chez E. coli, nous avons caractérisé un supercomplexe similaire qui inclut en plus de la RNase P: la PNPase, le complexe du RNA degradosome, l’ARN polymérase, quatre facteurs de transcription, neuf aminoacyl-tRNA synthétases, onze protéines ribosomiques, des chaperons et certaines protéines métaboliques. Ces résultats supposent l’association physique de la transcription, la voie de maturation et d’aminoacylation des ARNt, la dégradation des ARN. Le nombre de cas où les activités cellulaires sont fonctionnellement et structurellement associées est certainement à la hausse (ex, l’éditosome et le complexe de la glycolyse). En effet, l’organisation en supercomplexe multifonctionnel représente probablement l’unité fonctionnelle dans les cellules et les analyses de ces super-structures peuvent devenir la prochaine cible de la biologie structurale.

La RNase P mitochondriale chez Neurospora crassa

Résumé La Ribonucléase P (RNase P) est une enzyme principalement reconnue pour sa participation à la maturation en 5’des ARN de transfert (ARNt). Cependant, d’autres substrats sont reconnus par l’enzyme. En général, la RNase P est composée d’une sous-unité ARN (le P-ARN, codé par le gène rnpB) qui porte le centre actif de l’enzyme et d’une ou de plusieurs sous-unités protéiques (la P-protéine). Les P-ARN chez toutes les bactéries, la majorité des archéobactéries et dans le génome nucléaire de la plupart des eucaryotes, possèdent généralement une structure secondaire très conservée qui inclut le noyau (P1-P4); l’hélice P4 constitue le site catalytique de l’enzyme et l’hélice P1 apparie les extrémités du P-ARN en stabilisant sa structure globale. Les P-ARN mitochondriaux sont souvent moins conservés et difficiles à découvrir. Dans certains cas, les seules régions de structure primaire qui restent conservées sont celles qui définissent le P4 et le P1. Pour la détection des gènes rnpB, un outil de recherche bioinformatique, basé sur la séquence et le profil de structure secondaire, a été développé dans le laboratoire. Cet outil permet le dépistage de toutes les séquences eucaryotes (nucléaires et mitochondriales) du gène avec une très grande confiance (basée sur une valeur statistique, E-value). Chez les champignons, plusieurs ascomycètes encodent un gène rnpB dans leur génome mitochondrial y compris tous les membres du genre d’Aspergillus. Cependant, chez les espèces voisines, Neurospora crassa, Podospora anserina et Sordaria macrospora, une version mitochondriale de ce gène n’existe pas. Au lieu de cela, elles contiennent deux copies nucléaires du gène, légèrement différentes en taille et en contenu nucléotidique. Mon projet a été établi dans le but d’éclaircir l’évolution de la RNase P mitochondriale (mtRNase P) chez ces trois espèces voisines d’Aspergillus. En ce qui concerne les résultats, des modèles de structures secondaires pour les transcrits de ces gènes ont été construits en se basant sur la structure consensus universelle de la sous-unité ARN de la RNase P. Pour les trois espèces, par la comparaison de ces modèles, nous avons établi que les deux copies nucléaires du gène rnpB sont assez distinctes en séquence et en structure pour pouvoir y penser à une spécialisation de fonction de la RNase P. Chez N. crassa, les deux P-ARN sont modifiés probablement par une coiffe et les extrémités 5’, 3’ sont conformes à nos modèles, ayant un P1 allongé. Encore chez N. crassa, nous avons constaté que les deux copies sont transcrites au même niveau dans le cytoplasme et que la plus petite et la plus stable d’entre elles (Nc1) se retrouve dans l’extrait matriciel mitochondrial. Lors du suivi du P-ARN dans diverses sous-fractions provenant de la matrice mitochondriale soluble, Nc1 est associée avec l’activité de la RNase P. La caractérisation du complexe protéique, isolé à partir de la fraction active sur un gel non dénaturant, révèle qu’il contient au moins 87 protéines, 73 d’entre elles ayant déjà une localisation mitochondriale connue. Comme chez la levure, les protéines de ce complexe sont impliquées dans plusieurs fonctions cellulaires comme le processing de l’ADN/ARN, le métabolisme, dans la traduction et d’autres (par exemple : la protéolyse et le repliement des protéines, ainsi que la maintenance du génome mitochondrial). Pour trois protéines, leur fonction est non déterminée.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

Desenvolvimento de um sistema de expressão em Metarhizium anisopliae baseado no promotor homólogo do gene tef-1 α

Vetores de expressão são ferramentas moleculares úteis para investigar a função de genes tanto em sistemas procarióticos quanto eucarióticos. O fungo Metarhizium anisopliae, utilizado no controle biológico de artrópodes, é bem caracterizado em nível molecular. Genes candidatos a participar do processo de infecção de hospedeiros tem sido isolados utilizando estratégias em que o gene candidato é predefinido (enzimas hidrolíticas, por exemplo) ou estratégias mais globais como projetos de ESTs e a análise de bibliotecas de subtração. A superexpressão tem sido o método adotado para verificar a participação de genes isolados no processo de infecção. Esta estratégia tem sido baseada no promoter heterólogo PgpdA de Aspergillus nidulans. Neste trabalho, o gene que codifica o fator de alongamento de tradução tef-1 α de Metarhizium anisopliae foi clonado e a sua região promotora foi localizada e utilizada na construção de um vetor de expressão. Somente uma cópia do gene tef-1α está presente no genoma de M. anisopliae e o seu perfil de expressão foi analisado. Uma árvore filogenética foi construída baseada nos ortógos de tef-1 α e mostrou uma alta correlação com o fungo Cordyceps taii. A região de 639 pb à montante do codon de iniciação (ATG) foi utilizada com sucesso para a expressão do gene repórter sGFP e do gene bar, que confere resistência ao glifosinato de amônio, em M. anisopliae. Os transformantes construídos não apresentaram alteração na sua virulência em bioensaios com carrapatos, em relação a linhagem receptora. Além disso, demonstramos que o nível de expressão permite a detecção óptica da fluorescência de sGFP durante a infecção dos carrapatos. Desta forma, o vetor desenvolvido será uma ferramenta útil para a superexpressão em Metarhizium.

Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da hidrólise do bagaço de cana-de-açúcar por fungos filamentosos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)