Aspergillus fumigatus ist ein opportunistisches fungales Humanpathogen, das ein breites Erkrankungsspektrum von der invasiven Aspergillose (IA) in immunkompromittierten Patienten bis zu einer Reihe von Hypersensitivitätserkrankungen in immunkompetenten Individuen hervorrufen kann. Die Diagnostik für A. fumigatus assoziierte Krankheitsbilder beruht auf mehreren diagnostischen Tests, die auch in ihrer Kombination oft zu späten und unzuverlässigen Diagnosen führen, was wiederum zu einer suboptimalen Patientenversorgung, erhöhter Mortalität und gesteigerten Kosten für das Gesundheitssystem führt. Es besteht daher die unbedingte Notwendigkeit, neue und bessere diagnostische Tests zur Detektion von A. fumigatus zu entwickeln. T Zell Assays sind vielversprechende, innovative diagnostische Tests, die bereits für andere Infektionskrankheiten in der Routinediagnostik eingesetzt werden. Erste Versuche wurden bereits unternommen, diese Assays auch für A. fumigatus assoziierte Erkrankungen einzusetzen. Die gängigsten, auf mononukleären Zellen des peripheren Blutes (PBMC)-basierten T Zell Assays sind der Enzyme-linked Immunosorbent Assay (ELISA), Enzyme-linked Immuno Spot Assay (ELISPOT) und die Durchflusszytometrie. Das Ziel dieser Dissertation war die Entwicklung eines klinisch einsetzbaren T-Zell-Assays für A. fumigatus assoziierte Erkrankungen. Die in der Literatur beschriebenen Assays zeigten in unseren Experimenten bei der Anwendung für mykologische Fragestellungen eine hohe Suszeptibilität gegenüber bereits kurzen präanalytischen Lagerzeiten und Krykonservierung, was einen klinischen Einsatz erschwerte. Wir entwickelten deshalb einen Vollblut basierten ELISA (VB-ELISA) mit dualer Kostimulation (α-CD28 und α-CD49d), hoher Reproduzierbarkeit und verbesserter Robustheit gegenüber präanalytischen Einflussfaktoren. Der VB ELISA konnte hohe Differenzen zwischen Typ 1 T Helferzellen (Th1) , Th2 und Th17 Zytokinkonzentrationen bei Patienten mit Aspergillus assoziierten Hypersensitivitätskrankheitsbildern und Kontrollpatienten feststellen. Um zu testen, ob dieser Anstieg auf die Erkrankung zurückzuführen ist oder auch bei hoher Aspergillus-Umweltexposition vorzufinden ist, wurde der Assay in Aspergillus exponierten gesunden ökologischen Landwirten getestet. In dieser Gruppe fanden wir ebenfalls eine erhöhte Th1 und Th2 Expansion und Zytokinsekretion gegenüber gesunden Kontrollspendern, jedoch wurde nur ein geringer Anstieg des Th17 Signalzytokines IL-17 detektiert. Die Detektion von IL-17 im VB-ELISA in Kombination mit anderen Zytokinmarkern ist daher ein vielversprechender Biomarker für die Diagnose von A. fumigatus assoziierten Hypersensitivitätserkrankungen. Neben diesen Hypersensitivitätserkrankungen haben wir den VB-ELISA auch in immunkompromittierten Patienten nach allogener Stammzelltransplantation (alloSZT), einer Hochrisikogruppe für die IA und die durch das humane Cytomegalovirus (HCMV) ausgelöste Zytomegalie, evaluiert. Während in unserer monozentrischen Pilotstudie aufgrund der geringen Inzidenz keine Evaluation an IA-Patienten erfolgen konnte, wurde mittels VB-ELISA eine hohe Konkordanz der HCMV-spezifischen T Zell Antwort mit der HCMV Serologie sowie eine vergleichbare Leistung zum ELISPOT, dem am häufigsten eingestetzen Assay für diese Fragestellung, festgestellt. Zusammenfassend haben wir mit dem VB ELISA einen vielversprechenden und breitflächig im Spektrum A. fumigatus assoziierter Erkrankungen einsetzbaren T Zell Assay entwickelt, der in der Zukunft in großen Studien mit klar definierten Patientenkohorten getestet werden sollte. Auf Grund von Daten aus Folgestudien, die auf dieser Arbeit basieren, ist des Weiteren davon auszugehen, dass der VB-ELISA auf Grund seiner Stärken potenziell in einer Vielzahl von Anwendungsgebieten und Pathogenen (eine Folgestudie mit SARS-CoV-2 wurde vor kurzem veröffentlicht) universell eingesetzt werden kann. Neben der Immundiagnostik für diverse Infektionserkrankungen könnte der Assay außerdem für T Zell Antworten auf Vakzinierungen und Immuntherapien, in vivo Experimente und in vitro Toxizitätstests verwendet werden. / Aspergillus fumigatus is an opportunistic human pathogen, which is the cause of a wide disease spectrum. The spectrum ranges from invasive Aspergillosis (IA) in immunocompromised patients to diverse hypersensitivity diseases in immunocompetent individuals. Diagnostic assays of A. fumigatus have to be combined for efficient detection and still lead to unreliable and late diagnosis, resulting in suboptimal patient care, increased mortality and public health costs. There is, therefore, a great need to develop novel diagnostics for the detection of A. fumigatus. T-cell assays are promising, innovative diagnostic assays, which are used in routine diagnostics for certain infectious diseases. First affords have been made to adapt T-cell assays for the diagnosis of A. fumigatus-associated diseases. The most common T-cell assays are based on the isolation and stimulation of peripheral blood mononuclear cell (PBMC) and relay on Enzyme-linked Immunosorbent Assay (ELISA), Enzyme-Linked Immuno Spot Assay (ELISPOT) and flow cytometry as their read-out platforms. The aim of this dissertation was to develop a clinically feasible T-cell assay for A. fumigatus-associated diseases. We were able to demonstrate that all of these assays have high susceptibility towards pre-analytic factors like cryopreservation and shortly extended pre-analytic blood storage periods, hampering clinical feasibility. Thus, we developed a whole blood based ELISA (WB-ELISA) with dual co-stimulation (α-CD28 and α-CD49d), which showed high reproducibility, increased robustness towards pre-analytic factors and increased cytokine read-outs. The WB-ELISA was able to quantify large differences of T helper cell 1 (Th1), Th2 and Th17 cytokine concentrations in patients suffering from Aspergillus-associated hypersensitivity diseases compared to healthy controls. To analyze, whether these increased cytokine concentrations were the result of the pathology or could also be found in heavily Aspergillus-exposed individuals, we examined cytokine concentration in heavily Aspergillus-exposed organic farmers. We quantified increased Th1 and Th2 cytokine concentrations, however, we only found a minimal increase in the Th17 signal cytokine IL-17. Interleukin (IL)-17 (most likely in combination with other cytokines) is therefore a promising potential biomarker for the diagnosis of Aspergillus-associated hypersensitivity diseases. In addition to Aspergillus-associated hypersensitivity diseases, we tested the feasibility of the WB ELISA in immunocompromised patients after allogeneic stem cell transplantation (alloSCT). These patients are at high-risk for infections like IA as well as cytomegalovirus (CMV) disease, which is caused by CMV. Although, IA-specific evaluation could not be conducted, due to the low IA-incidence in these patients, the WB-ELISA showed high concordance of HCMV-specific T-cell responses with HCMV-serology as well as comparable performance to the ELISPOT, a commonly used T-cell assay for HCMV, in alloSCT patients. In conclusion, the successful development of the WB-ELISA has led to a promising and widely applicable T-cell assay for Aspergillus-associated diseases. In the future, the WB-ELISA should be evaluated in larger, multi-centric studies in well-defined patient cohorts suffering from Aspergillus-associated diseases. Furthermore, the WB-ELISA might be useful for a wide range of areas and pathogens (a follow-up study in COVID-19 patients was recently published). Besides the use in the immune diagnostics of infectious diseases, the WB-ELISA might also be applicable in the quantification of T-cell responses in vaccination- and immune therapy studies, in-vivo experiments and in-vitro toxicity testing.

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus during infection. This siderophore includes N5-hydroxylated L-ornithine in the peptide backbone that serve as iron chelators. Af SidA is the L-ornithine N5-hydroxylase, which performs the first enzymatic step in the biosynthesis of ferrichrome. In this study, Af SidA was recombinantly expressed and purified as a soluble tetramer with a bound FAD cofactor. The enzyme demonstrated typical Michaelis-Menten kinetics in a product formation assay with respect to L-ornithine, but similar experiments as a function NADH and NADPH indicated inhibition at high coenzyme concentrations. Af SidA is highly specific for its substrate; however, it is promiscuous with respect to its coenzyme requirement. A multi-functional role of NADPH is observed since NADP+ is a competitive inhibitor with respect to NADPH and steady-state kinetic experiments indicate that Af SidA forms a ternary complex with NADP+ and L-ornithine for catalysis. Furthermore, in the absence of substrate, Af SidA forms a stable C4a-(hydro)peroxyflavin intermediate that is stable on the second time scale. Af SidA is also inhibited by several halides and the arginine-reactive reagent, phenylglyoxal. Biochemical comparison of Af SidA to other flavin-containing monooxygenases reveal that Af SidA likely proceeds by a sequential-ordered mechanism. / Master of Science in Life Sciences

La première partie de ce travail se centre sur la recherche de tests sanguins (reposant sur les granulocytes) de dépistage, de suivi et d’efficacité thérapeutique dans différentes maladies allergiques. Elle débute par un travail réalisé dans la maladie asthmatique. Elle se poursuit par l’étude du rôle des granulocytes (polynucléaires neutrophiles et/ou polynucléaires éosinophiles et/ou basophiles) dans deux maladies allergiques : l’oesophagite à éosinophiles et les allergies alimentaires. Chez des patients atteints d’oesophagite à éosinophiles, nous avons étudier le profil d’activation des éosinophiles sanguins et de l’œsophage. Nous nous sommes focalisés sur certains marqueurs d’activation de surface (CD66b) et sur certains phosphoépitopes d’intérêt (Ph-STAT1 et Ph-STAT6). Nous avons démontré que les éosinophiles sanguins avaient un profil spécifique dans cette maladie. Enfin, cette première partie s’achève par deux études portant sur les basophiles sanguins dans les allergies alimentaires, plus précisément, dans les allergies aux fruits à coques ; Nous avons développé de nouveaux potentiels tests de dépistage de patients atteints d’allergies alimentaires, d’identification des allergènes responsables ainsi que de potentiels marqueurs d’évaluation d’efficacité de nouvelles thérapeutiques dans les allergies alimentaires par l’utilisation des marqueurs d’activation de surface des basophiles.La deuxième partie se centre principalement sur le rôle des polynucléaires neutrophiles (PNN) du sang et des voies aériennes des patients atteints de mucoviscidose. La mucoviscidose est la maladie génétique autosomique récessive la plus fréquente dans l’Europe du nord. Son pronostic est étroitement lié à l’atteinte pulmonaire, qui se caractérise par des infections à répétition, une obstruction et une inflammation à PNN. Notre équipe a tout d’abord démontré que les PNN sanguins de patients atteints de mucoviscidose présentaient un déficit en glutathion. Nous avons donc réaliser un essai clinique de phase IIa ou, N-acétyl-cystéine (précurseur du glutathion) était donné, à forte dose et par voie orale à des patients atteints de mucoviscidose. Ce traitement par N-acétyl-cystéine pendant douze semaines a permis une correction du déficit des PNN sanguins en glutathion. Cette correction de l’excès de stress oxydatif au sein des PNN a permis une diminution significative du nombre d’exacerbation chez les patients atteints de mucoviscidose. En revanche, possiblement en raison de la courte durée de ce traitement, aucune différence significative ne fut observée au niveau des paramètres de fonction respiratoire, tels que le VEMS. De plus, nous avons démontré que les PNN des voies aériennes des patients atteints de mucoviscidose dysfonctionnaient. En effet, alors qu’il était rapporté dans la littérature que les PNN nécrotiques des voies aériennes larguaient de manière passive l’élastase et la myélopéroxidase, nous avons démontré que les PNN vivants larguaient de manière active ces deux enzymes, dont la présence est en étroite corrélation avec le déclin de la fonction pulmonaire.. Nous avons alors émis l’hypothèse que les PNN, lors de leur migration du sang vers les voies aériennes, s’activaient anormalement. Nous avons donc décider d’étudier leurs cascade d’activations intracellulaire. Nous avons ainsi démontré que les PNN des voies aériennes des patients atteints de mucoviscidose présentaient une régulation positive de la voie mTOR. mTOR étaient possiblement activé par la pléthore d’acides aminés présents dans le poumon mucoviscidosique. mTOR pourrait refléter une possible survie prolongée des PNN des voies aériennes du poumon mucoviscidosique. Ces PNN pourraient aussi secréter de nouvelles protéines, alors même qu’ils sont « conventionnellement » définis comme cellules terminallement différenciées. [...] / Summary of the first part. We hypothesized that granulocytes were not only playing an effector role in atopic diseases, but also a regulatory role. Furthermore, we proposed that granulocytes, due to their rapid activation response, could be used in rapid non-invasive whole blood assays for Allergic Asthma (AA), Food Allergy (FA) and Eosinophillic Esophagitis (EoE), three allergic diseases. We first studied asthma. Then, we explored the profil of activation of blood eosinophils in patients with EoE. We explored some activation surface markers (CD66b) and some intracellular phosphoepitopes of interest (Ph-STAT1 and Ph-STAT6). We then focused our attention on blood basophils in food allergy. We developped a potential blood basophil assay (based on two basophil activation surface markers, CD203c and CD63), which could discriminate a patient with food allergy, which could also identify the offending allergen and, which could monitor the effect of new therapy.Summary of the second part. We focused our attention on the role of the blood and sputum neutrophils in cystic fibrosis (CF). Cystic fibrosis is the most frequent disease in Caucasians. While CF affects all exocrine organs throughout the body, its lung manifestation represents the main cause of morbidity and mortality. We first discovered that blood neutrophils were deficient in glutathion. We therefore started a clinical phase IIa, where N-acetyl-cystein were given orally in high dose to patients with CF for twelve weeks. Thanks to this regimen, the deficit in glutathion in blood PNN disappeared. The number of exacerbations significantly decreased, however, no positive effect were observerd on the lung function. Furthermore, we demonstrated that profound functional and signaling changes readily occur within viable PNN recruited to CF airways, compared to their blood counterparts. For a long time, neutrophil dysfunction in CF airways has been equated with necrosis and passive release of elastase, DNA and, actin. However, we established recently by direct ex vivo analysis of airway neutrophils from CF patients that a large fraction of these cells are viable and appear to actively release these enzymes-containing granules. We also show that neutrophils that entered CF airways have increased phosphorylation of key effectors in the amino acid-regulated mammalian target of rapamycin (mTOR) pathway. An upregulation of the mTOR pathway might reflect an increase of the survival of the neutrophils in the airways. Another common view of peripheral neutrophils is that of terminally differentiated population, with little if any ability to become anabolic. However, we outlined the ability of human neutrophils to modify their transcriptional profile upon migration to the lung in CF. The last part of these thesis is a combination of knowledge that we acquired on the blood basophils in food allergy and on the neutrophils from the airways of patients with CF. We are currently trying to develop an unmet need blood (basophil) test which could discrimate the CF patients with allergic bronchopulmonary aspergillosis. We are also trying to understand the role of airways neutrophils and eosinophils in the pathogenese of these disease.

Due au champignon Aspergillus fumigatus, l’aspergillose pulmonaire invasive représente une grave menace pour les individus souffrant d’immunodépression sévère. En parallèle d’un diagnostic manquant de spécificité, les traitements actuels présentent une forte toxicité. Ces travaux se sont dans un premier temps intéressés au développement d’anticorps thérapeutiques dirigés contre les protéines pariétales Chitin ring formation du champignon. Le ciblage de ces protéines impliquées dans la croissance fongique a permis la mise en évidence d’effets modérés in vitro, et ont induit, in vivo, un recrutement significatif de cellules immunitaires impliquées dans la défense anti-aspergillaire. Dans un second temps, ces travaux se sont intéressés au ciblage du microenvironnement et de la réponse de l’hôte au cours de l’aspergillose, afin de mieux comprendre les processus physiopathologiques induits au cours de la maladie, et de permettre l’identification de nouveaux biomarqueurs et cibles thérapeutiques. L’utilisation de la spectrométrie de masse iTRAQ®, chez des rats et des manchots, a permis la mise en évidence de plusieurs voies de signalisation surreprésentées. Ces travaux se sont également intéressés à la caractérisation immunologique d’un modèle rat d’API. En plus de la mise en évidence des effets du champignon sur le recrutement de certaines populations de cellules immunitaires, l’utilisation de l’iTRAQ® a permis la mise en évidence de la surexpression de l’interleukine-33 et de son récepteur ST2 au cours de la maladie. Ces travaux ouvrent d’intéressantes perspectives dans la mise en place de nouveaux traitements contre l’API. / Caused by the fungus Aspergillus fumigatus, invasive pulmonary aspergillosis is a serious threat for individuals suffering from severe immunosuppression. In parallel of a diagnosis lacking specificity, current treatments present a high toxicity. This work first focused on the development of therapeutic antibodies directed against cell wall proteins Chitin ring formation of the fungus. Targeting of these proteins involved in fungal growth highlighted moderate effects in vitro, and induced, in vivo, a significant recruitment of immune cells involved in anti-aspergillary defense. In a second time, this work focused on targeting the microenvironment and the host response during aspergillosis, in order to better understand pathophysiological processes induced during the disease, and allow the identification of new biomarkers and therapeutic targets. Use of iTRAQ® mass spectrometry in rat and penguins allowed the identification of several overrepresented signaling pathways. This work also focused on the immune characterization of a rat model of IPA. In addition of highlighting the effects of the fungus in the recruitment of some immune cell populations, use of iTRAQ® exhibited an overexpression of interleukin-33 and its receptor ST2 during the disease. Overall, this work is bringing interesting insights in the establishment of new treatments against IPA.

Peroxirredoxinas (Prxs) são peroxidases muito eficientes que dependem de uma tríade catalítica composta por uma Thr (ou Ser em alguns casos), uma Cys e uma Arg para decompor peróxidos. Elas podem ser classificadas em 1-Cys Prx e 2-Cys Prx, de acordo com o número de Cys envolvidas na catálise, ou de acordo com características estruturais que dividem as Prxs em 6 subfamílias. A grande maioria das enzimas da subfamília Prx6 é composta por 1-Cys Prx. As Prx6 são ainda pouco caracterizadas e a identidade biológica de seu (s) redutor (es) ainda é controversa. Alguns dos redutores candidatos são a Trx e o ascorbato. As Prx6 de mamíferos também possuem atividade de fosfolipase do tipo A2 (PLA2), que depende de uma tríade catalítica composta por uma His, uma Ser e um Asp. Nessa tese, investigamos aspectos bioquímicos e estruturais de duas enzimas da subfamília Prx6 de Aspergillus fumigatus: a AfPrx1 citossólica e a AfPrxC mitocondrial. A. fumigatus é o mais importante fungo patogênico transmitido pelo ar. Inicialmente, caracterizamos as cinéticas de oxidação de AfPrx1 e AfPrxC por H2O2, t-BOOH, CuOOH, LAOOH e ONOO-, monitorando alterações redox-dependentes das fluorescência intrínseca dessas proteínas. Adicionalmente, avaliamos as reduções de AfPrx1 e AfPrxC por Trx, Grx, ascorbato, ergotioneína, GSH e H2S. Apenas H2S reduziu eficientemente AfPrx1 e AfPrxC (κAfPrx1 ≈ 103 M-1 s-1 e κAfPrxC ≈ 104 M-1 s-1). Além da atividade peroxidásica, utilizamos lipossomos radioativos para caracterizar pela primeira vez atividade fosfolipásica (PLA2) para uma Prx de não-mamífero que AfPrx1 e AfPrxC possuem. Esta atividade (≈ 200 nmol/ h/ mg de proteína) pode ser inibida por MJ33 (aproximadamente 75 %) e aumentada pela fosforilação através de MAPK. Adicionalmente, estas proteínas estão envolvidas na sobrevivência do fungo durante interação com macrófagos. Utilizando microeletrodo, pudemos verificar que AfPrx1 é importante para detoxificar o fungo de H2O2 exógeno. A caracterização bioquímica destas atividades catalíticas das Prxs pode abrir novas perspectivas para tratamentos, já que pelo menos AfPrx1 está envolvida na virulência de A. fumigatus em ensaios com camundongos / Peroxiredoxins (Prxs) are highly efficient peroxidases that depend on a catalytic triad composed of Thr/Ser, Cys and Arg residues. These enzymes can be classified as 2-Cys Prx and 1-Cys Prx, according to the number of Cys residues involved in catalysis or according to structural characteristics that divide the Prxs in 6 subfamilies. The Prx6 subfamily is almost exclusively composed by 1-Cys Prx enzymes. This subfamily is still poorly characterized and the identities of their biological reductants are still controversial. Some of the reductant candidates are thioredoxin (Trx) and ascorbate. The mammalian members of this subfamily possess an additional phospholipase A2 (PLA2) activity that relies on another catalytic triad that is composed by His, Ser and Asp residues. In this thesis, we investigated biochemical and structural aspects of two enzymes belonging to the Prx6 subfamily from Aspergillus fumigatus: the cytosolic AfPrx1 and the mitochondrial AfPrxC. A. fumigatus is the most important pathogenic fungus transmitted through the air. Initially, we characterized the oxidation of AfPrx1 and AfPrxC by H2O2, t-BOOH, CuOOH, LAOOH and ONOO-, monitoring redox dependent changes in the intrinsic fluorescence of these enzymes. Additionally, we characterized the reduction of AfPrx1 and AfPrxC by Trx, Grx, ascorbate, ergothioneine, GSH and H2S. Interestingly, only H2S reduced these proteins efficiently κAfPrx1 ≈ 103 M-1 s-1 and κk;AfPrxC ≈ 104 M-1 s-1). In addition to the peroxidase activity, we determined for the first time the phospholipase activity (PLA2) for a non-mammalian Prx, using liposomes radioactive-labeled. AfPrx1 and AfPrxC displayed PLA2 activity (≈ 200 nmol/ h/ mg of protein) that was inhibited by MJ33 (about 75 %) and enhanced after phosphorylation by MAPK. Moreover, we also showed that these proteins were important for fungus survival during studies co-cultures with macrophages. Furthermore, AfPrx1 was important to detoxify A. fumigatus from exogenous added H2O2 as observed through an electrochemical approach. The biochemical and structural characterization of these Prxs described herein can open new therapeutic strategies, since at least AfPrx1 is involved in fungus virulence in a mouse model

A agroindústria da cana-de-açúcar no Brasil foi um dos setores que mais se desenvolveu nas últimas 4 décadas. Do caminho trilhado desde os canaviais até as bombas de combustível, cerca de 30% da massa de cana-de-açúcar utilizada para a produção de etanol é perdida na forma de palha e bagaço. O resíduo de natureza lignocelulósica, apresenta composição aproximada de 32-48% de celulose, 23-27% de hemicelulose e 19-32% de lignina. Em virtude da resistência física e mecânica oferecida por esse tipo de material, diversas metodologias como pré-tratamentos químicos ou enzimáticos têm sido propostos visando tornar mais acessível a fração polissacarídica do bagaço à quebra em açúcares fermentescíveis passíveis a conversão em etanol de segunda geração. Nesse contexto, o fungo Aspergillus fumigatus apesar de patogênico, apresenta enorme relevância no cenário da desconstrução de resíduos de origem lignocelulósica como o bagaço. Com a recém descoberta das chamadas mono-oxigenases líticas de polissacarídeos (LPMOs), metaloenzimas auxiliares à degradação de compostos lignocelulósicos que apresentam notoriedade por conta de seus mecanismos de ação oxidorredutivos, as mesmas já foram encontradas em diversos tipos de microorganismos, incluindo o próprio A. fumigatus. Nesse sentido, o projeto em questão realizou a clonagem e expressão heteróloga na cepa E. coli Rosetta(TM)(DE3)pLysS dos genes AFUA_1G12560 e AFUA_4G07850 responsáveis por codificar respectivamente as enzimas AfuLPMO9A e AfuLPMO9C de A. fumigatus Af293 identificadas em análises de secretoma e RNA-Seq previamente obtidos pelo nosso grupo de pesquisa. Através da análise desses dois genes, foi verificado que na presença de diferentes fontes de carbono, ambos foram induzidos, de modo que na presença de SEB, CMC e Avicel, o gene AFUA_4G07850 foi em até 3500X, 2000X e 1000X mais induzido, enquanto que o AFUA_1G12560 apresentou um aumento moderado de 16X, 13X e 7X, respectivamente. A avaliação da conformação tridimensional, bem como o alinhamento múltiplo com enzimas de maior grau de homologia revelaram algumas características estruturais importantes como a presença de núcleos ricos em estruturas ? em conformação de sanduíche e que importantes resíduos conservados como H20; H105; Y194 e H22; H107; Y196 encontravam-se coordenados ao centro metálico das enzimas AfuLPMO9A e AfuLPMO9C, respectivamente. Uma análise mais profunda baseada em DFT revelou que o sítio ativo de ambas assume uma geometria piramidal de base quadrada. Após purificação das LPMOs, ensaios de atividade revelaram que as enzimas AfuLPMO9A e AfuLPMO9C aumentaram respectivamente em até 1,26X e 1,20X a atividade do coquetel enzimático quando nas concentrações de 10 mg/g Avicel e presença de 0,02% NaN3. Análises mais profundas dessas enzimas auxiliares vão permitir uma maior compreensão sobre o papel desempenhado por elas na desconstrução do bagaço de cana-de-açúcar. / The sugarcane agroindustry in Brazil was one of the most developed sectors in the last 4 decades. In the path from cane fields to the gas stations, about 30% of the sugarcane mass used for ethanol production is lost in form of straw and bagasse. The lignocellulosic residue presents approximately 32-48% cellulose, 23-27% hemicellulose and 19-32% lignin. Due to the physical and mechanical resistance offered by this type of material, several methodologies such as chemical or enzymatic pre-treatments have been proposed to make the polysaccharide fraction of the bagasse accessible to the break in fermentable sugars that can be converted into second generation ethanol. In this context, the fungus Aspergillus fumigatus, despite being pathogenic, presents enormous relevance in the scenario of deconstruction of lignocellulosic residues such as bagasse. With the recently discovery of the so-called polysaccharide lytic monooxygenases (LPMOs), metalloenzymes that help the degradation of lignocellulosic compounds and are notorious for their oxidoreductive mechanisms of action, they have already been found in several types of microorganisms, including A. fumigatus. In this regard, this project performed the heterologous cloning and expression in E. coli Rosetta(TM)(DE3)pLysS of the AFUA_1G12560 and AFUA_4G07850 genes responsible for respectively encoding the A. fumigatus Af293 enzymes AfuLPMO9A and AfuLPMO9C identified in secretome and RNA-Seq results previously obtained by our research group. By analyzing these two genes, it was found that in the presence of different carbon sources, both were induced, so that in the presence of SEB, CMC and Avicel, the AFUA_4G07850 gene was up to 3500X, 2000X and 1000X more induced while AFUA_1G12560 presented a lower increase of 16X, 13X and 7X, respectively. The evaluation of the three-dimensional conformation as well as the multiple alignment with higher degree homology enzymes revealed some important structural features such as the presence of cores rich in ? structures in sandwich conformation and that important conserved residues as H20; H105; Y194 and H22; H107; Y196 were coordinated to the metal center of the enzymes AfuLPMO9A and AfuLPMO9C, respectively. Further analysis based on DFT revealed that the active site of both assumes a square-based pyramidal geometry. After purification of LPMOs, activity assays revealed that the enzymes AfuLPMO9A and AfuLPMO9C were able to increase cocktail activity up to approximately 1,26X and 1,20X at concentrations of 10 mg/g Avicel and presence of 0.02% NaN3. Further analysis of these auxiliary enzymes will allow a better understanding of their role in sugarcane bagasse deconstruction.

Aspergillus fumigatus é um fungo filamentoso e saprofítico encontrado em todas as regiões do mundo. A principal forma de infecção ocorre através da inalação de conídios do fungo com predominância de infecções no trato respiratório, principalmente em pacientes imunocomprometidos. Foi caracterizada a função mitocondrial de A. fumigatus e sugeriu-se a presença de vias alternativas, dentre elas, a presença da NADH desidrogenase alternativa. A fim de colaborar com estudos para elucidação do papel desta enzima, foi realizada a clonagem dos genes das NADH desidrogenase alternativa interna e externa de A. fumigatus. A análise da seqüência de aminoácidos revelou um perfil de hidropaticidade com a presença de quatro regiões hidrofóbicas, semelhante às outras seqüências já descritas. Além disso, foram identificados dois motivos (GXGXXG) altamente conservados para ligações a nucleotídeos, dentro de um domínio os quais estão relacionados com a estrutura e atividade da enzima. A seqüência de cDNA do gene ndhiAf foi clonada em plasmídeo pYES2 e expressa em S. cerevisiae cepa CEN.PK873-2B; a expressão da NDHIAf foi verificada por técnica de Western-blot. A proteína foi expressa de forma ativa, conferindo à levedura uma respiração e a formação de um potencial de membrana resultantes da oxidação de substratos clássicos do complexo I, sugerindo sua localização na membrana mitocondrial interna. Além disso, as cepas expressando essa proteína foram capazes de crescer em meio contendo apenas lactato como fonte de carbono, uma característica atribuída à presença da NADH desidrogenase alternativa interna (NDHi). Considerando que a atividade das NDHIAf e NDHEAf estão sob controle metabólico, nos avaliamos o efeito de diferentes concentrações de glucose como única fonte de carbono no meio de cultura. Durante a germinação conidial, não foi observada expressão da NDHIAf e NDHEAf na presença de baixas concentrações de glucose. Entretanto, durante a fase de hifas foi observado um aumento na expressão de NDHIAf e NDHEAf. Por outro lado, em alta concentrações de glucose, durante a germinação conidial, foi observado expressão da NDHIAf mas nenhuma expressão da NDHEAf. Na fase de hifas observou-se um aumento da expressão da NDHIAf e uma diminuição da NDHEAf. / Aspergillus fumigatus is a filamentous and saprophytic fungus found in all regions of the world. The main form of infection occurs through inhalation of fungi conidial, with predominance of infections in the respiratory treat, mainly in immunocompromised host. It was characterized the mitochondrial function of A. fumigatus and suggested the presence of alternative pathways, including the alternative NADH dehydrogenase. In order to elucidate its role, genes of the external and internal enzymes were cloned. Analysis of the amino acid sequence and hydropathy plot revealed a profile with four hydrophobic regions, similar to other already described sequences. Moreover, two highly conserved motifs (GXGXXG) for the nucleotides interaction, related with the structure and activity of the enzyme, were identified inside the a domain. The sequence of DNA of the ndhiAf gene was cloned in an expression plasmid pYES2 and expressed in CEN.PK873-2B S. cerevisiae strain; the expression was confirmed by Western-blot analysis. The protein was expressed in an active form, providing to the yeast an oxygen consumption and a potential membrane formation due to oxidation of the complex I substract, suggesting its localization in the inner mitochondrial membrane. Moreover, strains expressing this protein were capable of growing in medium with only lactate as a carbon source, a characteristic associated with the presence of internal alternative NADH dehydrogenase (NDHI). Considering that NDHI and NDHE activities are under metabolic control, we evaluated the effect of different concentrations of glucose as the only carbon source in the medium of culture. During conidia germination, it was not observed neither an expression of NDHI nor NDHE in the presence of low concentrations of glucose. However, in hyphae phase was observed an increase an expression of NDHI and NDHE. On the other hand, in high concentration of glucose, during conidia germination was observed an expression of NDHI and not expression of NDHE. In hyphae phase was observed a decrease an expression of NDHI and an increase of NDHE.

A percepção e aquisição de nutrientes essenciais do meio ambiente estão associadas com o desenvolvimento do fungo saprófita A. fumigatus em um ambiente hostil. O fosfato inorgânico é um dos nutrientes essenciais para o desenvolvimento deste fungo. Os sistemas para aquisição de fosfato em células eucarióticas têm sido caracterizados como sistema de alta afinidade, o qual é ativado em resposta a ausência externa de fosfato e sistema de baixa afinidade, o qual assegura um suprimento de fosfato em concentrações normais ou altas de fosfato extracelular. Como um passo inicial para o entendimento da via PHO em A. fumigatus, caracterizou-se o homólogo PHO80, PHO84 e PHO85 aqui denominados phoBPHO80, phoDPHO84 e phoAPHO85, respectivamente. Resultados mostraram que o mutante phoBPHO80 apresenta um defeito no crescimento polarizado e que existe uma interação entre o metabolismo de PhoBPHO80, da calcineurina e do cálcio. As hibridações de microarray realizadas com RNA obtido das cepas mutante phoBPHO80 e selvagem mostraram que os genes Afu4g03610 (phoDPHO84), Afu7g06350 (phoEPHO89), Afu4g06020 (phoCPHO81) e Afu2g09040 (transportador vacuolar Vtc4) estão mais expressos no background do mutante phoBPHO80 ou em concentração de 0.1 mmol/L de fosfato. Nossos resultados indicam que a mutação phoBPHO80 pode afetar o acúmulo de polifosfato em vacúolos em alta ou baixa concentração de fosfato extracelular. Não obteve-se êxito na deleção da quinase phoAPHO85 desta forma pode-se levar em consideração que o gene phoAPHO85 parece ser essencial em A. fumigatus. Surpreendentemente, a cepa com a deleção de phoDPHO84 não apresentou nenhum fenótipo diferente da sua cepa selvagem. Além do mais, as cepas mutantes phoBPHO80 e phoDPHO84 não apresentaram redução na virulência em um modelo murino de aspergilose invasiva. Nossos resultados sugerem também, que a deleção da proteína quinase A está contribuindo para um decréscimo na expressão de genes PHO em A. fumigatus. / Environmental sensing and retrieval of nutrients from the environment are associated with growth of A. fumigatus in inhospitable environments. Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80, PHO84 and PHO85 homologues, here denominaded phoBPHO80, phoDPHO84 and phoAPHO85, respectively. We show that the phoBPHO80 mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoBPHO80, calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and phoBPHO80 mutant cells identify Afu4g03610 (phoDPHO84), Afu7g06350 (phoEPHO89), Afu4g06020 (phoCPHO81), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the phoBPHO80 mutant background and under phosphate-limiting conditions of 0.1 mM Pi. Epi-fluorescence microscopy revealed accumulation of poly-phosphate in phoBPHO80 vacuoles, which was independent of extracellular phosphate concentration. We also tried to isolate the phoAPHO85 deletion strain without succes after several times what raises the interesting possibility that the phoAPHO85 null mutant might be essential in Aspergillus fumigatus. Surprisingly, phoDPHO84 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, phoBPHO80 and phoDPHO84 mutant are fully virulent in a murine low dose model for invasive aspergillosis.

Aspergillus fumigatus é um fungo patogênico oportunista capaz de infectar pacientes imunocomprometidos causando eventualmente infecções disseminadas difíceis de serem controladas e com alta taxa de mortalidade dos indivíduos infectados.. Para um melhor entendimento de como esse fungo age no hospedeiro é importante saber como as vias de sinalização que regulam esses fatores de virulência são orquestradas. Proteínas fosfatases são centrais em uma grande variedade de vias de transdução de sinal. Neste trabalho, nós caracterizamos a proteína fosfatase 2A SitA, a proteína homóloga de Sit4 em Saccharomyces serevisiae. O gene sitA não é essencial e por isso fomos capazes de construir um mutante nulo em A. fumigatus. A cepa ?sitA apresenta aumento na fosforilação da MpkA, é mais sensível à agentes que causam dano na parede celular, tem um aumento na quantidade de ?-1,3 glicano e quitina, e também tem problemas na adesão e formação de biofilme. O mutante ?sitA é mais sensível a vários metais e íons, como MnCl2, CaCl2, LiCl, entretanto é mais resistente à ZnSO4. O mutante ?sitA é avirulento em modelo de aspergilose pulmonar invasiva em camundongos. Esses resultados revelam que a fosfatase SitA está envolvida na via de integridade da parede celular de A. fumigatus possivelmente modulando a atividade de PkcA/MpkA / Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients causing eventually disseminated infections that are difficult to be controlled, and lead to high mortality rates. It is important to understand how are orchestrated the signalling pathways that regulate these factors involved in virulence. Protein phosphatases are central to numerous signal transduction pathways. Here we characterize A. fumigatus protein phosphatase 2A SitA, the S. cerevisiae Sit4p homologue. The sitA gene is not an essential gene and we were able to construct an A. fumigatus null mutant. The ?sitA strain had increased MpkA phosphorylation, was more sensitive to cell wall damaging agents, had increased ??1,3?glican and chitin, and was impaired in biofilm formation. The ?sitA strain is more sensitive to several metals and ions such as MnCl2, CaCl2, and LiCl, however, it is more resistant to ZnSO4. The ?sitA strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway

O Aspergillus fumigatus é um fungo filamentoso e saprofítico, encontrado em todas as regiões do mundo, desempenhando um importante papel na reciclagem de carbono e nitrogênio do solo. A principal forma de infecção ocorre através da inalação dos conídios com predominância de infecções no trato respiratório, principalmente em pacientes imunocomprometidos. A mitocôndria de A. fumigatus foi caracterizada em nosso laboratório, que revelou a presença de uma respiração resistente a cianeto mediada pela oxidase alternativa. A clonagem e sequenciamento deste gene foram realizadas através de screening de uma biblioteca de DNA genômico. O alinhamento das sequências genômica e de cDNA mostrou a presença de dois introns, que após splicing codifica uma proteína contendo 352 aminoácidos, possuindo uma massa molecular estimada de 40,84 kDa e um pI teórico de 9,51. Além disso, foram identificados domínios altamente conservados (LET, NERMHL, LEEA e RADEH) que interagem com átomos de ferro e estão contidos em -hélices propostas como responsáveis pela organização estrutural da enzima. A fim de caracterizar bioquimicamente esta proteína, a sequência de cDNA do gene foi clonada em plasmídeo pYES2 e expressa em S. cerevisiae INVSc1 como um modelo eucarioto. Após expressão, a proteína encontrou-se de forma ativa, conferindo à levedura uma respiração resistente a cianeto. Esta característica herdada provocou uma discreta diminuição na taxa de crescimento em meio não-fermentativo e uma capacidade de sobrevivência na presença de KCN. Acredita-se que a atividade das AOXs esteja diretamente relacionada com a presença de diferentes espécies reativas de oxigênio (ERO). Neste contexto, a avaliação do efeito de diferentes agentes pró-oxidantes provocou um aumento na atividade e na expressão da enzima. Paralelamente, a caracterização funcional do gene foi realizada através da técnica de interferência por RNA, utilizando o vetor de expressão pALB1. Em meio contendo maltose, as cepas pALB1/aoxAf apresentaram coloração branca devido ao silenciamento do gene alb1. Os níveis de mRNA do gene aoxAf foram determinados por Real time RT-PCR, mostrando o eficiente silenciamento do gene alvo com a construção utilizada. Devido à relação já descrita entre ERO e a atividade das AOXs, avaliou-se a produção de espécies reativas de oxigênio nas cepas silenciadas utilizando-se a sonda CM-H2DCFDA, observando maior produção na cepa pALB1/aoxAf. Além disso, a viabilidade destas cepas foi determinada por citometria de fluxo após a exposição com agentes pró-oxidantes, a qual indicou maior letalidade na cepa pALB1/aoxAf, quando comparada com CEA e pALB1. Da mesma forma, após a incubação dos conídios das cepas silenciadas com macrófagos de camundongos foi verificada uma maior atividade microbicida dos macrófagos na cepa duplamente silenciada pALB1/aoxAf, quando comparada com as outras cepas. Com estes resultados podemos concluir que a oxidase alternativa apresenta uma importante atividade antioxidante, além de contribuir nos mecanismos de defesa durante o processo de infecção de A. fumigatus. / The saprophytic species Aspergillus fumigatus is a deuteromycete fungus found worldwide, which has an essential role in recycling carbon and nitrogen. Following inhalation of conidia by the immunocompetent host, the innate cellular immune system is responsible for killing the conidia, exposing them to reactive oxygen. However, A. fumigatus is capable of surviving and replicating within the phagolysosomal compartment of immunocompromised macrophages. It was previously demonstrated that A. fumigatus mitochondria possess an alternative oxidase (aoxAf) wich is a cyanide-resistant protein. A partial genomic DNA library was screened to cloning an aoxAf gene. The alignment between the cDNA and genomic DNA sequences revealed the existence of two introns which after splicing encodes a 352 amino acid sequence with a calculated molecular mass of 40 kDa and a theoretical pI of 9.51. The deduced amino acid sequence revealed four regions completely conserved among the AOXs sequences (LET, NERMHL, LEEA and RADE-H), where six conserved amino acids residues are proposed to be a metal ligand site. To characterize the AOX protein, a cDNA of aoxAf gene was cloned into pYES2 plasmid and transformed in S. cerevisiae INVSc1. After the incubation of the cells in a nonfermentable medium in the presence of KCN, S. cerevisiae expressing AOX was able to grow, while it was lethal for the control yeast. These results suggest that the recombinant AOXAf is properly targeted to the S. cerevisiae mitochondria where it has functional activity. Studies with different species demonstrated that AOX is induced by a variety of treatments usually labeled as stresses. To verify the function of AOX in A. fumigatus under oxidative stress conditions, conidia were treated with different donors of ROS. These treatments caused an increase in aoxAf activity and transcription levels. To identify genetically attributes of virulence and oxidative defense in A. fumigatus, we construct a RNA interference plasmid. Two inverted repeated sequences of conserved region of an interest gene were amplified and cloned in pALB1 plasmid. In maltose medium pALB1 and pALB1/aoxAf transformants demonstrated white colonies, attributable to the reduction of alb1 gene expression. The aoxAf mRNA levels were analyzed by Real time RT-PCR, showing an efficient alternative oxidase gene silencing in pALB1 plasmid construction. It was previously demonstrated that ROS can stimulate the AOXs activity, so, we used the dye CM-H2DCFDA to measure ROS production in RNAi transformants, showing that the decrease in aoxAf gene expression caused an increase in ROS production. After incubation with ROS donors the viability of these strains was determined by flow cytometry analysis. The pALB1/aoxAf strain showed higher lethality, when compared with CEA and pALB1, suggesting the involvement of AOX in antioxidant defense in A. fumigatus. Besides, ROS produced by alveolar macrophages play an essential role in the killing of A. fumigatus conidia. In the same way, phagocytosis assay revealed that pALB1/aoxAf strain was more lethal than CEA and pALB1. With these results, we concluded that alternative oxidase is an efficient antioxidant system and might contribute with defense mechanism of A. fumigatus.