Techn. Universiẗat, Diss., 2005--München.

Aspergilose é a doença fúngica mais comum em cães com alterações clínicas nasais e Aspergillus fumigatus é a espécie mais encontrada em animais com envolvimento primário do trato respiratório superior. Por serem agentes cosmopolitas, diversas espécies de Aspergillus estão freqüentemente presentes na biota anemófila e, portanto, estão também comumente presentes no trato respiratório superior de cães sadios. Devido a isto, resultados positivos no exame de cultivo não são suficientes para firmar o diagnóstico de aspergilose nasal. Já o exame sorológico, serve de bom suporte ao clínico para o diagnóstico de aspergilose nasal canina, principalmente com o emprego da técnica de eletrosinerese, a qual apresenta alta sensibilidade e especificidade. Apesar da aspergilose nasal canina ser relatada com relativa freqüência em diversos países, a literatura veterinária brasileira não registra a ocorrência desta doença. Esta situação sugere que a mesma não está sendo colocada como casuística diferencial no diagnóstico das prováveis moléstias de cães portadores de descarga nasal em âmbito nacional. Os objetivos principais deste trabalho foram a pesquisa de anticorpos circulantes anti-Aspergillus fumigatus através do teste sorológico de eletrosinerese e o cultivo fúngico de amostras obtidas de 83 cães com descarga nasal, atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, no período de agosto de 2006 a julho de 2007. O objetivo secundário foi verificar uma possível concordância entre os resultados obtidos por estas duas técnicas laboratoriais. Doze cães (14,4%) tiveram resultados positivos no teste sorológico de eletrosinerese. Não houve nenhuma associação significativa entre a variável sorologia (positiva e negativa) com as demais variáveis (raça, sexo, conformação craniana e faixa etária). Quatorze gêneros foram isolados no cultivo fúngico. Não houve concordância entre os exames sorológico e cultivo, posto que dos 12/83 animais que tiveram exame sorológico positivo, apenas 2/12 (16,6%) cães tiveram cultivo fúngico positivo para Aspergillus fumigatus. Cultivo fúngico realizado a partir de amostras colhidas por swab direto da descarga nasal deve ser analisado com cautela para se estabelecer o diagnóstico de aspergilose nasal canina. A partir dos resultados obtidos neste trabalho e dos relatos encontrados em vários outros países, pode-se concluir que a doença provavelmente não seja rara no Brasil, e que os clínicos veterinários deveriam incluir esta entidade nosológica como casuística potencial no diagnóstico diferencial das doenças do trato respiratório superior de cães, o que certamente possibilitará estudos comparativos (clínicos e epidemiológicos) de dados obtidos de diversas regiões do Brasil. / Aspergillosis is the most common fungal disease in dogs with nasal disorders and Aspergillus fumigatus is the most often specie found in animals with primary involvement of the upper respiratory tract. As cosmopolitans agents, diverse species of Aspergillus are frequently found in the anemophilous biota, therefore, it is also found in the upper respiratory tract of healthy dogs. Due this, fungal culture positive results are not sufficient to confirm nasal aspergillosis in dogs. Nevertheless, serological tests can serve as a good support to the clinicians for the diagnosis of canine nasal aspergillosis, specially with electrosyneresis technique, which presents high sensitivity and specificity. Despite canine nasal aspergillosis have been frequently described in different countries, in the Brazilian veterinary literature does not register any occurrence. This finding can suggest that nasal aspergillosis has not been included in the differential diagnosis of dogs with nasal discharge in this country. The main aims of this study were the search for the presence of anti-Aspergillus fumigatus circulating antibodies means electrosyneresis of technique and to obtain positive cultures of Aspergillus fumigatus from 83 dogs with nasal discharge, presented at the Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, from August, 2006 to July, 2007. Another purpose of this study was to observe whether a correlation could be established between the methods. Twelve dogs (14,4%) were identified as serological positive. No significant association was observed among the serological test (positive and negative) and dog’s features (breed, sex, head conformation and age). Fourteen fungi genus were isolated in cultures. There was no correlation between the serological test and the culture. Only 2 from a contingent of 12 dogs with positive serological test had simultaneous positives cultures to Aspergillus fumigatus. Cultures obtained from samples collected with swab from nasal discharge must be analyzed with caution to establish the diagnosis of canine nasal aspergillosis. Taken into account the herein presented results and looking into the results from reports elsewhere, we conclude that canine nasal aspergillosis probably is not rare in our environment, and veterinarians must include this entity in the differential diagnosis of dog´s upper respiratory tract disorders. The data generated by the present dissertation will undoubtedly contribute to further studies and for comparative trials about the frequency of the disease in dogs from other regions of Brazil.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, infecção fúngica oportunista com altas taxas de mortalidade afetando, principalmente, pacientes com neutropenia profunda e prolongada. Durante o processo de invasão e disseminação características desta infecção sistêmica, os conídios do fungo inalados e não eliminados pelas células do sistema imune inato diferenciam-se em hifas que, por sua vez, são angioinvasivas. Pouco se conhece sobre as moléculas da parede celular envolvidas na patogênese do A. fumigatus e/ou secretadas por este patógeno. Neste contexto, este trabalho procura ampliar o entendimento desta doença através do estudo de proteínas diferencialmente expressas na superfície de A. fumigatus durante a morfogênese. Foi utilizada uma abordagem proteômica e foram estudados extratos de superfície de células de A. fumigatus em diferentes estágios durante o processo de filamentação. Estas células foram denominadas, de acordo com o tempo de cultivo e a morfologia, como: TG6h (tubo germinativo), H12h ou H72h (hifas). As proteínas de superfície celular foram extraídas, a partir de células intactas, por tratamento brando com o agente redutor DTT (ditiotreitol). Observou-se que o perfil funcional das proteínas expressas por H12h e H72h foi similar, com exceção de proteínas relacionadas à resposta ao estresse, enquanto o perfil para TG6h apresentou diferenças significativas para vários grupos funcionais de proteínas quando comparado às hifas. Desta forma, foram realizados experimentos de proteômica diferencial entre tubo germinativo (TG6h) e a hifa madura (H72h), pela técnica de DIGE (differential gel electrophoresis). Os resultados revelaram que entre as proteínas diferencialmente expressas, aquelas relacionadas às vias de biossíntese e outras denominadas multifuncionais encontraram-se superexpressas em TG6h. Em relação às proteínas de resposta a estresse, observou-se que algumas HSPs eram mais expressas neste morfotipo, enquanto a MnSOD, relativa à resposta ao estresse oxidativo, era mais abundante na hifa. Com exceção da PhiA, integrante da parede celular, as proteínas identificadas como diferencialmente expressas na superfície do A. fumigatus não possuem sinal para secreção identificável, enquadrando-se nas proteínas atípicas de superfície. Foi verificada a integridade da membrana celular após tratamento com DTT, bem como a marcação por biotina das proteínas extraídas, o que comprovou sua localização superficial na célula fúngica. Hipóteses de que estas proteínas sejam endereçadas à parede celular por via secretória alternativa sustentam estes dados. Estas evidências foram confirmadas pelo fato de não terem sido encontradas as mesmas proteínas da superfície na análise do secretoma do A. fumigatus. Além disso, todas as proteínas caracterizadas no secretoma apresentavam sinal de secreção determinado pelo FunSecKB (www.proteomics.ysu.edu/secretomes/fungi.php). A análise do secretoma foi realizada utilizando-se a cepa selvagem AF293 e a mutante ∆prtT, mutante para um fator de transcrição que atua na regulação da secreção de proteases. Os resultados revelam a ALP1 como expressa na cepa selvagem, assim como outras proteases importantes para virulência e desenvolvimento da célula fúngica, estando suprimidas quando o gene prtT foi deletado. / Aspergillus fumigatus is the main etiologic agent of invasive aspergillosis (IA), a opportunistic a life-threatening disease for immunocompromised hosts, especially those with acute and prolonged neutropenia. During the invasion and dissemination, which occurs in this systemic infection, the A. fumigatus conidia, after its inhalation, germinates into angioinvasive hyphae in case the innate immune response fails in eliminate these cells. Little is known about the cell wall molecules and/or the secreted proteins involved on the A. fumigatus pathogenesis, at this context the present work aims to amplify the knowledge about the aspergillosis by studying the differentially surface proteins of A. fumigatus during the filamentation process. These cells were denominated according to their morphology and their growth time as: TG6h (germ tubes), H12h and H72h (hyphae). The surface proteins were mildly extracted from intact cells using the reducing agent DTT (dithiothreitol). The functional profile of the H12h and H72h were similar except for the stress response proteins, while the TG6h presented significant differences for several functional groups. On this base, the DIGE (differential gel electrophoresis) was performed using the surface extracted proteins of the germ tubes (TG6h) and mature hyphae (H72h) cells. The results indicate that multiple functional proteins and proteins related to the biosynthesis pathways were overexpressed at TG6h. Some stress response proteins as the HSPs were overexpressed on this morphotype while the MnSOD, oxidative stress responsive protein, was most abundant at the hyphae. PhiA, an integrant protein of the cell wall, was the only protein with a secretion signal sequence. All other proteins identified on the cell surface lack an identifiable secretion sign, and are denominated atypical proteins. The plasma membrane integrity was verified after the mild extraction using DTT, and also the biotinylation of the cell extracted proteins, which ensured these proteins surface localization on the fungal cell. There are hypothesis that some proteins can be transport to the cell wall by alternative pathways and support the results found for this work. These evidences were confirmed by the fact that the proteins identified at the surface were not the same identified on the A. fumigatus secretome. Besides, all proteins identified on the secretome presented secretion signal determinate by the FunSecKB (www.proteomics.ysu.edu/secretomes/fungi.php). The secretome analysis was performed using the wild type AF293 and the ∆prtT mutant, lacking a transcription factor that regulates proteases secretion. The results demonstrate ALP1 overexpressed on the wild type and also other proteases important to virulence and development of the fungal cell suppressed when prtT was deleted.

Aspergilose é a doença fúngica mais comum em cães com alterações clínicas nasais e Aspergillus fumigatus é a espécie mais encontrada em animais com envolvimento primário do trato respiratório superior. Por serem agentes cosmopolitas, diversas espécies de Aspergillus estão freqüentemente presentes na biota anemófila e, portanto, estão também comumente presentes no trato respiratório superior de cães sadios. Devido a isto, resultados positivos no exame de cultivo não são suficientes para firmar o diagnóstico de aspergilose nasal. Já o exame sorológico, serve de bom suporte ao clínico para o diagnóstico de aspergilose nasal canina, principalmente com o emprego da técnica de eletrosinerese, a qual apresenta alta sensibilidade e especificidade. Apesar da aspergilose nasal canina ser relatada com relativa freqüência em diversos países, a literatura veterinária brasileira não registra a ocorrência desta doença. Esta situação sugere que a mesma não está sendo colocada como casuística diferencial no diagnóstico das prováveis moléstias de cães portadores de descarga nasal em âmbito nacional. Os objetivos principais deste trabalho foram a pesquisa de anticorpos circulantes anti-Aspergillus fumigatus através do teste sorológico de eletrosinerese e o cultivo fúngico de amostras obtidas de 83 cães com descarga nasal, atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, no período de agosto de 2006 a julho de 2007. O objetivo secundário foi verificar uma possível concordância entre os resultados obtidos por estas duas técnicas laboratoriais. Doze cães (14,4%) tiveram resultados positivos no teste sorológico de eletrosinerese. Não houve nenhuma associação significativa entre a variável sorologia (positiva e negativa) com as demais variáveis (raça, sexo, conformação craniana e faixa etária). Quatorze gêneros foram isolados no cultivo fúngico. Não houve concordância entre os exames sorológico e cultivo, posto que dos 12/83 animais que tiveram exame sorológico positivo, apenas 2/12 (16,6%) cães tiveram cultivo fúngico positivo para Aspergillus fumigatus. Cultivo fúngico realizado a partir de amostras colhidas por swab direto da descarga nasal deve ser analisado com cautela para se estabelecer o diagnóstico de aspergilose nasal canina. A partir dos resultados obtidos neste trabalho e dos relatos encontrados em vários outros países, pode-se concluir que a doença provavelmente não seja rara no Brasil, e que os clínicos veterinários deveriam incluir esta entidade nosológica como casuística potencial no diagnóstico diferencial das doenças do trato respiratório superior de cães, o que certamente possibilitará estudos comparativos (clínicos e epidemiológicos) de dados obtidos de diversas regiões do Brasil. / Aspergillosis is the most common fungal disease in dogs with nasal disorders and Aspergillus fumigatus is the most often specie found in animals with primary involvement of the upper respiratory tract. As cosmopolitans agents, diverse species of Aspergillus are frequently found in the anemophilous biota, therefore, it is also found in the upper respiratory tract of healthy dogs. Due this, fungal culture positive results are not sufficient to confirm nasal aspergillosis in dogs. Nevertheless, serological tests can serve as a good support to the clinicians for the diagnosis of canine nasal aspergillosis, specially with electrosyneresis technique, which presents high sensitivity and specificity. Despite canine nasal aspergillosis have been frequently described in different countries, in the Brazilian veterinary literature does not register any occurrence. This finding can suggest that nasal aspergillosis has not been included in the differential diagnosis of dogs with nasal discharge in this country. The main aims of this study were the search for the presence of anti-Aspergillus fumigatus circulating antibodies means electrosyneresis of technique and to obtain positive cultures of Aspergillus fumigatus from 83 dogs with nasal discharge, presented at the Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, from August, 2006 to July, 2007. Another purpose of this study was to observe whether a correlation could be established between the methods. Twelve dogs (14,4%) were identified as serological positive. No significant association was observed among the serological test (positive and negative) and dog’s features (breed, sex, head conformation and age). Fourteen fungi genus were isolated in cultures. There was no correlation between the serological test and the culture. Only 2 from a contingent of 12 dogs with positive serological test had simultaneous positives cultures to Aspergillus fumigatus. Cultures obtained from samples collected with swab from nasal discharge must be analyzed with caution to establish the diagnosis of canine nasal aspergillosis. Taken into account the herein presented results and looking into the results from reports elsewhere, we conclude that canine nasal aspergillosis probably is not rare in our environment, and veterinarians must include this entity in the differential diagnosis of dog´s upper respiratory tract disorders. The data generated by the present dissertation will undoubtedly contribute to further studies and for comparative trials about the frequency of the disease in dogs from other regions of Brazil.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, infecção fúngica oportunista com altas taxas de mortalidade afetando, principalmente, pacientes com neutropenia profunda e prolongada. Durante o processo de invasão e disseminação características desta infecção sistêmica, os conídios do fungo inalados e não eliminados pelas células do sistema imune inato diferenciam-se em hifas que, por sua vez, são angioinvasivas. Pouco se conhece sobre as moléculas da parede celular envolvidas na patogênese do A. fumigatus e/ou secretadas por este patógeno. Neste contexto, este trabalho procura ampliar o entendimento desta doença através do estudo de proteínas diferencialmente expressas na superfície de A. fumigatus durante a morfogênese. Foi utilizada uma abordagem proteômica e foram estudados extratos de superfície de células de A. fumigatus em diferentes estágios durante o processo de filamentação. Estas células foram denominadas, de acordo com o tempo de cultivo e a morfologia, como: TG6h (tubo germinativo), H12h ou H72h (hifas). As proteínas de superfície celular foram extraídas, a partir de células intactas, por tratamento brando com o agente redutor DTT (ditiotreitol). Observou-se que o perfil funcional das proteínas expressas por H12h e H72h foi similar, com exceção de proteínas relacionadas à resposta ao estresse, enquanto o perfil para TG6h apresentou diferenças significativas para vários grupos funcionais de proteínas quando comparado às hifas. Desta forma, foram realizados experimentos de proteômica diferencial entre tubo germinativo (TG6h) e a hifa madura (H72h), pela técnica de DIGE (differential gel electrophoresis). Os resultados revelaram que entre as proteínas diferencialmente expressas, aquelas relacionadas às vias de biossíntese e outras denominadas multifuncionais encontraram-se superexpressas em TG6h. Em relação às proteínas de resposta a estresse, observou-se que algumas HSPs eram mais expressas neste morfotipo, enquanto a MnSOD, relativa à resposta ao estresse oxidativo, era mais abundante na hifa. Com exceção da PhiA, integrante da parede celular, as proteínas identificadas como diferencialmente expressas na superfície do A. fumigatus não possuem sinal para secreção identificável, enquadrando-se nas proteínas atípicas de superfície. Foi verificada a integridade da membrana celular após tratamento com DTT, bem como a marcação por biotina das proteínas extraídas, o que comprovou sua localização superficial na célula fúngica. Hipóteses de que estas proteínas sejam endereçadas à parede celular por via secretória alternativa sustentam estes dados. Estas evidências foram confirmadas pelo fato de não terem sido encontradas as mesmas proteínas da superfície na análise do secretoma do A. fumigatus. Além disso, todas as proteínas caracterizadas no secretoma apresentavam sinal de secreção determinado pelo FunSecKB (www.proteomics.ysu.edu/secretomes/fungi.php). A análise do secretoma foi realizada utilizando-se a cepa selvagem AF293 e a mutante ∆prtT, mutante para um fator de transcrição que atua na regulação da secreção de proteases. Os resultados revelam a ALP1 como expressa na cepa selvagem, assim como outras proteases importantes para virulência e desenvolvimento da célula fúngica, estando suprimidas quando o gene prtT foi deletado. / Aspergillus fumigatus is the main etiologic agent of invasive aspergillosis (IA), a opportunistic a life-threatening disease for immunocompromised hosts, especially those with acute and prolonged neutropenia. During the invasion and dissemination, which occurs in this systemic infection, the A. fumigatus conidia, after its inhalation, germinates into angioinvasive hyphae in case the innate immune response fails in eliminate these cells. Little is known about the cell wall molecules and/or the secreted proteins involved on the A. fumigatus pathogenesis, at this context the present work aims to amplify the knowledge about the aspergillosis by studying the differentially surface proteins of A. fumigatus during the filamentation process. These cells were denominated according to their morphology and their growth time as: TG6h (germ tubes), H12h and H72h (hyphae). The surface proteins were mildly extracted from intact cells using the reducing agent DTT (dithiothreitol). The functional profile of the H12h and H72h were similar except for the stress response proteins, while the TG6h presented significant differences for several functional groups. On this base, the DIGE (differential gel electrophoresis) was performed using the surface extracted proteins of the germ tubes (TG6h) and mature hyphae (H72h) cells. The results indicate that multiple functional proteins and proteins related to the biosynthesis pathways were overexpressed at TG6h. Some stress response proteins as the HSPs were overexpressed on this morphotype while the MnSOD, oxidative stress responsive protein, was most abundant at the hyphae. PhiA, an integrant protein of the cell wall, was the only protein with a secretion signal sequence. All other proteins identified on the cell surface lack an identifiable secretion sign, and are denominated atypical proteins. The plasma membrane integrity was verified after the mild extraction using DTT, and also the biotinylation of the cell extracted proteins, which ensured these proteins surface localization on the fungal cell. There are hypothesis that some proteins can be transport to the cell wall by alternative pathways and support the results found for this work. These evidences were confirmed by the fact that the proteins identified at the surface were not the same identified on the A. fumigatus secretome. Besides, all proteins identified on the secretome presented secretion signal determinate by the FunSecKB (www.proteomics.ysu.edu/secretomes/fungi.php). The secretome analysis was performed using the wild type AF293 and the ∆prtT mutant, lacking a transcription factor that regulates proteases secretion. The results demonstrate ALP1 overexpressed on the wild type and also other proteases important to virulence and development of the fungal cell suppressed when prtT was deleted.

Aspergilose é a doença fúngica mais comum em cães com alterações clínicas nasais e Aspergillus fumigatus é a espécie mais encontrada em animais com envolvimento primário do trato respiratório superior. Por serem agentes cosmopolitas, diversas espécies de Aspergillus estão freqüentemente presentes na biota anemófila e, portanto, estão também comumente presentes no trato respiratório superior de cães sadios. Devido a isto, resultados positivos no exame de cultivo não são suficientes para firmar o diagnóstico de aspergilose nasal. Já o exame sorológico, serve de bom suporte ao clínico para o diagnóstico de aspergilose nasal canina, principalmente com o emprego da técnica de eletrosinerese, a qual apresenta alta sensibilidade e especificidade. Apesar da aspergilose nasal canina ser relatada com relativa freqüência em diversos países, a literatura veterinária brasileira não registra a ocorrência desta doença. Esta situação sugere que a mesma não está sendo colocada como casuística diferencial no diagnóstico das prováveis moléstias de cães portadores de descarga nasal em âmbito nacional. Os objetivos principais deste trabalho foram a pesquisa de anticorpos circulantes anti-Aspergillus fumigatus através do teste sorológico de eletrosinerese e o cultivo fúngico de amostras obtidas de 83 cães com descarga nasal, atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, no período de agosto de 2006 a julho de 2007. O objetivo secundário foi verificar uma possível concordância entre os resultados obtidos por estas duas técnicas laboratoriais. Doze cães (14,4%) tiveram resultados positivos no teste sorológico de eletrosinerese. Não houve nenhuma associação significativa entre a variável sorologia (positiva e negativa) com as demais variáveis (raça, sexo, conformação craniana e faixa etária). Quatorze gêneros foram isolados no cultivo fúngico. Não houve concordância entre os exames sorológico e cultivo, posto que dos 12/83 animais que tiveram exame sorológico positivo, apenas 2/12 (16,6%) cães tiveram cultivo fúngico positivo para Aspergillus fumigatus. Cultivo fúngico realizado a partir de amostras colhidas por swab direto da descarga nasal deve ser analisado com cautela para se estabelecer o diagnóstico de aspergilose nasal canina. A partir dos resultados obtidos neste trabalho e dos relatos encontrados em vários outros países, pode-se concluir que a doença provavelmente não seja rara no Brasil, e que os clínicos veterinários deveriam incluir esta entidade nosológica como casuística potencial no diagnóstico diferencial das doenças do trato respiratório superior de cães, o que certamente possibilitará estudos comparativos (clínicos e epidemiológicos) de dados obtidos de diversas regiões do Brasil. / Aspergillosis is the most common fungal disease in dogs with nasal disorders and Aspergillus fumigatus is the most often specie found in animals with primary involvement of the upper respiratory tract. As cosmopolitans agents, diverse species of Aspergillus are frequently found in the anemophilous biota, therefore, it is also found in the upper respiratory tract of healthy dogs. Due this, fungal culture positive results are not sufficient to confirm nasal aspergillosis in dogs. Nevertheless, serological tests can serve as a good support to the clinicians for the diagnosis of canine nasal aspergillosis, specially with electrosyneresis technique, which presents high sensitivity and specificity. Despite canine nasal aspergillosis have been frequently described in different countries, in the Brazilian veterinary literature does not register any occurrence. This finding can suggest that nasal aspergillosis has not been included in the differential diagnosis of dogs with nasal discharge in this country. The main aims of this study were the search for the presence of anti-Aspergillus fumigatus circulating antibodies means electrosyneresis of technique and to obtain positive cultures of Aspergillus fumigatus from 83 dogs with nasal discharge, presented at the Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, from August, 2006 to July, 2007. Another purpose of this study was to observe whether a correlation could be established between the methods. Twelve dogs (14,4%) were identified as serological positive. No significant association was observed among the serological test (positive and negative) and dog’s features (breed, sex, head conformation and age). Fourteen fungi genus were isolated in cultures. There was no correlation between the serological test and the culture. Only 2 from a contingent of 12 dogs with positive serological test had simultaneous positives cultures to Aspergillus fumigatus. Cultures obtained from samples collected with swab from nasal discharge must be analyzed with caution to establish the diagnosis of canine nasal aspergillosis. Taken into account the herein presented results and looking into the results from reports elsewhere, we conclude that canine nasal aspergillosis probably is not rare in our environment, and veterinarians must include this entity in the differential diagnosis of dog´s upper respiratory tract disorders. The data generated by the present dissertation will undoubtedly contribute to further studies and for comparative trials about the frequency of the disease in dogs from other regions of Brazil.

Aspergillus fumigatus is a filamentous fungus and the main pathogen responsible for the often fatal respiratory condition, aspergillosis. Airway epithelial cells (AECs) are likely to be the first line of host defence that come into contact with the inhaled conidia of A. fumigatus. Recent evidence strongly suggests that the response of the airway epithelium to inhaled pathogens is pivotal in orchestrating immune responses by inducing phagocytic-like reactions and the secretion of inflammatory cytokines and antimicrobial peptides. However, the majority of previous work investigating A. fumigatus-host interactions has been performed using macrophages and neutrophils, thereby neglecting the epithelium. AECs have been shown to secrete inflammatory cytokines in response to A. fumigatus although these studies predominantly used transformed AEC lines that lack tight junctions and do not fully differentiate. Furthermore, most studies used culture filtrate or extract of A. fumigatus rather than live, whole organism and as a result, the direct interaction of the germinating fungus and the airway epithelium has been overlooked. During the early germination and growth period, the cell wall composition of A. fumigatus is dynamic, with various antigens exposed at different morphological stages. The aim of this thesis was to determine whether AECsare able to alter the germination and growth rate of A. fumigatus, and, conversely, if A. fumigatus affects AECs in terms of the secretion of inflammatory mediators. These studies used live, germinating A. fumigatus, and human primary differentiated AECs to obtain a more realistic in vitro model than those used in previous studies. Data showed that AECs are able to significantly inhibit the germination and growth of A. fumigatus, although this effect was less pronounced in differentiated primary AEC than in transformed AEC lines. A. fumigatus also significantly inducedthe expression and secretion of the inflammatory cytokines, IL-6 and IL-8, probably via the interaction of fungal cell wall β-glucans, and as of yet unidentified AEC receptor. The A1160pyrG+ strain of A. fumigatus secreted factors capable of inducing cytokine secretion whereas Af293 strain did not, highlighting diverse mechanisms of action for different strains. Upregulation of both cytokines was dependent on the stage of A. fumigatus growth with induction synchronous with germination. Despite being associated with fungal sensitisation in asthmatics, AEC-derived cytokines associated with this disease, namely TSLP, IL33 and IL25,did not appear to be upregulated by transformed AECs in response to A. fumigatus. Similarly, A. fumigatus did not seem to induce synthesis and secretion of the acute phase response protein, fibrinogen above baseline levels. The data presented in this thesis confirms the importance of the airway epithelium in directing anti-A. fumigatus immunity and the involvement of complex ligand-receptor interactions.

Sialic acids are a family of 9 carbon acidic sugars that are often part of glycans that decorate glycoproteins and glycolipids. N-acetylneuraminic acid, or Neu5Ac, is the predominant sialic acid in Homo sapiens and is most commonly found on the terminus of glycan chains where it functions as a signalling molecule or providing a negatively-charged mask to cells. Neu5Ac is less commonly found in microbes. A number of pathogens, including the fungus Aspergillus fumigatus and the parasite Trypanosoma cruzi, present Neu5Ac on their surface which aids evasion of the host immune system. A. Fumigatus has no known sialic synthesis genes however a single sialidase, AfS, has been identified. This sialidase is relatively poor at cleaving Neu5Ac in comparison with other members of the sialidase family. Here we present the structure of AfS to 1.8Å, the first fungal sialidase structure published. The overall topology of AfS shares the 6-bladed β-propellor fold common to all known sialidases and has an rmsd of only 1.44 Å over 309 C[sub]αs with the bacterial sialidase from Micromonospora viridifaciens. The active site of AfS also contains all the residues necessary for efficient hydrolysis of sialic acids. Despite repeated attempts. It was not possible to crystallize AfS in complex with Neu5Ac. Closer inspection of the AfS active site revealed that the pocket that usually accommodates the N-acetyl moiety of Neu5Ac contained more polar residues and was shallower than that of other sialidases. The apo structure suggested that substitution of the N-acetyl group on C5 of Neu5Ac with a smaller group may be accommodated better in the active site of AfS. 2-Keto-3-deoxy-D-galactononulosonic acid (KDN), which has a hydroxyl group at this position, is capable of binding in the active site and is the preferred substrate of AfS. The structure of AfS in complex with KDN was determined to 1.45 Å and is the first KDN specific sialidase structure determined. Crystal complexes with either a di-fluoro-KDN or KDN2en allowed atomic snapshots to be obtained of the catalytic cycle from Michaelis complex, through transition state to covalent intermediate. Several KDN binding sites on the surface of the enzyme were discovered suggesting that the enzyme may be specific for polyKDN, which intruigingly has been found in the human lung. Mutations were made to AfS to enlarge the cavity around C5 to see if the enzyme could accommodate Neu5Ac-related substrates. Identification and mutation of arginine 171 in the active site to leucine increased the depth and hydrophobicity of the cavity and improved the ability of AfS to utilize Neu5Ac, albeit poorly. The structure AfS[sub](R171L) in complex with Neu5Ac2en was solved to 1.8 Å. Trypanosoma cruzi is the etiological agent of Chagas disease. The trans sialidase from T. cruzi, TcTs, is a verified virulence factor. Here we detail a fragment-based search for novel inhibitors of TcTs using a fluorogenic assay, STD-NMR and WaterLOGSY-NMR, in silico screening and X-ray crystallography. There was poor agreement between the techniques used and only fragment hits validated by X-ray crystallography were pursued. All protein-ligand complexes shared two common interactions. The verified compounds interacted with the arginine triad via a carboxylic acid, as is the case with the sialic acid substrate. Secondly, all compiunds possessed an amine that was within hydrogen bonding distance of the TcTs catalytic aspartic acid. One of these compunds, 3-piperidinic acid, was capable of binding in the TcTs active site in both of its anomeric forms. Interestingly, each anomer binds in 2 similar but distinct positions in the active site. In one of these conformations Tyr119 moves into the active site and hydrogen bonds with the fragment's amine. In an attempt to combine the interaction seen in both the R and S anomers of 3-piperidinic acid, (R)-2-piperazine carboxylic acid was soaked into TcTs crystals. This compound made additional interactions with the protein and demonstrated a 4-fold improvement in the IC50. A known sialidase inhibitor, Siastatin B, based on a 3-piperidinic acid frame was also investigated and we present a TcTs-siastatin B complex structure to 2.1 Å, the first structure of Siastatin B in complex with a glycoside hydrolase. There is much work to be done to these preliminary inhibitors however here we have identified 3 druggable sites within the TcTs active site. The research presented herein provides a platform for future drug design studies on TcTs.

The lung was recently identified to consist of a complex microenvironment made up of microorganisms that interact with one another and the host cells via direct and indirect interactions. As a result, understanding the dynamic of the microbiome in chronic respiratory diseases has become the focus of pulmonary researches. In cystic fibrosis (CF), chronic infections are a comorbidity associated with the genetic disorder. Recently, it was noted that the interactions of the fungus, Aspergillus fumigatus, and the bacterium, Pseudomonas aeruginosa together contribute to more severe disease outcomes in CF patients. In vitro co-cultures show that P. aeruginosa and A. fumigatus can affect one another’s growth and pathogenicity, but very few studies have attempted to model interactions of these microorganisms in vivo. Based on clinical and basic research, we developed a co-exposure model in which we could compare non-allergic and allergic animals co-exposed to Pseudomonas aeruginosa and Aspergillus fumigatus. While both groups had significant neutrophilia and production of acute phase response cytokines and chemokines, the allergic co-exposed group had a greater mortality with 34.8% of the animals expiring by 24h in comparison to 12.5% for the non-allergic co-exposed animals and 100% survival in the controls. A contributing factor to the more severe disease outcomes in the allergic co-exposed group is the increase in eosinophilic inflammation and IL-17A production, which only occurs when both microorganisms are viable. In addition, it was found that viable P. aeruginosa but not A. fumigatus causes interstitial inflammation, significant neutrophilia, and even death during co-exposures. The decline in health of animals co-exposed to the fungus and bacteria could be attributed not only to the host’s inflammatory response, but also to the spatial and temporal co-localization in the lung. To address this, we performed in vitro studies finding an aggregation of the microorganisms that could also be identified in vivo. This current research emphasizes the need for in vivo studies on polymicrobial interactions. / ND Agricultural Experiment Station; National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R155AI137886

Ziel der vorliegenden Arbeit war die Charakterisierung von Punktmutationen in ifng und ncam1 in Hinblick auf eine veränderte Funktionalität von NK-Zellen bei der Interaktion mit A. fumigatus. Die gewonnenen Erkenntnisse sollen langfristig zur Verbesserung der Diagnostik, Prophylaxe und Therapie einer Invasiven Aspergillose, die zum Beispiel im Rahmen einer Stammzelltransplantation auftreten könnte, beitragen. In dieser Arbeit wurde die DNA von zwanzig gesunden Spendern auf einen ifng-SNP (rs2069705) und einen ncam1-SNP (rs10502171) untersucht. Von je drei ausgewählten Spendern mit SNP und sechs Kontrollspendern wurden NK-Zellen isoliert. Diese wurden unstimuliert belassen, mit Interleukin 2/15 oder A. fumigatus stimuliert. Bei der Versuchsreihe zum ifng-SNP wurde eine qPCR zur Ermittlung der relativen Expression von ifng und ccl4, bei den Versuchen zum ncam1-SNP eine durchflusszytometrische Analyse zur Messung der Expression verschiedener Oberflächenmarker durchgeführt. Bei beiden wurde mittels ELISA die Freisetzung von IFN-gamma bzw. CCL4/MIP-1ß bestimmt. Die in dieser Arbeit gewonnenen Ergebnisse zum ifng-SNP lassen vermuten, dass das Vorliegen dieses ifng-SNP keine durch NK-Zellen vermittelten Auswirkungen auf das Risiko der Patienten, an einer Invasiven Aspergillose zu erkranken, hat. In Bezug auf den ncam1-SNP konnte die Hypothese bestätigt werden, dass der SNP die Interaktion zwischen der NK-Zelle und A. fumigatus verändert. Der SNP korreliert zwar mit einer erhöhten Grundaktivierung von NK-Zellen, jedoch auch mit einem schwächeren Aktivierungspotential bei Stimulation mit dem Pilz. / The aim of this thesis was the characterization of point mutations in ifng and ncam1 with respect to an altered functionality of NK cells during interaction with A. fumigatus. In the long term, the findings should contribute to the improvement of the diagnosis, prophylaxis and therapy of invasive aspergillosis, which can occur, for example, after stem cell transplantation. In this work, the DNA of twenty healthy donors was examined for one ifng-SNP (rs2069705) and one ncam1-SNP (rs10502171). NK cells were isolated from three donors for each SNP and six control donors. These were left unstimulated, stimulated with interleukin 2/15 or A. fumigatus. In the ifng-SNP series qPCR was performed to determine the relative expression of ifng and ccl4, in the ncam1-SNP series flow cytometric analysis was performed to measure the expression of different surface markers. In both cases the release of IFN-gamma and CCL4/MIP-1ß was determined by ELISA. The results obtained in this study on ifng-SNP suggest that the presence of this ifng-SNP has no NK cell mediated effects on the risk of patients suffering from invasive aspergillosis. With regard to the ncam1-SNP, the hypothesis that the SNP alters the interaction between the NK cell and A. fumigatus was confirmed. The SNP correlates with an increased basic activation of NK cells, but also with a weaker activation potential when stimulated with the fungus.