Mechanisms of an Aspergillus fumigatus virulence mutant

描述（由申请人提供）：子囊菌属曲霉属的成员是常见的动植物病原体。烟曲霉引起大多数人类曲霉感染，然而，无法确定真正的毒力因素阻碍了这种顽固性霉菌的治疗进展。最近，一种烟曲霉突变体，DlaeA，已被表征为毒性降低。LaeA是曲霉次生代谢的转录调节因子，而烟曲霉的LaeA突变体在许多次生代谢物的产生中受损，包括真菌毒素（包括胶质毒素、helvolic acid、fumigatus）和孢子代谢物。在肺小鼠模型中，毒力的丧失与胶质毒素水平的丧失、死亡率和生长的降低、对巨噬细胞吞噬的易感性增加和杀死中性粒细胞的能力下降有关。本研究的总体目标是阐明导致DlaeA突变体毒力下降的机制，重点是确定与致病性有关的次级代谢物。具体目标包括：目标1。确定胶质毒素产生损失对DlaeA表型的贡献。这将通过检查胶质毒素突变体DgliZ，并将该突变体与DlaeA和WT在肺小鼠模型中的毒力以及巨噬细胞和中性粒细胞测定进行比较。目标2。鉴定与烟曲霉致病性有关的其他菌丝体和分生孢子次生代谢产物的基因。该方法将包括通过微阵列分析在体外（培养基）和体内（小鼠肺）鉴定参与菌丝代谢物生物合成的基因。对孢子代谢物进行化学纯化，然后进行反向遗传，以确定编码基因。目标3。烟曲霉次生代谢物的功能分析。在Aim 2中发现的基因将被破坏，化学定义突变，并检查Aim 1中的毒力。这项工作的进展将导致鉴定赋予烟曲霉致病性的毒力因素。对毒力因素的了解将导致治疗方法，以帮助保护公众免受这种威胁生命的霉菌的感染。

DESCRIPTION (provided by applicant): Members of the Ascomycete genus Aspergillus are common animal and plant pathogens. Aspergillus fumigatus causes the majority of Aspergillus human infections, however, inability to identify bona-fide virulence factors has impeded therapeutic advances of this recalcitrant mold. Recently an A. fumigatus mutant, DlaeA, has been characterized that is reduced in virulence. LaeA is a transcriptional regulator of secondary metabolism in the Aspergilli and the A. fumigatus DlaeA mutant is impaired in the production of numerous secondary metabolites including mycotoxins (including gliotoxin, helvolic acid, fumagillin) and spore metabolites. Loss of virulence is associated with loss of gliotoxin levels and associated reduced mortality and growth in a pulmonary murine model, increased susceptibility to macrophage phagocytosis and decreased ability to kill neutrophil cells. The overall goal of this research is to elucidate the mechanisms leading to decreased virulence of the DlaeA mutant with emphasis on identifying the secondary metabolites contributing to pathogenicity. Specific aims include: Aim 1. Determine the contribution of loss of gliotoxin production to the DlaeA phenotype. This will be achieved through examination of a gliotoxin mutant, DgliZ, and comparison of this mutant to DlaeA and WT for virulence in pulmonary murine model and macrophage and neutrophil assays. Aim 2. Identify genes of other mycelial and conidial secondary metabolites involved in A. fumigatus pathogenicity. The method will consist of identifying the genes involved in biosynthesis of mycelial metabolites via microarray analysis in vitro (media) and in vivo (mouse lung). Chemical purification of spore metabolites followed by reverse genetics will be employed to identify encoding genes. Aim 3. Functional analysis of A. fumigatus secondary metabolites. Genes identified in Aim 2 will be disrupted, mutant chemically defined and examined for virulence as in Aim 1. Advances from this work will result in identification of virulence factors conferring pathogenicity to Aspergillus fumigatus. Knowledge of virulence factors will lead to therapies to help protect the public from infections from this life threatening mold.