Mechanisms of an Aspergillus fumigatus virulence mutant

描述(申请人提供)：曲霉属的成员是常见的动植物病原体。烟曲霉是人类大多数曲霉菌感染的原因，然而，无法识别真正的毒力因子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被鉴定为毒性降低。LaeA是曲霉次生代谢的转录调节因子，而烟曲霉LaeA突变体在产生包括真菌毒素(包括胶质毒素、Helvolic酸、烟青素)和孢子代谢物在内的大量次生代谢产物方面受到损害。在肺小鼠模型中，毒力的丧失与胶质毒素水平的丧失和相关的死亡率和生长减少有关，增加了对巨噬细胞吞噬的敏感性，并降低了杀死中性粒细胞的能力。这项研究的总体目标是阐明导致DlaeA突变体毒力降低的机制，重点是鉴定有助于致病性的次生代谢物。具体目标包括：目的1.确定胶质毒素产生的损失对DlaeA表型的贡献。这将通过检测一种胶质毒素突变体DgliZ，并在小鼠肺模型以及巨噬细胞和中性粒细胞检测中将该突变体与DlaeA和WT进行毒力比较来实现。目的2.鉴定与烟曲霉致病相关的其他菌丝和分生孢子次生代谢物基因。该方法将包括通过微阵列分析在体外(介质)和体内(小鼠肺)鉴定参与菌丝代谢物生物合成的基因。孢子代谢产物的化学纯化和反向遗传学将被用来鉴定编码基因。目的3.烟曲霉次级代谢产物的功能分析。与目标1一样，目标2中确定的基因将被干扰，突变的化学定义和毒力检测。这项工作的进展将导致鉴定与烟曲霉致病有关的毒力因子。对致病因子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.