Genetic regulation of invasive hyphal growth of Aspergillus fumigatus

描述（由申请方提供）：我们建议鉴定烟曲霉侵入性菌丝生长所需的遗传位点。这种无处不在的真菌是侵袭性曲菌病的主要病原体，侵袭性曲菌病是一种全球性疾病，特别威胁白血病、肺病、癌症或实体器官移植患者。尽管进行了积极的抗真菌治疗，侵袭性曲霉病的总死亡率仍大于50%。这些患者最统一的临床特征是曲霉菌菌丝侵入破坏肺组织和血管。由于完全避免这种普遍存在的真菌几乎是不可能的，预防或限制曲霉侵入性菌丝生长是控制侵袭性曲霉病的关键。 在适当的环境中，A.烟曲霉发出称为菌丝的长管状细胞，并通过尖端延伸寻找营养物质而生长。当A.烟曲霉孢子通过吸入进入易感宿主的呼吸系统，它们形成高度极化的菌丝，侵入肺部和其他器官，并导致这些宿主组织的破坏。我们假设，侵入性菌丝生长，使A。烟曲霉能够有效地在环境中的固体介质中探测潜在的食物来源，也是真菌侵入人体组织并引起侵袭性曲霉病的重要特征。侵入性菌丝生长所需的分子和生化途径，因此，代表有前途的抗真菌靶点。然而，潜在的A.烟曲霉侵入性菌丝生长仍然不清楚。 我们建议使用正向遗传学方法系统地揭示调节A.烟曲霉侵入性菌丝生长。我们将：（1）生成额外的30，000个随机插入A。烟曲霉突变体;（2）体外筛选突变体收集物的侵入性菌丝生长缺陷，并使用反向PCR和测序鉴定所选突变体中的突变基因。(3)通过目的基因的缺失和互补，确定了一个与侵入性菌丝生长有关的候选基因子集。我们最近发展了一种有效而简单的方法来产生大量的随机插入突变体。通过农杆菌介导的转化法转化烟曲霉。初步研究使用8000突变体产生的这种方法表明，我们提出的目标可以实现。虽然这超出了本提案的范围，但所有确定的有希望的遗传位点都将通过靶基因缺失和互补来确认，并且它们在发病机制中的作用将在未来使用动物模型进行检查。 我们提出的研究将在功能上揭示这种重要的人类病原体中控制侵入性菌丝生长的遗传成分。这个项目是一个垫脚石，为发展一个更全面的了解生长特性，使A。烟曲霉，一种生活在堆肥中的腐殖真菌，在人类中引起入侵性疾病。 公共卫生相关性：烟曲霉是侵袭性曲霉病的主要病原体，侵袭性曲霉病是免疫功能低下或免疫抑制个体的常见致命疾病。这种真菌通过菌丝侵入宿主组织和器官引起侵袭性真菌病。这项拟议中的研究将确定允许A.烟曲霉以菌丝形式侵入性生长，这是对其致病性重要的生长特征。从拟议的研究中获得的信息将被利用来开发新的机制来控制侵袭性曲霉病，并允许发现有前途的新的抗真菌药物靶点。 .

DESCRIPTION (provided by applicant): We propose to identify genetic loci required for invasive hyphal growth in Aspergillus fumigatus. This ubiquitous fungus is the leading causative agent of invasive aspergillosis, a global disease that particularly threatens individuals with leukemia, lung disease, cancer, or solid organ transplantation. Despite aggressive antifungal therapy, the overall mortality rate of invasive aspergillosis is greater than 50%. The most unifying clinical feature among those afflicted individuals is destruction of lung tissue and blood vessels by Aspergillus hyphal invasion. Because complete avoidance of this ubiquitous fungus is nearly impossible, prevention or restriction of Aspergillus invasive hyphal growth is essential in controlling invasive aspergillosis. In the proper environment, spores of A. fumigatus send out long tubular cells known as hyphae and grow by tip extension searching for nutrients. When A. fumigatus spores enter the respiratory system of susceptible hosts through inhalation, they form highly polarized hyphae, invade the lungs and other organs, and cause destruction of these host tissues. We hypothesize that invasive hyphal growth, which enables A. fumigatus to efficiently probe potential food sources in solid media in the environment, is also an important characteristic for the fungus to invade human tissues and cause invasive aspergillosis. Molecules and biochemical pathways required for invasive hyphal growth, therefore, represent promising antifungal targets. However, the molecular details of underlying A. fumigatus invasive hyphal growth have remained unclarified. We propose to use forward genetic approaches to systematically uncover genetic loci that regulate A. fumigatus invasive hyphal growth. We will: (1) Generate additional 30,000 random insertional A. fumigatus mutants; (2) Screen the mutant collection in vitro for defective invasive hyphal growth and identify mutated genes in selected mutants using inverse PCR and sequencing. (3) Confirm a subset of candidate genes involved in invasive hyphal growth by target gene deletion and complementation. We have recently developed an efficient and simplified method to generate a large number of random insertional mutants of A. fumigatus via Agrobacterium-mediated-transformation. A pilot study using 8000 mutants generated by this method indicates that our proposed aims can be accomplished. Although it is beyond the scope of this proposal, all the promising genetic loci identified will be confirmed by target gene deletion and complementation, and their roles in pathogenesis will be examined in the future using animal models. Our proposed research will functionally uncover genetic components that govern invasive hyphal growth in this important human pathogen. This project is a stepping stone for the development of a more comprehensive understanding of growth characteristics that enable A. fumigatus, a saprophytic fungus living on compost, to cause invasive diseases in humans. PUBLIC HEALTH RELEVANCE: Aspergillus fumigatus is the leading causative agent of invasive aspergillosis, an often fatal disease in immuno-compromised or immuno-suppressed individuals. This fungus causes invasive mycoses through hyphal invasion of host tissue and organs. The proposed research will identify genes that allow A. fumigatus to grow invasively in the hyphal form, a growth characteristic important for its pathogenicity. Information obtained from the proposed research will be harnessed to develop novel mechanisms to control invasive aspergillosis and allow for the discovery of promising new antifungal drug targets. .