Genetic regulation of invasive hyphal growth of Aspergillus fumigatus

描述(由申请人提供)：我们建议鉴定烟曲霉侵袭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霉病的主要病原体，侵袭性曲霉病是一种全球性疾病，尤其对患有白血病、肺部疾病、癌症或实体器官移植的人构成威胁。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超过50%。在这些患者中，最统一的临床特征是由曲霉菌丝入侵造成的肺组织和血管的破坏。由于完全避免这种无处不在的真菌几乎是不可能的，防止或限制曲霉侵袭性菌丝生长是控制侵袭性曲霉病的关键。在适当的环境中，烟曲霉的孢子发出被称为菌丝的长管细胞，并通过尖端延伸生长以寻找营养。当烟曲霉的孢子通过吸入进入易感宿主的呼吸系统时，它们会形成高度极化的菌丝，入侵肺部和其他器官，并对这些宿主组织造成破坏。我们推测，侵袭性菌丝生长使烟曲霉菌能够有效地在环境中的固体介质中寻找潜在的食物来源，这也是该真菌入侵人体组织并导致侵袭性曲霉病的重要特征。因此，侵袭性菌丝生长所需的分子和生化途径是很有希望的抗真菌靶标。然而，烟曲霉菌侵袭性菌丝生长的分子细节仍不清楚。我们建议使用正向遗传方法系统地发现调节烟曲霉侵袭性菌丝生长的遗传位点。我们将：(1)产生额外的30,000个随机插入烟曲霉突变体；(2)在体外筛选突变体库，寻找有缺陷的侵袭性菌丝生长，并利用反向PCR和测序技术在所选突变体中鉴定突变基因。(3)通过目的基因的缺失和互补，确定与侵袭性菌丝生长相关的候选基因的子集。我们最近开发了一种高效而简单的方法，通过农杆菌介导的转化产生大量的随机插入突变体。用这种方法产生的8000个突变体进行的初步研究表明，我们提出的目标是可以实现的。虽然这超出了这一建议的范围，但所有已确定的有希望的遗传位点将通过靶基因的缺失和互补来确认，并将在未来使用动物模型来研究它们在发病机制中的作用。我们提议的研究将从功能上揭示控制这种重要人类病原体侵袭性菌丝生长的遗传成分。该项目是开发更全面地了解生长特性的垫脚石，这些特性使寄生在堆肥上的腐生真菌烟曲霉菌能够在人类中引起侵袭性疾病。 公共卫生相关性：烟曲霉是侵袭性曲霉病的主要病原体，侵袭性曲霉病是一种免疫受损或免疫抑制的人通常会致命的疾病。这种真菌通过菌丝侵入宿主组织和器官而引起侵袭性真菌病。这项拟议的研究将确定允许烟曲霉菌以菌丝形式侵袭生长的基因，这是一种对其致病性至关重要的生长特征。从拟议的研究中获得的信息将被利用来开发控制侵袭性曲霉病的新机制，并允许发现有前景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. .