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，这是一种全球性疾病，尤其威胁白血病、肺病、癌症或实体器官移植的个体。尽管有积极的抗真菌治疗，侵袭性曲霉病的总死亡率仍大于50%。在这些受折磨的个体中，最统一的临床特征是由曲霉菌丝入侵破坏肺组织和血管。由于完全避免这种无处不在的真菌几乎是不可能的，预防或限制曲霉侵入性菌丝的生长对控制侵入性曲霉病至关重要。在适当的环境下，烟曲霉的孢子发出被称为菌丝的长管状细胞，并通过尖端延伸寻找营养物质生长。烟曲霉孢子经吸入进入易感寄主的呼吸系统后，形成高度极化的菌丝，侵入肺部等器官，对寄主的这些组织造成破坏。我们推测，侵袭性菌丝生长使烟曲霉能够有效地探测环境中固体介质中潜在的食物来源，这也是真菌侵入人体组织并引起侵袭性曲霉病的重要特征。因此，侵入性菌丝生长所需的分子和生化途径代表了有希望的抗真菌靶点。然而，烟曲霉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揭示烟曲霉侵袭性菌丝生长的遗传位点。我们将：(1)随机增加3万个插入的烟熏假单胞菌突变体；(2)对收集的突变体体外筛选侵入性菌丝生长缺陷，并利用反相PCR和测序技术鉴定所选突变体中的突变基因。(3)通过靶基因的缺失和互补，确定了参与侵袭性菌丝生长的候选基因子集。我们最近开发了一种高效、简化的方法，通过农杆菌介导的转化产生大量随机插入突变体。一项使用该方法产生的8000个突变体的初步研究表明，我们提出的目标是可以实现的。虽然这超出了本提案的范围，但所有被发现的有希望的基因位点都将通过靶基因的缺失和互补来证实，并在未来使用动物模型来研究它们在发病机制中的作用。我们提出的研究将从功能上揭示在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。该项目为更全面地了解烟曲霉（a . fumigatus，一种生活在堆肥上的腐生真菌）在人类中引起侵袭性疾病的生长特性奠定了基础。

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. .