Ribosome Heterogeneity in Cryptococcus neoformans

侵袭性真菌感染是免疫缺陷和移植患者并发症和死亡的主要原因。有限的抗真菌药物库和抗真菌药物耐药性的出现使得对新型抗真菌治疗的需求变得至关重要。由核糖体进行的细胞蛋白质合成是原核生物和真核生物中药物干预的已知且经过充分审查的靶标，但尚未在抗真菌开发中利用，因为假定不能靶向具有真菌特异性的核糖体的核心催化功能。使用人类病原体新型隐球菌，我们已经利用两种方法来确定与宿主中遇到的应激条件下的真菌核糖体相关的因子。使用基于同源性的方法，我们已经确定了隐球菌特异性同源物的保守真核生物mRNA帽结合复合物（eIF4F），我们有名称替代eIF4G（AFG1）和替代eIF4E（AFE 1）。AFG1的缺失导致缺氧适应受损，缺氧适应是主要人类真菌病原体中的主要发病相关途径。关于Afg1和Afe1作为新型eIF4F复合物组分的功能的进一步研究概述于目标1，它们结合的mRNA靶点的鉴定概述于目标3。我们的第二种方法开发了一种新的蛋白质组学管道，我们称之为RiboPROT，以确定在适应温度应激和氧化应激期间与翻译核糖体相关的因素。本提案的目的2将研究这些蛋白在应激反应翻译和发病机制中的作用。我们已经优先考虑了我们最初的RiboPROT实验中的命中，以进一步研究，并建议在与隐球菌发病机制和生物学相关的其他条件下确定其他因素。对于具有RNA结合结构域的那些，将在目标3中鉴定同源mRNA靶标，鉴定应激反应性翻译调节子。在这些研究的结论，我们将有一套全面的核糖体相关因子，以研究在未来的工作作为抗真菌的目标。这些独特的胁迫特异性和真菌特异性核糖体相关因子可能为特异性靶向真菌核糖体提供新的途径。

Invasive fungal infections are a major cause of co-morbidity and mortality in patients living with immunodeficiency and transplantation. A limited antifungal arsenal and emergence of antifungal drug resistance have made the need for novel antifungal therapies paramount. Cellular protein synthesis carried out by the ribosome is a known and well-vetted target for pharmaceutical intervention in prokaryotes and eukaryotes, but has not been exploited in antifungal development because of the presumed inability to target the core catalytic function of the ribosome with specificity in fungi. Using the human pathogen Cryptococcus neoformans, we have leveraged two approaches to identify factors that associate with the fungal ribosome under stress conditions encountered in the host. Using a homology based approach, we have identified Cryptococcus-specific homologues of the conserved eukaryotic mRNA cap-binding complex (eIF4F) that we have names alternative eIF4G (AFG1) and alternative eIF4E (AFE1). Deletion of AFG1 results impairs hypoxia adaptation, which is a major pathogenesis-associated pathway in the major human fungal pathogens. Further work on the function of Afg1 and Afe1 as components of a novel eIF4F complex are outlined in Aim 1, and the identification of the mRNA targets they bind is outlined in Aim 3. Our second approach developed a novel proteomic pipeline that we’ve termed RiboPROT to identify the factors associating with translating ribosomes during adaptation to temperature stress and oxidative stress. Aim 2 of this proposal will investigate the role of these proteins in stress-responsive translation and pathogenesis. We have prioritized hits from our initial RiboPROT experiments to investigate further, and propose to identify additional factors under additional conditions relevant to cryptococcal pathogenesis and biology. For those with RNA-binding domains, cognate mRNA targets will be identified in Aim 3, identifying stress responsive translational regulons. At the conclusion of these studies, we will have a comprehensive set of ribosome-associating factors to investigate in future work as antifungal targets. These unique stress-specific and fungi-specific ribosome-associating factors may provide a novel pathway to targeting the fungal ribosome with specificity.