Ribosome Heterogeneity in Cryptococcus neoformans

侵袭性真菌感染是免疫缺陷患者和移植患者共同发病和死亡的主要原因。有限的抗真菌武器库和出现的抗真菌耐药性使得对新的抗真菌疗法的需求变得至关重要。核糖体进行的细胞蛋白质合成是原核生物和真核生物药物干预的已知和经过广泛审查的靶点，但由于假定无法针对真菌中核糖体的核心催化功能而被开发，因此尚未被开发用于抗真菌开发。使用人类病原体新生隐球菌，我们利用两种方法来确定在宿主遇到的应激条件下与真菌核糖体相关的因素。利用基于同源性的方法，我们已经鉴定了保守的真核生物mRNA帽结合复合体(EIF4F)的隐球菌特异性同源物，我们将其命名为替代eIF4G(AFG1)和替代eIF4E(AFE1)。AFG1缺失导致缺氧适应受损，这是人类主要真菌病原体的一个主要致病相关途径。目标1概述了Afg1和Afe1作为新的eIF4F复合体组件的功能，目标3概述了它们结合的mRNA靶标的鉴定。我们的第二种方法开发了一种新的蛋白质组流水线，我们称之为RiboPROT，以确定在适应温度应激和氧化应激过程中与核糖体翻译相关的因素。该提案的目的2将研究这些蛋白在应激反应翻译和发病机制中的作用。我们已经将最初的RiboPROT实验的命中结果放在了优先位置，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。对于那些具有RNA结合结构域的人，将在目标3中识别同源mRNA靶标，识别应激反应的翻译调节因子。在这些研究的结论中，我们将有一套全面的核糖体相关因子作为抗真菌靶点进行研究。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了一条新的途径。

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.