CAREER: Cellular Mechanisms of Killer Toxin Resistance in Yeasts

该奖项的全部或部分资金来自《2021年美国救援计划法案》(公法117-2)。真菌细胞通过构建由碳水化合物和蛋白质组成的坚固的细胞壁和底层膜来将自己与环境隔离开来。这使得新陈代谢过程能够被区分开来，并保护真菌细胞免受外部应激源的影响，包括被广泛用于控制有害真菌生长的商业杀菌剂。不同种类的真菌产生的抗真菌蛋白可以攻击细胞壁和细胞膜，导致敏感真菌细胞的伤害和死亡。这些抗真菌药物被认为可能有助于控制不良真菌的生长。这项研究将探索真菌抵抗抗真菌蛋白中毒的基本细胞机制。具体地说，该项目将测试用于细胞壁和膜构建和稳定性的基因突变如何导致对抗真菌蛋白的抗药性。抗真菌蛋白是由很大一部分真菌产生的，特别是与昆虫、水果和发酵(如酿造和烘焙)有关的酵母菌。公众对酵母的普遍熟悉将使学童和当地农贸市场的顾客能够参与分离产生新的抗真菌蛋白的酵母。这些外展活动将通过本科生的参与得到加强，以便能够识别新的抗真菌蛋白及其应用，以进一步研究真菌膜和细胞壁的功能和组织。来自爱达荷大学代表性不足群体的学生也将被纳入研究活动。这种方法的中心目标是增加大学生的保留率，提高高等教育中大专和本科毕业生的入学率，并改善公共STEM教育。本研究的目标是发现真菌对抗真菌“杀手”毒素产生耐药性的重要细胞机制。这将发现对细胞表面功能和弹性很重要的新途径。研究假设是ACE2和细胞形态发生(RAM)信号网络在细胞壁和细胞膜组织中发挥着未知的作用，并且Kre1p的定位和多样性是杀手毒素中毒所必需的。这些假说是基于初步数据提出的，这些数据已经确定了RAM网络中的突变，这些突变导致杀手毒素抗性以及膜和细胞壁完整性的丧失。为了验证这些假设，将确定RAM网络中导致杀手毒素抗性的突变如何改变与细胞质分裂和细胞极性有关的网络组织、定位和功能。具体地说，将测试这些突变对杀手毒素膜受体(Kre1p)定位的影响。这一方法将得到对Kre1p多样性对杀手毒素抗性的影响以及杀手酵母防止自身中毒的免疫机制的深入研究。这项研究的结果将证实RAM网络在细胞表面组织中的新角色。这将得到对RAM网络和Kre1p的调查及其在抵抗杀手毒素方面的作用的补充。这一裁决反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).Fungal cells separate themselves from their environment by constructing a robust cell wall made of carbohydrates and proteins with an underlying membrane. This enables the compartmentalization of metabolic processes and protects fungal cells from external stressors, including commercial fungicides that are used extensively to control unwanted fungal growth. Antifungal proteins produced by different species of fungi can attack the cell wall and membrane causing injury and the death of susceptible fungal cells. These antifungals have been cited as potentially being useful to control the growth of undesirable fungi. This research will explore the fundamental cellular mechanisms that are employed by fungi to resist intoxication by antifungal proteins. Specifically, the project will test how mutations in genes used for cell wall and membrane construction and stability result in resistance to antifungal proteins. Antifungal proteins are produced by a significant proportion of fungi, especially by yeasts that are associated with insects, fruits, and fermentation (e.g. brewing and baking). The general familiarity of the public with yeasts will enable schoolchildren and patrons of the local farmer’s market to participate in the isolation of yeasts that produce novel antifungal proteins. These outreach activities will be enhanced by the participation of undergraduate research students to enable the identification of novel antifungal proteins and their application to further investigate the function and organization of the fungal membrane and cell wall. Students from underrepresented groups at the University of Idaho will also be included in research activities. The central goal of this approach is to increase retention of university students, enrollment in higher education of postsecondary and postbaccalaureate students, and to improve public STEM education.The goal of this research is to discover the cellular mechanisms that are important for fungal resistance to antifungal “killer” toxins. This will uncover novel pathways that are important for cell surface function and resilience. The research hypotheses are that the Regulator of Ace2 and Cell Morphogenesis (RAM) signaling network plays an undescribed role in cell wall and membrane organization, and that the localization and diversity of the killer toxin receptor Kre1p is essential for intoxication by killer toxins. These hypotheses were formulated based on preliminary data that has identified mutations in the RAM network that result in killer toxin resistance and the loss of membrane and cell wall integrity. To test these hypotheses, it will be determined how mutations in the RAM network that cause killer toxin resistance alter the networks organization, localization, and function relating to cytokinesis and cell polarity. Specifically, the effect of these mutations on the localization of the killer toxin membrane receptor (Kre1p) will be tested. This approach will be complemented by an in-depth study of the effect of Kre1p diversity on killer toxin resistance and the immunity mechanisms of killer yeasts that prevent self-intoxication. The outcome of this research will be to confirm the novel role of the RAM network in cell surface organization. This will be complemented by the investigation of the RAM network and Kre1p and their role in killer toxin resistance.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.