Adaptive Evolution of Candida Biofilms

项目总结 迫切需要确定与真菌环境胁迫适应有关的重要参数。 生物膜，以开发有效的治疗策略来对抗真菌感染。真菌的治疗 假丝酵母菌的感染，包括新出现的光滑念珠菌，仍然是临床上的 挑战，特别是在免疫功能低下的人中。光滑线虫现在是第二个最常见的分离物。 假丝酵母菌在北美。在体内，微生物主要存在于生物膜中，作为保护层。单元格 在生物膜中表现出对环境压力的更强的抵抗力。由于生物膜是生物膜的组成部分 发病机制，真菌病原体在生物膜内的适应是一个重要的方面，需要更深层次的研究 理解。然而，现有的关于真菌在生物膜中适应的知识是有限的，部分原因是缺乏 建立了真菌生物膜长期繁殖的实验方法。有一个长期目标是 为制定念珠菌病的治疗策略，该项目的总体目标是 开发一种用于实验适应性进化的真菌生物膜繁殖方法，并应用于该系统 目的：鉴定光肩星毛虫生物膜中的必需基因。在目标1中，建立了一种适合于长时间培养的生物膜培养系统。 术语的体外进化将被开发和表征。正在开发的方法的一个关键特征是 能够从同一种子生物膜中培养多个生物膜，允许多个程序进行分析和 体外进化过程中每一代生物膜的特性。AIM 2将实施真菌生物膜 光叶锦鸡儿对环境胁迫的体外进化繁殖体系。关键属性，例如 将监测生物膜的生成量和生物膜结构的变化。分子机制 真菌生物膜对环境应激源的适应将从基因组的角度得到阐明。 测序和表型分析。目标3将生物膜繁殖方法与转座子相结合 对光肩星天牛生物膜形成过程中的必需基因进行测序。正在开发的方法 可广泛应用于其他微生物病原体，以更好地识别病原体如何适应和进化到更 与宿主相关的环境，并能够确定针对难治性肺炎的潜在治疗策略。 处理生物膜。R16将在四年内资助大约20名本科生(UGS)的研究， 包括许多少数民族实习生。UGS将执行大部分拟议工作，并提供培训和 一名技术人员和私家侦探的指导。这笔资金将使PI能够继续发展强有力的轨道 记录研究，为未被充分代表的少数族裔学生提供有意义的研究经验，并提高 圣何塞州立大学的研究能力。

PROJECT SUMMARY There is a critical need to identify important parameters involved in adaptation to environmental stresses in fungal biofilms, in order to develop effective therapeutic strategies against fungal infections. Treatment of fungal infections by Candida spp, including the emerging pathogen Candida glabrata (C. glabrata), remains a clinical challenge especially in immunocompromised individuals. C. glabrata is now the second most frequently isolated Candida spp in North America. In vivo, microbes mostly exist in biofilms, which serve as protective layers. Cells in biofilms exhibit increased resistance to environmental stresses. Since biofilms are an integral part of pathogenesis, the adaptation of fungal pathogens inside a biofilm is an important aspect that requires a deeper understanding. However, existing knowledge of fungal adaptation in biofilms is limited, partly due to the lack of established experimental methods for the long-term propagation of fungal biofilms. With a long-term goal of contributing to the development of therapeutic strategies for candidiasis, the overall objective of the project is to develop a fungal biofilm propagation method for use in experimental adaptive evolution, and to apply the system to identify essential genes involved in C. glabrata biofilms. In Aim 1, a biofilm culture system suitable for long- term in vitro evolution will be developed and characterized. A key feature of the method being developed is the ability to grow multiple biofilms from the same seed biofilm, allowing multiple procedures for analysis and characterization of biofilms at each passage during in vitro evolution. Aim 2 will Implement the fungal biofilm propagation system for in vitro evolution of C. glabrata to environmental stressors. Key properties, such as amount of biofilm formation and changes in biofilm structure will be monitored. Molecular mechanisms associated with adaptation to environmental stressors in fungal biofilms will be elucidated based on genome- sequencing and phenotypic analyses. Aim 3 will combine the biofilm propagation method with transposon sequencing to identify essential genes involved in biofilm formation in C. glabrata. The method being developed can be broadly applied to other microbial pathogens to better identify how pathogens adapt and evolve in a more host-relevant environment, and enables the identification of potential therapeutic strategies against difficult-to- treat biofilms. This R16 will fund the research of approximately 20 undergraduates (UGs) over four years, including many minority trainees. UGs will perform the majority of the proposed work, with training and mentorship from a technician and the PI. This funding would allow the PI to continue to develop a strong track record in research, give meaningful research experiences to underrepresented minority students, and enhance the research capacity at San Jose State University.