Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly.  After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites.  While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and post-translational mechanisms. One mRNA sample from each of Purine-Starved and Purine-Replete cells were analyzed using SL RNA-Seq methodology

利什曼原虫（Leishmania）在昆虫宿主或哺乳动物宿主体内的存活能力，依赖于其感知并适应微环境变化的能力。然而，目前对于该寄生虫响应环境变化（如营养可获得性）的分子机制，尚缺乏深入认知。为阐明杜氏利什曼原虫（Leishmania donovani）的营养应激响应通路，我们以嘌呤饥饿作为研究范式。寄生虫从宿主环境中获取嘌呤是其复制所必需的，但经嘌呤饥饿处理的寄生虫仍可在无外源补充嘌呤的培养基中存活超过3个月，这表明其对嘌呤饥饿的响应极为稳健，可使寄生虫在极端营养匮乏条件下维持存活。为解析嘌呤饥饿过程中的代谢重编程事件，我们采用了全局组学研究策略。在6至48小时的时间周期内，对嘌呤饥饿组与嘌呤充足组寄生虫开展全蛋白质组比较分析，结果揭示了寄生虫对嘌呤饥饿的时序性协同响应。在培养基移除嘌呤后的数小时内，参与细胞表面嘌呤摄取的嘌呤转运蛋白及相关酶即被上调；而其他关键嘌呤补救合成组分则在后续时间点以相对温和的幅度被上调。至处理后48小时，嘌呤饥饿寄生虫的蛋白质组已发生广泛重塑，其对嘌呤应激的适应似乎同时针对嘌呤匮乏与一般应激两种场景进行了适配。为探究介导嘌呤饥饿响应的蛋白质组重塑的分子机制，我们对嘌呤饥饿组与嘌呤充足组寄生虫开展了比较RNA测序（RNA-seq）、实时定量反转录PCR（qRT-PCR）及荧光素酶报告基因检测实验。尽管少数蛋白质的调控水平可与mRNA水平的变化保持一致，但对于多数受调控的蛋白质而言，嘌呤应激过程中的蛋白质组重塑主要通过翻译及翻译后调控机制实现。我们分别采集嘌呤饥饿组与嘌呤充足组细胞的mRNA样本，采用剪接前导RNA测序（SL RNA-Seq）方法完成分析。