Hard-wiring in the Plasmodium falciparum transcriptome 2

To help malaria parasites survive unpredictable host immune responses, it is known that genes for surface proteins express stochastically in Plasmodium falciparum. Here, we demonstrate that gene expression for intracellular metabolic functions may be preordained and insensitive to specific metabolic perturbations. In a tightly-controlled, large microarray study involving over 100 hybridizations to isogenic drug-sensitive and drug-resistant parasites, the lethal antifolate WR99210 failed to over-produce RNA for the biochemically and genetically proven target dihydrofolate reductase-thymidylate synthase (DHFR-TS). Beyond the target, this transcriptional obstinacy carried over to the rest of the parasite genome, including genes for target pathways of folate and pyrimidine metabolism. Even 12 hours after commitment to death, the transcriptome remained faithful to evolutionarily entrained paths. A system-wide transcriptional disregard for metabolic perturbations in malaria parasites may contribute to selective vulnerabilities of the parasite to lethal antimetabolites. While large protective metabolic responses were not detected, DNA microarrays helped capture small, but reproducible drug-dependent perturbations within hours of drug exposure. In addition, in Plasmodium cells that had adapted to long-term drug exposure, DNA microarrays revealed new, large genome-wide transcriptional adjustments in the hard-wired transcriptional program itself. Keywords: Plasmodium falciparum treated with pyrimethamine RNA from pyrimethamine-treated parasite vs RNA from untreated control, Pyr-sensitive TM4/8.2 strain, pyrimethamine concentration at IC50 and treated for 0 h and 24 h, microarray data were obtained from at least four hybridizations using RNA from at lease two independent parasite cultures

为帮助疟原虫应对宿主不可预测的免疫应答，目前已知恶性疟原虫（Plasmodium falciparum）的表面蛋白基因会以随机方式表达。本研究证实，细胞内代谢功能相关的基因表达或已预先设定，且不受特定代谢扰动的影响。在一项严格受控的大型微阵列（microarray）研究中，我们对同基因的药物敏感型与耐药型疟原虫完成了超100次杂交实验，结果显示致死性抗叶酸剂WR99210并未使经生化与遗传学验证的靶标——二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）——的RNA转录水平上调。除靶标基因外，这种转录层面的固有惰性还延伸至疟原虫基因组的其余区域，包括叶酸与嘧啶代谢的靶标通路相关基因。即便疟原虫进入死亡程序后的12小时内，其转录组仍严格遵循进化上固有的表达路径。疟原虫中存在的全系统转录不响应代谢扰动的现象，或会使该寄生虫对致死性抗代谢物产生选择性易感弱点。尽管未检测到大规模的保护性代谢应答，但DNA微阵列（DNA microarray）技术可在药物暴露后数小时内捕捉到微小但可重复的药物依赖性转录扰动。此外，在适应了长期药物暴露的疟原虫细胞中，DNA微阵列技术还揭示了其固有转录程序本身发生了全新的大规模全基因组转录调控变化。关键词：经乙胺嘧啶（pyrimethamine）处理的恶性疟原虫：乙胺嘧啶处理组疟原虫RNA与未处理对照组RNA， 嘧啶敏感型TM4/8.2菌株，采用半数抑制浓度（IC50）的乙胺嘧啶处理0小时与24小时，微阵列数据来自至少两次独立疟原虫培养的RNA所完成的至少四次杂交实验。