PDF(Pubmed)
Abstract：
Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs\' mode of action, with possible implications for target identification and drug development.
摘要：
热带疟疾,由寄生虫恶性疟原虫(P.恶性疟原虫)，仍然是人类最大的公共卫生负担之一。由于它在寄生虫生存中的关键作用，恶性疟原虫的能量代谢是药物设计的一个有趣的目标。为此,中心代谢物三磷酸腺苷（ATP）的分析是非常感兴趣的。到目前为止,该系统中只有细胞破坏性或增强ATP测定可用，机械解释的各种缺点和部分不一致的结果。为了解决这个问题，我们已经建立了荧光探针，基于Förster共振能量转移（FRET），被称为ATeam，用于血液阶段的寄生虫。ATEAM能够以比率计量的方式测量MgATP2-水平，从而有助于使用荧光显微镜和读板器检测实时测量ATP动力学，并克服已建立的ATP分析方法的许多障碍。此外,我们在恶性疟原虫中建立了比例pH传感器pHluorin（sfpHluorin）的超折叠变体，以监测pH稳态并控制pH波动，这可能会影响ATeam测量。我们在体外表征了重组ATeam和sfpHluorin蛋白，并将传感器稳定地整合到恶性疟原虫NF54attB细胞系的基因组中。使用这些新工具，我们发现了由几种不同药物类别引起的不同的传感器反应模式.芳基氨基醇增加，氧化还原循环减少ATP；多西环素引起第一个周期的胞质溶胶碱化；4-氨基喹啉引起异常的蛋白水解。我们的结果为药物的作用模式开辟了一个全新的视角，可能对靶标识别和药物开发产生影响。
