Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, Giessen, Germany.
INRES-Chemical Signaling, University of Bonn, Bonn, Germany.
PLoS Pathog. 2013;9(12):e1003782. doi: 10.1371/journal.ppat.1003782. Epub 2013 Dec 5.
In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be -314.2±3.1 mV and -313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.
在疟原虫恶性疟原虫中，细胞内氧化还原电势影响信号事件、抗氧化防御以及药物作用和耐药机制。到目前为止，由于传统方法会破坏亚细胞完整性，因此一直限制了对疟原虫内氧化还原电势的实时测定。我们使用由与人谷胱甘肽还原酶 1 连接的氧化还原敏感的绿色荧光蛋白 (hGrx1-roGFP2) 组成的谷胱甘肽生物传感器，系统地描述了对药物敏感 (3D7) 和耐药 (Dd2) 恶性疟原虫寄生虫细胞溶质谷胱甘肽依赖性氧化还原电势 (EGSH) 的基础值和药物诱导的变化。通过共焦显微镜，我们证明 hGrx1-roGFP2 可以快速检测氧化和硝化应激诱导的 EGSH 变化。3D7 和 Dd2 的细胞溶质基础 EGSH 分别估计为-314.2±3.1 mV 和-313.9±3.4 mV，表明这是一个高度还原的隔室。我们还监测了用各种氧化还原活性化合物和抗疟药物孵育后 EGSH 的短期、中期和长期变化。有趣的是，亚甲蓝和绿脓菌素这两种氧化还原循环剂迅速改变了恶性疟原虫细胞质中 hGrx1-roGFP2 的荧光比值，但这可以部分解释为与探针的直接相互作用。相比之下，奎宁和基于青蒿素的抗疟药物在较长的孵育时间 (24 小时) 后对寄生虫的 EGSH 表现出强烈的影响。在各种条件下测试时，这些影响伴随着用替代方法平行测定的总谷胱甘肽浓度下降。值得注意的是，这些影响在氯喹敏感的 3D7 株中比在耐药的 Dd2 株中更为明显。基于这些结果，hGrx1-roGFP2 可以作为一种可靠且特异的生物传感器，用于实时时空监测恶性疟原虫细胞内 EGSH。在进一步的研究中应用这项技术将增强我们对疟原虫氧化还原调节和药物作用与耐药机制的理解，也可能刺激其他病原体的氧化还原研究。
