Although QN was the first therapeutic compound used to treat malaria infection , its mechanism of action has never been fully resolved [6–8]. Some evidence suggests that parasite resistance to QN is associated with mutations and/or elevated copy number of the pfmdr1 gene, which encodes for a transporter protein found in the membrane of the parasite food vacuole [9, 10, 12–14, 16, 18, 20]. Here, the natural fluorescent properties of QN were exploited to obtain insight into the mechanism of action of the drug. Although knowledge of QN’s fluorescent properties has been around since the late-1800s , this is the first study to employ the QN’s fluorescence for imaging in the malaria parasite.
Fluorescence microscopy was employed to image QN subcellular localization in two P. falciparum strains that contained different pfmdr1 copy numbers. Quinine consistently overlapped with the haemozoin crystals in both strains when evaluated by 2-D microscopy (Figure 1). However, upon 3-D reconstruction of serial z-stack images, QN was found to reside in a distinct compartment, which is contiguous to, but separate from, the compartment stained by LysoTracker Red. The lack of co-localization with the acidotropic dye suggests that QN resides in a non-acidic compartment within the food vacuole, possibly the same one occupied by haemozoin. This would be consistent with previous reports that quinolone compounds including QN interact with haemozoin crystals directly or with enzymes involved in the haemozoin crystallization process, as previously reported [7, 8, 29–33]. In summary, these findings suggest that QN is localized in a non-acidic compartment in the food vacuole, possibly that which contains haemozoin.
This study underscores the importance of utilizing the 3-D reconstruction software in imaging studies, since the localization of QN into this novel compartment would not have been detected otherwise. A recent study revealed that QN-haem adducts exhibit fluorescence at least seven-fold greater than QN alone . Thus, it is possible that QN exists in other areas of the food vacuole but cannot be visualized due to the fluorescence intensity of the QN-haem adducts present in these parasites.
Although there was no apparent difference in localization in strains containing different pfmdr1 copy numbers, the possibility that the pfmdr1 gene has a role cannot be ruled out. Single nucleotide polymorphisms in pfmdr1 have also been associated with decreased sensitivity to QN [10, 13, 14]. Because the protein encoded by pfmdr1 is a membrane transporter that pumps solutes into the food vacuole, it is possible that mutations within the pfmdr1 gene could affect the transporter function of the protein by altering the conformation or function of the transporter protein.
In summary, this study is novel because it is the first to exploit quinine fluorescence to study the intracellular distribution of the drug. Here, QN was shown to enter a distinct, non-acidic compartment inside the parasite food vacuole. These results are important because they provide visual support for the hypothesis that QN interferes with haemozoin production, which could guide future studies to investigate a possible interaction between QN and enzymes involved in the haemozoin formation process.