Background
Quinine (QN) was isolated from the bark of the Peruvian Cinchona tree in the mid-1800s [
1] and quickly became the treatment of choice for intermittent fever worldwide [
2]. Although efficacious, QN use as an anti-malarial requires a relatively long treatment regimen and has substantial side effects. As a result, artemisinin-based combination therapy (ACT) has now been implemented as first-line treatment regimens due to drug efficacy and better patient tolerance. However, QN remains an important treatment option for severe malaria infections [
3‐
5]. Despite the longevity of its use as an anti-malarial, the mechanism of action of QN has not yet been fully resolved.
Early investigations into the anti-malarial activity of QN demonstrated the ability to inhibit chloroquine-induced clumping of haemozoin in the parasite food vacuole [
6]. Subsequent evidence found that, like chloroquine, QN interferes with haem crystallization [
7,
8], indicating that QN acts in the food vacuole. In recent years, parasite resistance to QN has been reported in Africa and Southeast Asia, prompting investigations into resistance mechanisms with a focus on the food vacuole [
4,
9‐
13]. Decreased QN sensitivity has been strongly linked to a protein on the food vacuolar membrane, encoded by the
Plasmodium falciparum multidrug resistance (
pfmdr1) gene [
10‐
12,
14‐
16]. Whereas some studies have found that
pfmdr1 mutations exert a significant effect on
in vitro QN sensitivity [
10,
13,
14], other studies have found no association between
pfmdr1 mutations and QN sensitivity [
17‐
19]. Elevated
pfmdr1 gene copy number, however, has been linked to reduced parasite susceptibility to QN in both
in vitro and clinical studies [
12,
16,
18‐
20]. Thus,
pfmdr1 has an effect on quinine activity.
Quinine is an aromatic alkaloid that naturally fluoresces under ultraviolet (UV) light. Because of its relatively constant fluorescence quantum yield, QN is commonly used in photochemistry as a fluorescence standard [
21]. Surprisingly, the inherent fluorescence of QN has not previously been exploited to investigate its mechanism of action. In this study, the fluorescent properties of QN were exploited to evaluate subcellular localization of the drug by fluorescence microscopy in parasites containing different
pfmdr1 copy numbers in order to determine if copy number of the gene affects drug localization.
Discussion
Although QN was the first therapeutic compound used to treat malaria infection [
1], 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 [
28], 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 [
34]. 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.
Conclusions
Quinine localizes to a non-acidic compartment within the food vacuole and pfmdr1 copy number does not affect QN subcellular localization. These results support previous findings that QN acts in the parasite food vacuole, perhaps interfering with haemozoin formation. This is the first study to provide evidence for QN localization in a sub-compartment of the food vacuole.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EBB participated in the design of the study, carried out all of the fluorescence microscopy experiments and data analysis, and drafted the manuscript. MC participated in the acquisition and analysis of microscopy data. SRM conceived of the study and participated in its design. All authors read and approved the final manuscript.