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Pfcrt AND CHLOROQUINE RESISTANCE IN Plasm o d iu m
falcipa r u m
The disease malaria, which is caused by single - celled parasites of the genus Plasmodiu m , is one of the greatest causes of human suffering in the world today. Indeed, every year 500 million people are infected with malaria and 2,500,000 of these, mostly children, will die from the disease (Ursos & Roepe, 2002). This has come after the develop me n t of the anti - malarial drug chloroquine (Fig 1). The result of a medical breakth r o u g h that took place during the 1940’s, chloroquine was inexpen sive, relatively nontoxic, and very effective in treating patients with malaria (Warhurs t et al, 2002). It worked well for about ten years (Warhurst et al, 2003). After that, strains of Plasmodiu m that were resistan t to chloroq uine began emerging. (Warhurst et al, 2002). By 1985, such strains were foun d in most parts of the world, making chloroquine far less effective than it once had been (Warhurst, et al, 2002). Given the harm that malaria causes and the poten tial of chloroquine to fight it, a great deal of research has been under take n to unders ta n d how Plasmodiu m defeats the drug. Within the past few years, a memb ra ne protein foun d in Plasmodiu m falciparu m (the most harmful species of malaria) has been identified as a critical compo ne n t of chloroquine resistance (Djimde et al, 2001). Much has been learned about this protein, called pfcrt , and how it may sup pre ss the Figure 1: Chloroq uine Figure 2: Fe(III) Protoporp hyrin IX /Ferric To begin, chloroq uine (CQ) acts on Plasmodiu m while it is infecting red blood cells (RBC’s). Inside of RBC’s, the parasite ingests hemoglobin and digests the molecule within its digestive vacuole or lysoso me by separating the globin chains from heme(Ursos & Roepe, 2002). The globin chains are a crucial source of amino acids for the parasite, but the detach m e n t of the heme group causes the iron boun d to heme to be oxidized from Fe2+ to Fe3+ (Ursos & Roepe, 2002). This compo u n d, called Fe(III) proto p o r p h y rin IX (FPIX), shown in Fig. 2, is highly toxic to the parasite and can peroxidize membr an e lipids and inhibit critical enzymes like proteases (Ursos & Roepe, 2002). In order to prevent this, the parasite converts FPIX into a benign compo u n d called hemo zoin (Ursos & Rope, 2002). Through a mechanis m that has not been entirely explained, chloroq uine blocks the formatio n of hemo zoin which, it seems, causes the build - up of a toxic FPIX derivative (Ursos & Roepe, 2002). The identity of the toxic FPIX derivative is though t to be a compo u n d called hematin (Warhurst et al, 2002 Waller et al, 2003). This process is illustrate d in Fig. Figure 3: Current model of CQ function in P. falciparu m digestive vacuole According to Ursos and Roepe (2002), it is believed that chloroquine resistance is due to either altered cellular transp o r t that lowers CQ concentration or to some process which lowers the availability of iron - bearing heme to the drug. With regard to the first theory, recent studies cited by Ursos and Roepe (2002) indicate a possible mechanis m of CQ resistance involving pH levels within the digestive vacuoles of P. falciparu m . Given the small size of these vacuoles, relevant analysis of their pH and membr a ne voltage potentials has been very difficult to carry out (Ursos & Roepe, 2002). One possible metho d, which utilizes a weakly basic mono p r o tic fluorescent probe called acridine orange (AO), has been devised and tested on CQ resistan t and nonresistan t strains of P. falciparu m (Ursos & Roepe, 2002). In theory, AO accumulates in acidic environ me n t s and prod uces greater fluorescen t intensity when concentration increases (Ursos & Roepe, 2002). Thus, levels of AO fluorescence indicate pH (Ursos & Roepe, 2002). The results of one study were that drug resistan t Dd2 strains displayed more AO concentratio n within their vacuoles than non - resistan t HB3 strains, indicating higher acidity in Dd2 vacuoles (Ursos & Roepe, 2002). Since CQ is a weak diprotic base, a lower pH would appear to induce the concentra tio n of CQ within the vacuole, rather than lower it. This increased concentr atio n would seem to make the parasite more sensitive to the drug (Ursos & Roepe, 2002).
Ursos and Roepe (2002), however, postulate that this higher vacuole acidity may, in fact, reduce the effectiveness of CQ. Here, it is stated that CQ acts upon FPIX in a dimer form. It is thought that low pH promo tes the formation of an aggregated form of FPIX, resulting in a significant decrease in FPIX dimer (Ursos & Roepe, 2002). Coupling this with evidence that 90% of CQ within a vacuole is boun d to heme, it stand s to reason that a lower concentration of FPIX dimer in the vacuole would both reduce the accumulation of CQ and make the drug less effective (Ursos & Roepe, 2002). This model is outlined in Fig. 4. Figure 4: Ursos & Roepe (2002) model of CQ resistance in P. falciparu m digestive vacuole. Other plausible explanation s for CQ resistance involve specific proteins and genes coded within the P. falciparu m genome. One protein that appears to play a significant role in CQ resistance is a trans me m b r a n e protein called pfcrt (Djimde et al, 2001). This protein is coded on chromo s o m e #7 of the P. falciparu m genome, and is found within the parasites’ digestive vacuoles (Djimde et al, 2001). Multiple studies have linked specific mutation s in the pfcrt gene to CQ resistance, and informatio n has emerged as to how the protein may function.
First of all, mutation s in the pfcrt gene appear to be prevalent in malaria infections that are CQ resistan t. One study reported by Djimde et al in 2001 using blood samples from malaria patients in Mali sup po r te d this hypothesis. In this case, DNA was isolated from the samples and genes encoding pfcrt mutation s and other proteins though t to be involved in CQ resistance were isolated and identified by polymerase chain reaction (PCR) and agarose - gel electro p h o r esis. The results showed that 92% of patients who displayed CQ resistance were infected by P. falciparu m that had a specifically mutated pfcrt gene (Djimde et al, 2001). This mutatio n was caused by a singe substitu tio n of threonine for lysine at position 76, which has been officially designated T76 (Djimde et al, 2002). Further analysis indicated that children under the age of ten were almost twice as likely to have CQ resistan t infections of T76 pfrct malaria than older patients infected with the same mutate d parasite (Djimde et al, 2001). Studies have shown older people who live in malaria - endemic areas can build up immu nity to the disease after repeated infections (Djimde et al, 2001). Taking this into accoun t, Djimde et al (2001) reasoned that this immu nity could explain why some patients who are infected with T76 malaria may seem to respon d to CQ when they are, in fact, resistan t to the Further evidence of the involvemen t of pfcrt in the mechanis m of CQ resistance was obtained by Sidhu et al in 2002. In their research, pfcrt alleles from resistan t strains of P. falciparu m were isolated and introd uced into the genome of CQ sensitive parasites to replace their original pfcrt genes (Sidhu et al, 2002). Successful implanta tion of the resistan t and mutan t pfcrt alleles was confirmed using Souther n blot, which is a gel electrop h o re sis test that identifies specific DNA sequences (Sidhu et al, 2002, Burgess, 1999). Transcrip tio n of the inserted genes and lack of transcrip tio n of the endogeno u s genes was confirmed by Western blot analysis, which is similar to Souther n blot except proteins are identified (Sidhu et al, 2002, Khalsa, 2001). Also, levels of CQ in the parasites was deter mined by its incorpor atio n with (3H)hypoxan t hine (Sidhu et al, 2002). These levels were used to calculate 50% inhibitory concentratio n values (IC50) of CQ for each cloned strain. IC50 values represen t the amoun t of a drug needed to reduce the growth of a pathogen by 50% (HIV and The results indicated that the P. falciparu m clones with resistan t pfcrt genes displayed IC50 values that indicate CQ resistance. (Sidhu et al, 2002). As a control, Sidhu et al (2002) exposed the recombinan t strains to a drug called verapa mil that is known to reduce CQ resistance and, sure enough, the drug lowered the IC50 values for the resistan t clones. In an interesting side experimen t, Sidhu et al (2002) also exposed the genetically modified parasites to other drugs, and noted the increased suscep tibility of artemisinin and quinine to the CQ resistan t clones. Such information, they argued, could be useful in controlling CQ resistan t malaria (Sidhu et Expanding on this research, a group of scientists, including Sidhu et al, sought to show that modifying pfcrt genes would have an effect on CQ resistance. First, various pfcrt gene sequences were linked to a reporter gene for the bioluminescen t marker luciferin and then they were introd uce d into the genome of P. falciparu m specimen s (Waller et al, 2003). From the luciferin assays, a very low functioning pfcrt sequence with 148 base pairs truncate d was discovered and introd uced into the genome of CQ resistan t malaria parasites via electro po re sis (Waller et al, 2003). Souther n blot analysis confirme d the truncated sequence had replaced endogeno u s sequences, and Northern blot analysis further illustrated the knockdown pfcrt gene’s inability to function (Waller et al, 2003). Like Souther n blot and Western blot, Norther n blot utilizes gel electro p h o r esis to identify the presence of a specific molecule, in this case, RNA (Burgess, 1999). Using the same (3H)hypoxant hine assay as Sidhu et al, 2002, it was noted that the CQ resistan t strains with the truncated pfcrt sequence displayed 38% lower IC50 values than the wild - type strain (Waller et al, 2003). This display of lower CQ resistance in the transfor m e d malarial strains further highlight pfcrt ’s probable role in fighting the effects of CQ.
Having establishe d pfcrt as a major factor in CQ resistance, some researcher s have turned their atten tion to how this protein may confer drug resistance to malaria. Ursos & Roepe (2002) cite studies suggesting pfcrt could be respon sible for, or at least involved in, the digestive vacuole acidification process described earlier. They also point out a study involving two CQ resistan t strains of P. falciparu m tested with the anti - CQ resistance drug verapa mil and AO. One of these strains displayed a lower than normal AO intensity, an indicator of increased alkalinity (Ursos and Roepe, 2002). The secon d strain was a control strain that is not suscep tible to the effects of verapamil, and there was higher AO intensity in this strain than in the first (Ursos & Roepe, 2002). Ursos & Roepe (2002) believe verapamil induced CQ sensitivity in the first strain by increasing digestive vacuole pH. As for the second strain, it was resistan t to verapa mil so, in accorda nce with their theory, digestive vacuole pH would not be expected to increase, which is consisten t with the higher AO intensity (Ursos & Roepe, 2002).
Waller et al (2003), further m o r e, perfor me d AO assays on their knockdown pfcrt clones. Here, the knockdown clones displayed intravacuolar and extravacuolar pH levels that are close to those seen in CQ suscep tible P. falciparu m strains (Waller et al, 2003). This would appear to show that acidic digestive vacuole pH is involved in CQ resistance and pfcrt mediates this pH change. Waller et al (2003), however, point out possible flaws in their research. For example, the knockdown clones displayed high IC50 values of 75 - 80 nM that bordered on minimu m CQ resistance levels of 80 to 100 nM. So, their clones barely met the requiremen t s for CQ resistance, meaning their results point to a connection between CQ resistance and pfcrt , but not conclusively. In addition, they cite research indicating pfcrt may actually trans p o r t AO, which would seriously decrease the accuracy of AO assays (Waller et al, 2003). Even more than this, Waller et al (2003) speculate that pfcrt might interact directly with CQ, perhap s by competing with hematin for binding with CQ, which would still be consisten t with curren t ideas of CQ Research cond ucted by Sanchez et al has directly contra dicted the digestion vacuole pH theory cited in Ursos & Roepe (2002) and Waller et al (2003). In this case, an experimen t was cond ucte d where CQ suscep tible and CQ resistan t P. falciparu m strains were first exposed to varying concentration s of unlabeled CQ for a period of fifteen minutes, washed, and then introd uce d to labeled (3H)- chloroq uine (Sanchez et al, 2003). Following this, deter min atio n s of intracellular and extracellular labeled CQ levels were made (Sanchez et al, 2003). The specific name for this kind of experimen t is a trans - stimulation assay. It involves measuring the influx of a substa nce into an enclosed area, and if the rate of influx is increased by something on the inside of the area, then trans - stimulation is said to be occurring (Stein, 2004). The data from this experimen t showed a direct inverse relations hip between preloade d CQ levels and intracellular levels of labeled CQ in the CQ susceptible strain (Sanchez et al, 2003). A curious anomaly was noted in the CQ resistan t strain. Intracellular levels of CQ first increased with increasing levels of preloade d CQ, and then decrease d as preloade d CQ levels became higher (Sanchez et al, 2003).
Sanchez et al (2003) interpreted the data as follows. First, the only way CQ could enter or leave the digestive vacuole in the CQ suscep tible strain was by simple diffusion. Once inside, the two forms of CQ would compete for a binding compo n en t, which, as stated earlier, could very well be heme /FPIX. Thus, as levels of preloade d CQ increased, levels of labeled CQ decreased. In the case of the CQ resistan t strain, there was competition between preloaded and labeled CQ for a carrier - mediated transp o r t system pumping CQ out of the vacuole. Membrane - boun d system s like this which pump drugs out of cells are normally referred to as drug efflux transp o r t er s (Carroll, 2003). When preloaded CQ levels were low, there was enough competition for the carrier to stop the efflux of labeled CQ from the vacuole. This, in turn, led to a build - up of labeled CQ entering by simple diffusion. At higher levels of preloade d CQ, the same binding - compo ne n t competition came into play as in the CQ susceptible strain, and intracellular levels of labeled CQ decreased. This pheno me n o n, illustrated in Fig. 5, is called trans - acceleration, and it is only observed in carrier - mediated trans p o r t systems (Sanchez et al, 2003). Since unlabeled CQ increased levels of labeled CQ, the results also meet the criteria of trans - stimulation (Stein, 2004). Sanchez et al (2003) conclude d that the model of lower digestive vacuole pH making CQ unable to bind to its target could not accoun t for the increase in labeled CQ accum ulatio n seen at low levels Figure 5: Trans - acceleration observed by Sanchez et al, 2003). In (A), labeled CQ enters the digestive vacuole by simple diffusion to compete with unlabeled preloade d CQ for a binding compo ne n t. When unlabeled CQ reaches a level high enough to cause competitio n for a carrier - mediated trans por t e r in (B), most efflux of labeled CQ is blocked, causing it to accum ulate. Eventually,as shown in (C), unlabeled CQ levels become so high that labeled CQ can not bind to the componen t, so its accum ulation decreases as it leaves the vacuole by simple diffusion.
Next, Sanchez et al (2003) tested the rate of CQ uptake of two CQ resistan t strains and two CQ susceptible strains of P. falciparu m exposed to various concentr atio n s of labeled CQ. They then curve fitted the data for the CQ susceptible strains to parameter s of simple diffusion, assu ming that any gain or loss of CQ in the digestive vacuole would be through diffusion (Sanchez et al, 2003). The rate of uptake from the CQ resistan t strains was curve fitted using an equation that deter mines the amou n t of a boun d substr ate, in this case, CQ and its binding compo ne n t in an environ me n t where substra te is pum pe d out throug h an efflux carrier (Sanchez et al, 2003). Both curves are shown on Fig. 6. The CQ suscep tible strains demon s t ra t e d uptake and loss kinetics that correlate with simple diffusion (Sanchez et al, 2003). The CQ resistan t strains, on the other hand, showed CQ uptake rates that are similar to those seen in drug efflux Chloroquine Conc.
Figure 6: Generalized curve fitting of CQ uptake kinetics in CQ resistant and CQ suscep tible strains of P. falciparu m from Sanchez et al, 2003.
Finally, analysis of CQ uptake in glucose - rich and glucose - poor media was carried out, and the trans - stimulation effect was only observed when CQ resistan t P. falciparu m was in a glucose - rich environ me n t (Sanchez et al, 2003). In fact, there was similar CQ uptake between resistan t and non - resistan t malaria strains in the low glucose environ me n t (Sanchez et al, 2003). Taking all of this evidence into accoun t, Sanchez et al (2003) conclude d that their findings indicate an energy - depen d e n t drug efflux system which pump s CQ out of the digestive vacuole is likely to be involved in CQ resistance. Sanchez et al (2003) agree that pfcrt could be an integral compo ne n t of this trans p o r t mechanis m. A third model of CQ resistance in P. falciparu m has been propose d by Warhurst et al (2002). They claim pfcrt resembles a chloride channel that has been studied in Salmonella typhi m u riu m . Also, it is noted from other research that the T76 pfcrt mutation discusse d earlier results in the loss of a strong positive charge on the interior side of the protein (Warhurst et al, 2002). In conjunction with an increase in pfcrt hydro p h o bicity caused by the T76 mutation, Warhurst et al (2002) have devised a system that accoun ts for both CQ resistance and the effects of verapa mil, which is described in Fig. 7. Here, the decreased positive charge on pfcrt diminishes unfavorable interactions with pfcrt and proton ate d CQ (Warhurst et al, 2002). This allows the CQ, apparen tly proton ate d by the high digestive vacuole acidity, to pass through the T76 pfcrt channel (Waller et al, 2002). Further m o r e, Warhurst et al (2002) state that verapa mil is known to be both hydrop h o bic and positively charged, making it attracted to mutan t pfcrt . Thus, verapamil would block the pfcrt channel, preventing the escape of CQ (Warhurst et al, 2002). So, not only does this hypothesis explain CQ resistance, it explains the how verapa mil reverses it (Warhurst et al, 2002). The latter is quite intriguing since, according to Ursos & Roepe (2002), the action of verapa mil has yet to be Figure 7: Model of CQ resista nce propo se d by Warhurst et al, 2003. Wild type Pfcrt in (A) has a strong positive charge (+) which prevents protona te d CQ2H+ from exiting the digestive vacuole. In CQ resistan t malarial strains (B) the charge is removed, allowing CQ to escape. In (C), verapamil (VE+) blocks Pfcrt with a positive charge in CQ resistan t malaria, preventing CQ from leaving the digestive vacuole, thereby reversing resistance.
In conclusion, bringing the inexpen sive and effective drug chloroq uine back into mankin d’s arsenal against malaria would be extremely beneficial. Current research has greatly expan de d the knowledge of how malaria can defeat this drug, and the possibility of finding a way aroun d CQ resistance increases with the collection of more informatio n. Some researchers believe a change in digestive vacuole pH, as indicated by AO, can render P. falciparu m resistan t to CQ. Others have foun d evidence of a cross - memb ra n e trans p o r t system that pulls the drug away from its target. Gene replacemen t research, knockdown gene insertion, and genetic analysis of parasites from malaria patients all indicate that the protein pfcrt plays an importan t part in neutralizing CQ.
Given the contradicting evidence of how pfcrt may function, more research is clearly necessary. Suppor ter s of the digestive vacuole pH theory would do well to further study AO uptake kinetics to prove or disprove questions of its effectiveness. They should also deter mine how CQ would be taken into the digestive vacuole under their model, in light of the informa tion collected by Sanchez et al, (2003). The theory put forwar d by Warhusrt et al (2002) explains much, but it is based mostly on informatio n compiled from previous research. Specific tests of their model are clearly called for. And, the evidence for the Sanchez et al (2003) model of drug efflux system moving CQ out of the digestive vacuole is compelling. Yet, it is only one study, and more research testing CQ resistance against trans m e m b r a n e drug trans p o r t parame ter s is warran te d to validate it. Overall, more research is needed to deter mine how CQ resistance comes about in P. falciparu m and how pfcrt contribu tes to this pheno m e n o n. Only then will chloroquine once again bring hope that one of the worst pathogen s to human beings may, some day, be controlled.
WORKS CITED
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