Localization of the ATP-binding cassette (ABC) transport proteins PfMRP1, PfMRP2, and PfMDR5 at the Plasmodium falciparum plasma membrane

Drug resistance is a major problem in malaria. Today only a limited number of effective anti-malarials is available. An important reason for therapeutic failure in malaria treatment could be that drugs do not reach their target sites, due to active extrusion by the parasite. The transport proteins responsible for this type of resistance are so-called multidrug resistance proteins (MDR/MRP), most of which belong to the superfamily of ATP binding cassette (ABC) proteins, one of the largest protein families. Many of these plasma membrane proteins actively pump out a wide range of structurally and functionally diverse amphipathic drugs, thereby decreasing the intracellular drug accumulation and resulting in drug resistance [1, 2]. The structure of a typical ABC transporter consists of six trans-membrane segments that form a trans-membrane domain (TMD) and the Walker A and Walker B motifs that form a nucleotide binding domain (NBD) (Figure 1). ABC transporters are either encoded as full transporters (TMD-NBD-TMD-NBD) or as half transporters (TMD-NBD) that upon translation combine to form a functional unit. Apart from their normal physiological role, ABC transporters are involved in various diseases either by a mutation or through an altered mode of their expression [3, 4].

Several polymorphisms and an increased copy number of the Plasmodium falciparum MDR1 have been associated with drug resistance [5]. Heterologous expression data indeed show that PfMDR1-mediated anti-malarial drug transport is affected by some of these polymorphisms [6, 7]. The other member of the PfABC B family that has been characterized is the half transporter PfMDR2 that confers heavy metal resistance to P. falciparum and most likely is not involved in drug resistance [8]. Finally, PfMRP1 polymorphisms have been associated with drug resistance and recently it has been shown that PfMRP1 transports anti-malarial drugs and glutathione [9]. PfMDR1 has been detected in the membrane of the food vacuole [10], which is in contrast to PfMRP1 that has been immunelocalized in the plasma membrane of the parasite [9, 11]. In previous studies the P. falciparum ABC family members were identified [12, 13] and categorized [14, 15]. In this study, the P. falciparum ABC transporter family has also been categorized and the database sequence of PfMRP1, PfMRP2, and PfMDR5 was confirmed. Moreover, with immunocytochemistry techniques it was shown that all three transporters are probably present in the plasma membrane of the parasite during the asexual erythrocytic stages.

In the plasmoDB database 16 members of the P. falciparum ABC family can be identified, 11 of which could be putative drug efflux transporters. A phylogenetic analysis of the PfABC genes was performed and the genes were categorized into seven subfamily groups. Three ABC family members were analysed in more detail and their expression in all erythrocytic stages as well as their localization at the plasma membrane of the parasite was demonstrated.

Development of resistance against newly introduced anti-malarials can be rapid especially in malaria endemic regions where drug pressure is high. For instance mefloquine was introduced in Thailand in 1984 as a very potent drug against multidrug resistant malaria, but six years later significant resistance had developed [23]. Multiple studies have demonstrated the role of PfMDR1 polymorphisms in multidrug resistant malaria. It was shown that the anti-malarial drugs halofantrine, quinine and chloroquine are transported by PfMDR1 [7]. Moreover, polymorphisms within PfMDR1 alter the substrate specificity for these anti-malarial drugs [7]. Recent association studies have linked single nucleotide polymorphisms on PfMRP1 with reduced parasite response to anti-malarial drugs [24, 25] and expression of both PfMRP1 and PfMRP2 were up-regulated by mefloquine and chloroquine in laboratory cultures of both drug sensitive and resistant strains [26]. This indicates that ABC transporters play a role in malaria chemotherapy [27]. The knowledge that other PfABC family members could be capable of drug transport indicates that it is likely that some of these transport proteins might play a role in emerging drug resistance. Further identification and characterization of these ABC transporters will provide information on their putative role in resistance and may provide novel targets to control and perhaps prevent spread of resistance against other efficacious drugs.

The PFAM search in plasmoDB resulted in 16 ABC family members that were also identified and catagorized by others [12–15]. ABC A subfamily members were not observed, but these seem to be absent in all Apicomplexa. The ABC B subfamily consists of seven members of which only PfMDR1 is a full transporter. The C subfamily contains two full transporters, whereas the G subfamily contains one half transporter. Next to these ABC family members there is one additional member that contains a TMD. As this member does not belong to the existing subfamilies, it was placed in the I family that was proposed by Verrier et al [20] to harbour "orphan" ABC components. Five ABC family members that do not contain TMDs are located in E, F, and I subfamilies.

Recently, Gangwar and colleagues [14] analysed the ABC family members of P. falciparum, their phylogenetic tree was, however, not based on an alignment of the conserved NDB regions but on the whole protein, which results in a phylogenetic tree where the subfamilies are not clustered. Moreover, they did not include ABCB4 (PFC0125w) in their analyses and included PFE0450w, which is not an ABC family member but a putative chromosome condensation protein. In a very recent review by Sauvage et al [15] the ABC families of different protozoan parasites were listed. They clustered the ABC family members in a way similar to our approach. In this study PF08_0078 was, however, assigned in the F family according to the phylogenetic analysis of both NBDs, whereas they categorized it as "other". As they did not show a phylogenetic analysis it is not clear why this discrepancy exists. Moreover, PFC0875w (PfABCI3), that does not possess a clear ABC signature motif, but according to the PFAM search belongs to the ABC family, is not listed in their overview. In this study the existing nomenclature for PfMDR1, PfMDR2, and PfMRP1, was not changed, but the nomenclature of Verrier et al [20] for plant ABC proteins that was adopted from the HUGO nomenclature and discussed in several international meetings was used. This nomenclature essentially constitutes a catalogue with numbered entries, where the numbers do not necessarily carry additional information, although the subfamily assignment does indeed convey information about phylogenetic relationships [20]. The gene nomenclature recently published by Sauvage et al [15] is based on the nomenclature for Toxoplasma gondii [28], which as a consequence in P. falciparum resulted in the absence of ABCB2, ABCG1, ABCG2, ABCH1, and ABCH3 and the relocation of PfMDR2 to ABCB3. Such a nomenclature is, however, impractical for a group with relatively few orthologous pairings, such as the ABCs [20].

It is known that the malaria parasite can express its membrane proteins on at least four different subcellular sites: on its membrane-bound organelles, the plasma membrane, the parasitophorous vacuolar membrane and on the plasma membrane of its host erythrocyte [29, 30]. The expression of PfMRP1, PfMRP2, and PfMDR5 was observed on the outside of the parasite. It is practically impossible to distinguish the plasma membrane from the parasitophorous vacuolar membrane by immunocytochemistry. However, a detailed look at the expression of all three ABC transporters shows that they are also present on membranes between parasitic nuclei in the multi-nuclear stages of development. As these are plasma membranes in the process of formation, it is concluded that all three ABC transport proteins are located on the plasma membrane in all asexual erythrocytic stages of P. falciparum. The localization of PfMRP1 at the plasma membrane has been shown by others [9, 11]. An elegant control was shown in the recent study of Ray et al [9], where a P. falciparum knock-out of PfMRP1 was used. In this study, knock-out parasites were not available and as an additional control the antibody was affinity purified with the peptide that was used to immunize the rabbits. In addition, background staining with the erythrocytes was not observed indicating that the staining was specific.

16 ABC proteins were categorized according the phylogenetic tree that was constructed from the aligned NBDs. PfMRP1, PfMRP2, and PfMDR5 were localized at the plasma membrane of the parasite throughout the asexual stages. This localization emphasizes the putative role of drug exporters of these ABC family members. Indeed, Ray et al [9] have shown that PfMRP1 plays a role in the efflux of glutathione, chloroquine, and quinine and contributes to parasite responses to multiple anti-malarial drugs, possibly by pumping drugs outside the parasite. PfMRP2 and PfMDR5 might have similar roles and thereby broadening the capacity of the parasite to extrude toxic compounds. Additional research is required to test this hypothesis.