Intracellular Localization of the ABCC Proteins of Leishmania and Their Role in Resistance to Antimonials

Abstract

The ABCC subfamily of proteins is composed of nine members in Leishmania. We report that all of these proteins have an intracellular localization and that the overexpression of at least four members, ABCC3, ABCC4, ABCC5, and ABCC7, can confer resistance to antimonials, the first-line drug against Leishmania.

The protozoan parasite Leishmania is responsible for a variety of clinical manifestations, ranging from mild cutaneous infections to life-threatening visceral diseases (12). Pentavalent antimony [Sb(V)] containing compounds such as sodium stibogluconate (Pentostam) and N-methylglucamine (Glucantime) remain the first-line drugs against all forms of Leishmania infections in developing countries (19), but their efficacies are threatened by resistant parasites in several regions where the disease is endemic (9, 17, 21). Our previous in vitro work on metal resistance in Leishmania led to the definition of a model of resistance involving proteins of the ATP-binding cassette (ABC) superfamily (20). ABC proteins form one of the largest families of transmembrane proteins and are characterized by the presence of the strongly conserved nucleotide-binding domain (NBD), which is composed of three major motifs. Along with the Walker A and B motifs found in many nucleotide-binding proteins (23), the NBD is composed of a characteristic ABC signature “C” motif located just upstream of the Walker B site (13). Eukaryotic ABC proteins can be divided into eight different subfamilies (ABCA to ABCH) on the basis of gene structure and NBD sequence homologies. A previous survey indicated the presence of 42 ABC protein-coding genes in the genomes of Leishmania major and Leishmania infantum (16), but the latest version of the L. infantum genome (GeneDB V3.0) revealed the presence of a new member of the ABC superfamily (LinJ24_V3.1510). This gene seems to be specific to L. infantum since it is absent from the genome of L. major (GeneDB V5.1) and is found as a pseudogene of low homology in the genome of Leishmania braziliensis (GeneDB V2.0). A phylogenetic analysis of the L. infantum ABC proteins revealed that LinJ24_V3.1510 is the most divergent member of the ABCC subfamily in Leishmania (results not shown) and has been named ABCC9. While the Walker A and B motifs are well conserved in the ABCC proteins of Leishmania, some conserved residues essential to the function of ABC proteins (7) are absent from the C motif of ABCC9 (Fig. 1), and the functionality of this protein in L. infantum still needs to be established.

FIG. 1.

Sequence alignment of the NBDs of L. infantum ABCC proteins. Shown are N-terminal NBDs (A) and C-terminal NBDs (B). The Walker A, Walker B, and signature C motifs of both NBDs are shown as boxes. An ABCC-like motif found in most ABCC proteins is underlined. The alignment was performed by using ClustalW (22), and the figure was formatted using Jalview (1).

Resistance to metalloids in Leishmania requires multiple steps, where Sb(V) is reduced to the trivalent form Sb(III), and the latter is conjugated to trypanothione (TSH), a bisglutathione-spermidine conjugate (6), before being transported inside an intracellular detoxification organelle by the ABC transporter MRPA/ABCC3 (15) or being extruded outside the cell by an ATP-dependent efflux pump of unknown identity (4). As the active extrusion of metalloids conjugated to thiols is a feature of the ABCC subfamily (14, 25), we hypothesized that the antimony efflux system located at the plasma membrane of Leishmania was encoded by one of the parasite ABCC homologs. However, transfection of ABCC proteins fused to the green fluorescent protein (GFP) into Leishmania parasites revealed that the entire ABCC subfamily is located to intracellular compartments (Fig. 2). Indeed, cells transfected with the ABCC1-GFP, ABCC2-GFP, and ABCC6-GFP fusions revealed a fluorescence signal located to a network of intracellular membranes. Cells transfected with the ABCC4-GFP and ABCC5-GFP fusions revealed a fluorescence signal located to a tubular compartment oriented along the longitudinal axis of the parasite. Cells transfected with the ABCC8-GFP fusion showed a punctuated fluorescence signal located near the posterior end of the parasite. We did not carry subcellular localization experiments for the MRPA/ABCC3 and the PRP1/ABCC7 proteins, as they are already known to be intracellular proteins (3, 15), nor for the ABCC9 protein, given the lack of antimony resistance following the overexpression of its gene (see below). The N-terminal and C-terminal GFP-tagged versions of ABCC5 localized to the same tubular compartment (results not shown), suggesting that the C-terminal GFP tagging strategy is probably not interfering with the localization of the fusion proteins.

FIG. 2.

Intracellular localization of the entire ABCC subfamily in Leishmania parasites. Shown are L. infantum promastigotes expressing fluorescence signals to a network of intracellular membranes (ABCC1-GFP, ABCC2-GFP, and ABCC6-GFP) to a tubular organelle oriented along the longitudinal axis of the parasite (ABCC4-GFP and ABCC5-GFP) and to the posterior end of the parasites (ABCC8-GFP). Shown are from differential interference contrast images (DIC1) (left), fluorescence only images (GFP) (middle), and a merge of the DIC1 and GFP images (right).

Several ABCC proteins have been shown to localize to the plasma membrane in many cell types, and it is intriguing that every Leishmania ABCC protein is located intracellularly. The ABCC proteins are often implicated in secretion of toxic metabolites and in cellular detoxification, and it could be advantageous for an intracellular parasite like Leishmania to transport toxic compounds and waste metabolites inside intracellular compartments instead of excreting them in the phagolysosome of the parasitizised macrophages of the mammalian host.

The involvement of the intracellular ABCC proteins MRPA/ABCC3 and PRP1/ABCC7 in antimony resistance has already been reported (2, 3, 15). However, only MRPA/ABCC3 has been shown to confer resistance both to Sb(V) and to Sb(III) (5) and to be amplified in field isolates derived from patients unresponsive to antimonials (18). Since none of the ABCC protein was located in the plasma membrane (Fig. 2), we next tested if the ABCC1, ABCC2, ABCC4, ABCC5, ABCC6, ABCC8, and ABCC9 proteins could act as intracellular transporters associated with resistance to Sb(III), the biologically active form of antimony. Growth curve experiments showed that the previously described MRPA/ABCC3 and PRP1/ABCC7 proteins were the only ABCC proteins associated with Sb(III) resistance when overexpressed in a wild-type (WT) background of L. infantum (not shown) or Leishmania tarentolae (Table 1). However, previous studies have shown that MRPA gave higher resistance levels when its gene was transfected in the cell line L. tarentolae As20.3rev (8, 11), and we thus transfected the various ABCC constructs in this partial revertant line. The L. tarentolae As20.3rev cell line was generated from the Sb(III)-resistant mutant L. tarentolae As20.3 by successive passages in the absence of antimony. The As20.3rev cell line is more susceptible than the parent mutant but remains considerably more resistant than its parental WT strain (Table 1). The overexpression of ABCC4-GFP and ABCC5-GFP in L. tarentolae As20.3rev resulted in a highly reproducible twofold increase in resistance to Sb(III) (Table 1). The ABCC4-GFP, ABCC5-GFP, and GFP-ABCC5 fusions were functional since similar resistance levels were observed with the unfused version of the proteins in L. tarentolae As20.3rev (Table 1). The other ABCC proteins were not associated with significant antimony resistance when overexpressed in L. tarentolae As20.3rev (apart from the previously described MRPA/ABCC3 and PRP1/ABCC7 proteins) (Table 1).

TABLE 1.

Antimony resistance in L. tarentolae transfectants

Cell line	Antimony resistance
	EC50 (μM)a	Fold increaseb
Tarll WT	0.14	1
Tarll WT + ABCC1-GFP	0.14	1
Tarll WT + ABCC2-GFP	0.14	1
Tarll WT + ABCC3/MRPA	0.37	2.5
Tarll WT + ABCC4-GFP	0.14	1
Tarll WT + ABCC5-GFP	0.15	1
Tarll WT + GFP-ABCC5	0.16	1
Tarll WT + ABCC5	0.15	1
Tarll WT + ABCC6-GFP	0.15	1
Tarll WT + ABCC7-GFP	0.24	1.7
Tarll WT + ABCC8-GFP	0.14	1
Tarll WT + liABCC9	0.15	1
Tarll As20.3rev	8	1
Tarll As20.3rev + ABCC1-GFP	8	1
Tarll As20.3rev + ABCC2-GFP	8	1
Tarll As20.3rev + ABCC3/MRPA	47	6
Tarll As20.3rev + ABCC4-GFP	17	2
Tarll As20.3rev + ABCC4	18	2
Tarll As20.3rev + ABCC5-GFP	20	2.5
Tarll As20.3rev + GFP-ABCC5	20	2.5
Tarll As20.3rev + ABCC5	20	2.5
Tarll As20.3rev + ABCC6-GFP	8	1
Tarll As20.3rev + ABCC7-GFP	14	1.7
Tarll As20.3rev + ABCC8-GFP	8	1
Tarll As20.3rev + liABCC9	9	1

^aAverage of three independent experiments.

^bFold increase compared with either the WT or As20.3rev.

It has been reported that increased levels of cellular thiols are required for antimony resistance in Leishmania. Accordingly, the TSH levels are increased 10 times in the L. tarentolae As20.3 resistant mutant and remain at least threefold higher in the partial revertant line L. tarentolae As20.3rev than in the WT L. tarentolae (8, 10, 11). By analogy with the GS-X system and as previously demonstrated for MRPA/ABCC3 (15), it might be possible that the ABCC4 and ABCC5 proteins transport Sb(III) as part of TSH complexes, where the increased thiol levels or the formation of the conjugates would be the rate-limiting steps of the transport process. This could explain the resistance phenotype conferred by the overexpresion of ABCC4 and ABCC5 being specific to the L. tarentolae As20.3rev background, but in light of recent results, an increase in TSH-transferase activity associated with Sb(III) resistance in Leishmania parasites is unlikely (24).

This study highlighted the role of the Leishmania ABCC protein subfamily in antimony resistance in vitro and suggested that the antimony efflux system previously described at the plasma membrane of the parasite is probably unrelated to ABCC proteins, given their intracellular localization. Further studies will be required to isolate this efflux system and to further study the role of ABCC proteins in resistance to antimonials in field isolates.