Characterization of an ATP-binding cassette transporter in Cryptosporidium parvum1

Molecular and Biochemical Parasitology 87 (1997) 117 – 122

Short Communication

Characterization of an ATP-binding cassette transporter in Cryptosporidium par6um 1 Margaret E. Perkins a,*, Sarah Volkman b, Dyann F. Wirth b, Sylvie M. Le Blancq a,c a

Di6ision of En6ironmental Health Sciences, Columbia Uni6ersity School of Public Health, 630 West 168th Street, VC15 -220, New York, NY 10032, USA b Department of Tropical Public Health, Har6ard Uni6ersity School of Public Health, Boston, MA 02115, USA c Center for En6ironmental Research and Conser6ation, Columbia Uni6ersity, New York, NY 10027, USA Received 4 March 1997; received in revised form 28 March 1997; accepted 30 March 1997

Keywords: Cryptosporidium par6um; ATP-binding cassette transporter; Intracellular parasite; Transport

The apicomplexan parasite Cryptosporidium par6um is the causative agent of cryptosporidiosis [1]. Despite its importance as an opportunistic infection in the acquired immune deficiency syndrome (AIDS) there is at present no reliable drug therapy for the treatment of cryptosporidiosis. One possible explanation is that drugs are poorly transported into this intracellular but extracytoAbbre6iations: ABC protein, ATP-binding cassette containing protein; CpABC, Cryptosporidium par6um ABC protein; Pgp, P-glycoprotein; Pgh1, Plasmodium falciparum P-glycoprotein homologue 1; MDR, multidrug resistance; H-MRP, human multidrug resistance-associated protein; PCR, polymerase chain reaction; SSC, standard saline citrate; SDS, sodium dodecyl sulfate. * Corresponding author. Tel.: + 1 212 3056727; fax: + 1 212 3054496; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank data base with the accession number U90628.

plasmic parasite. Knowledge of the pathways that regulate the influx and efflux of substrates and drug analogues in the C. par6um-infected enterocyte would be a first step in understanding drug accessibility in this parasite. To initiate studies on transport pathways in C. par6um-infected epithelial cells we have focused on the ATP-binding cassette (ABC) superfamily of transporters. ABC transporters translocate a wide variety of molecules across membranes against a concentration gradient. Members of the ABC family include P-glycoproteins (Pgp) and the multidrug resistance-associated proteins (MRP) which are involved in the multidrug resistance phenotype (MDR) in cancer cells [2,3]. Recent studies have shown that the mammalian MDR1 P-glycoprotein (MDR1 Pgp) functions as a lipid translocase [4], while human MRP (H-MRP) functions as an organic anion transporter [3]. The apicom-

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plexan parasite Plasmodium falciparum contains a P-glycoprotein homologue (Pgh), termed Pgh1, that has been proposed to have a role in resistance to both chloroquine and mefloquine [5 – 7]. Most ABC transporters consist of four domains, two hydrophobic membrane spanning regions which form the substrate translocation pathway and two ATP-binding domains [2,3]. In the present study we used the polymerase chain reaction (PCR) to amplify the ATP-binding domain of an ABC transporter from C. par6um. A set of degenerate oligonucleotide primers was designed based on highly conserved regions of the nucleotide (NTP) binding domain of the Pgh1 gene [5]. The PCR yielded a 405 bp product that encoded an open reading frame (ORF) of 135 amino acids (Fig. 1A). Alignment of the amino acid sequence with sequences in the GenBank database showed the highest sequence identity with the carboxy (C) terminal ATP-binding domain of human MRP [8]. There was 46% amino acid identity and an overall similarity of 72% between the C. par6um PCR product ORF and the H-MRP ATP-binding domain (Fig. 1A). Significant levels of identity and similarity were also seen in alignments with other members of the ABC transporter family, including the C-terminal nucleotide binding region of the P. falciparum Pgh1 [5]. There was 33% identity and 63% similarity between the C. par6um PCR product ORF and the Pgh1 nucleotide binding region [5]. We concluded from the high amino acid sequence similarity that we had amplified and cloned a nucleotide binding region of a C. par6um ABC transporter (CpABC). The PCR product is therefore referred to as CpABC-ATP. A cloned 900 bp EcoR1 restriction fragment from a C. par6um genomic library has shown that the sequence similarity with ABC transporters, particularly H-MRP, extends beyond the ATP-binding domain (data not shown). The current data suggest that CpABC is a member of the MRP subfamily. A single restriction fragment was observed to hybridize with the CpABC-ATP probe in genomic DNA from C. par6um digested by four restriction endonucleases indicating that the CpABC gene was present as a single copy (Fig. 1B). No additional cross-hybridizing restriction fragments were

seen after low stringency post-hybridizational washes. The 900 bp EcoR1 restriction fragment that hybridized with the CpABC-ATP probe (Fig. 1B) includes the single ATP-binding domain amplified by the PCR. The absence of a cross-hybridizing EcoR1 restriction fragment containing the second putative ATP-binding domain suggests either that the two domains have a low level of nucleotide sequence identity or that there is only one domain present in CpABC. Interestingly, the two ATP-binding domains of MRP transporters typically show less sequence identity than is seen in the Pgp transporters [3]. The CpABC-ATP probe hybridized with the smallest (0.95 megabase) chromosome on Southern blots of sizefractionated C. par6um chromosomes (data not shown). The nucleotide binding region is the most highly conserved region of the ABC protein genes and the absence of cross-hybridizing restriction fragments and chromosomes suggests that this region may exhibit considerable sequence divergence among other ABC protein genes in C. par6um. It is therefore possible that the CpABC gene is the only representative of its particular ABC transporter subfamily in C. par6um. The presence of a CpABC gene transcript in C. par6um sporozoites was demonstrated by Northern analysis of total RNA hybridized with the CpABC-ATP probe (Fig. 1C). Two transcripts were identified, a major hybridizing band of 4.6 kb and a minor band of 6.1 kb. The presence of CpABC gene transcripts in sporozoites suggests that the CpABC protein may be expressed in sporozoites prior to invasion of the host cell. We performed immunoblotting with antiserum generated against part of the C-terminal nucleotide binding region of Pgh1 [9] (indicated by asterisks in Fig. 1A). The Pgh1 antiserum detected a 190 kd protein in oocysts and excysted oocysts (Fig. 2). Several smaller proteins reacted weakly with the antiserum in the immunoblot and these were most likely products of proteolysis as there was a relatively greater amount of the 190 kd protein in samples pre-incubated with specific protease inhibitors (Fig. 2, lanes b–e). EDTA was the most effective protease inhibitor (lane e), indicating that a metalloprotease was active in the oocysts. Interestingly, a metallo-dependent cys-

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Fig. 1. Panel A, sequence alignment and comparison of the C. par6um PCR product CpABC-ATP and the extended nucleotide binding region of the carboxy termini of the human MRP and P. falciparum Pgh1 [5,8]. Genomic DNA (gDNA) was purified from excysted oocysts of the KSU-1 isolate of C. par6um kindly provided by Steve Upton (Kansas State University) [13,14]. PCR was performed with a pair of degenerate primers (Forward primer: 5%GT(T/A)GG(A/T)GAAAC(T/A)GG(T/A)AGTGG(A/ T)AAATC(A/T)AC3%; reverse primer: 5%ATC(T/A)AG(A/T)GA(A/T)GA(A/T)GT(A/T)GCTTCATC3%) with an annealing temperature of 42°C. No PCR product was seen in control reactions without C. par6um gDNA. The 405 bp PCR product was cloned into the pCRII vector using the TA cloning kit (Invitrogen) and both strands were sequenced by the dideoxy method. Standard protocols were used throughout [15]. Searches of the DNA databases were performed with the Blast server service, and sequence analysis and alignments were carried out with DNA Strider and GCG software (Madison, WI). The level of amino acid sequence identity between CpABC-ATP and H-MRP is 46% and the overall similarity is 72%. The level of sequence identity between CpABC-ATP and Pgh1 is 33% and the overall level of similarity is 63%. The regions corresponding to the PCR primers are underlined and were not included in the identity and similarity calculations. The insert unique to P. falciparum Pgh1 was similarly excluded. The NTP binding motif and the ABC transporters family motif are boxed. The sequence used to generate the Pgh1 antiserum is marked by asterisks. Panel B, Southern analysis of CpABC. Genomic DNA from excysted KSU-1 C. par6um oocysts was treated with B, BamHI; P, PstI; H, HindIII; and E, EcoR1 and fractionated on a 0.5% agarose gel, transferred to nylon and hybridized with a 32P-labeled CpABC-ATP probe [16]. Post-hybridization washes were to a final stringency of 0.3 × SSC, 0.1% SDS for 15 min at 55°C. Panel C, Northern analysis of CpABC. RNA was purified from excysted KSU-1 oocysts by lysis in guanidinium isothiocyanate followed by centrifugation through a CsCl cushion. Lane (a) Ethidium bromide staining of total RNA size fractionated on a denaturing agarose gel; lane (b) Northern blot of RNA in lane a hybridized with a 32P-labeled CpABC-ATP probe. Post-hybridization washes were to a final stringency of 0.3 × SSC, 0.1% SDS for 15 min at 55°C.

teine protease associated with the sporozoite surface has recently been identified [10]. The major breakdown product had a size of 140 kd. Pre-immune serum recognized minor proteins of low molecular size (data not shown). A protein of identical size to that in oocysts was present in a population of excysted oocysts which contained about 50% sporo-

zoites (Fig. 2, lane g). The protein is probably tightly membrane-bound since solubilization required boiling (compare lanes f and g). Extracts of P. falciparum (K1 strain) immunoblotted with the Pgh1 antiserum detected a high molecular size protein of 210 kd (Fig. 2, lane h) somewhat larger than that reported by Cowman et al. [11].

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M.E. Perkins et al. / Molecular and Biochemical Parasitology 87 (1997) 117–122

Fig. 1 (B, C).

There is significant sequence identity between CpABC-ATP and the 3% end of the nucleotide binding region of P. falciparum Pgh1 (Fig. 1A) that would account for the cross-genera reactivity

of the Pgh1 antiserum. It is therefore likely that the protein identified in C. par6um by the Pgh1 antiserum is an ABC protein. By analogy with other ABC proteins it is probable that CpABC is

M.E. Perkins et al. / Molecular and Biochemical Parasitology 87 (1997) 117–122

glycosylated, thereby accounting for the apparent discrepancy between the size of the major mRNA transcript (4.6 kb) and a 190 kd protein. The size of many post-translationally modified mammalian P-glycoproteins is in the range 160 – 170 kd but the H-MRP is a 190 kd protein [12] similar to the C. par6um ABC protein detected here. The Pgh1 antiserum also reacted strongly with intracellular stages of C. par6um cultured in Caco-2 cells by indirect immunofluorescence assay (data not shown). CpABC is the first transport protein to be identified in C. par6um. Future studies will examine its role in C. par6um-infected cells in order to

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identify drugs that may inhibit its transport properties.

Acknowledgements We wish to thank Dr Steve Upton for C. par6um oocysts and Dr Jeff Griffiths for Caco-2 cells and advice on in vitro cultivation of C. par6um. We thank Teresa Wu for technical assistance. This work was supported by N.I.H. grant AI26497 in the International Collaborative Infectious Disease Research Program, and grants from the Calderone Foundation and the Center of Environmental Research and Conservation (Columbia University) to S.M.L.B..

References

Fig. 2. Immunoblotting was performed using antibodies generated against part of the C-terminal nucleotide binding region of Pgh1, indicated by the asterisks in Fig. 1A [9]. The Pgh1 antiserum exhibits high specificity for Pgh1 (Volkman and Wirth, unpublished data). Intact oocysts (2 × 106) and excysted oocysts (4 ×106) were treated as described below and solubilized in sample buffer (2% SDS, 0.1 M Tris–HCl, pH 6.8, 10% glycerol, 0.1 M DTT, 0.1% bromophenol blue) by boiling and electrophoresed on a 6% SDS-PAGE gel. Intact oocysts were incubated in the following protease inhibitors for 15 min prior to solubilization in sample buffer: (a) no additions; (b) 1 mM PMSF; (c) 1 mM iodoacetamide; (d) 5 mM leupeptin; (e) 5 mM EDTA. Excysted oocysts [13] were extracted with either 2% SDS in phosphate buffered saline (PBS) (lane f) or boiled in 2% SDS in PBS (lane g). P. falciparum (K1 strain) were lysed in ice-cold 10 mM Tris buffer and solubilized in 2% SDS in PBS (lane h). The gel was processed for immunoblotting with Pgh1 antiserum essentially as described [17] except that the second antibody was alkaline phosphataseconjugated goat anti-rabbit antibody (Bio-Rad) and the incubation was for 2 h followed by washing in Tris saline-0.1% Tween. The color was developed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate in citrate buffer.

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