CHAPTER 4 The Plasmodium spp. putative copper transport protein: Native protein
4.2 Results
4.2.1 Confirmation of a putative Plasmodium copper transporter coding domain
Initial attempts at identifying a parasite copper transporter coding domain, as well as the encoded native protein, made use of the rodent-infecting malaria parasite model. This model parasite serves as a useful comparative tool as it is capable of reproducing much of the biology of human malaria parasites (Carlton et al., 2002; Hall et al., 2005; van Lin et al., 2001). In the first instance, P. yoelii parasite DNA was isolated from parasitised BALB/c mouse blood and used as a template for amplification of the putative copper transporter's full-length coding domain by PCR. The target sequence was predicted to contain two introns and three protein- coding exons (data not shown), producing a sequence approximately 1791 bp long. Agarose
gel analysis of the PCR amplicons revealed a product approximately 1800 bp in size (Figure 4.1a), corresponding to the predicted size. PCR amplification of the LDH coding domain was included as a positive control with an amplification product approximately 815 bp in size (Figure 4.1a). This amplicon was in agreement with the predicted size of the targeted LDH coding domain region. As a further control, DNA was isolated from uninfected BALB/c mouse blood and used as a template for PCR amplification. All reactions using this template DNA returned negative results (data not shown).
Figure 4.1 PCR and reverse transcriptase-PCR amplification of the coding domains for putative P. yoelii and P. berghei copper transporters
A PCR amplification, from P. yoelii genomic DNA, of the coding domains for lactate dehydrogenase (lane 1) and the putative copper transport protein (lane 3). Lanes 2 and 4 are controls using DNA isolated from uninfected mouse blood. B Nested PCR of the putative P. yoelii copper transport protein coding region. Lane 1 primary PCR product, lane 3 secondary PCR from lane 1. Lanes 2 and 4 negative controls. C BamHI digestion to support P. yoelii amplicon identity. Lane 1, gel purified amplicon of the putative P. yoelii copper transporter coding region. Lane 2, BamHI digestion of lane 1 amplicon with arrows highlighting digested products. The upper arrow in lane 2 is 1050 bp, lower arrow 750 bp. D Reverse transcriptase-PCR amplification, from P. berghei RNA, of the coding domains for the amino terminus of a putative P. berghei copper transporter (lane 1) and for LDH (lane 2). All PCR products were analysed on 1% (w/v) agarose gels. Molecular mass markers (M) are on the left of each gel (see materials and methods for details).
The identity of the P. yoelii 1800bp amplicon was supported by nested PCR (Figure 4.1b) and BamHI enzyme digestion (Figure 4.1c). For nested PCR, a 2700bp region encompassing the predicted copper transporter coding domain was amplified from genomic DNA and from this the 1800bp product was amplified (Figure 4.1b). The 1800bp amplicon was subsequently gel purified and digested with BamHI. BamHI digestion yielded two products (Figure 4.1c), of 1050bp and 750bp, which corresponded well with the predicted sizes. For functional studies of the murine malaria parasite's copper transporter, the predicted amino terminus was to be recombinantly expressed and its copper binding ability assessed. Since the number of copper transporters encoded by the P. berghei and P. falciparum genomes is similar, P. berghei was selected as the murine malaria parasite model organism. The relevant portion of the P. berghei
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copper transporter's coding domain was therefore amplified by reverse transcription-PCR (RT- PCR) from a P. berghei RNA pool. Amplification yielded a 450bp product (Figure 4.1d), indicating native transcription of the targeted coding domain. As before, amplification of the LDH coding domain served as a positive control and yielded a product of the expected size.
The putative copper transporter amino terminal amplicon was ligated into the pGEM®-T vector and sequenced. Sequencing data correlated well with the P. berghei nucleotide sequence available at PlasmoDB, showing 99% identity (Figure 4.2). In silico translation of this sequence yielded an amino acid sequence with 98% identity to the PlasmoDB sequence (data not shown).
PbCtrNt_vect ATGAATATATGGAAAATTATATATATTGTTTTAATTATAAACTCTTCATTTGTGGTGAAT 60 PbCtrNt_plas AAGAATATATGGAAAATTATATGCATTGTTTTAATTATAAACTCTTCATTTGTGGTAAAT 60 * ******************** ******************************** ***
PbCtrNt_vect GTAAATTGTGAGTCCAATGATAAAAATGATGATACTAAAAACAACAAAAATATGGGTTGT 120 PbCtrNt_plas GTAAATTGTGAGTCCAATGATAAAAATGATGATACTAAAAACAACAAAAATATGGGTTGT 120 ************************************************************
PbCtrNt_vect GGTGGTGGTACTAATAAATCACTGCCATCCGTAAACAAACCGCTAAATAGTTGTTGCAGT 180 PbCtrNt_plas GGTGGTGGTACTAATAAATCACTGCCATCCGTAAACAAACCGCTAAATAGTTGTTGCAGT 180 ************************************************************
PbCtrNt_vect GGTAAACGATCGGGAAATGATGAGTGCAAACCAATATTAGATCTCAATCACATAGGAAGT 240 PbCtrNt_plas GGTAAACGATCGGGAAATGATGAGTGCAAACCAATATTAGATCTCAATCACATAGGAAGT 240 ************************************************************
PbCtrNt_vect GAAGGCAAAAACAAAATTCCATTTATTTACAAATGCTGTATAAATCATGATACATATGAA 300 PbCtrNt_plas GAAGGCAAAAACAAAATTCCATTTATTTACAAATGCTGTATAAATCATGATACATATGAA 300 ************************************************************
PbCtrNt_vect AACATAATTAATGAGCACTTTAATGAAGAAAATCAAAACAATGCAACCGATGCCCCCAAA 360 PbCtrNt_plas AACATAATTAATGAGCACTTTAATGAAGAAAATCAAAACAATGCAACCGATGCCCCCAAA 360 ************************************************************
PbCtrNt_vect AAATTGAATATGATGATGAGTGATTTATCTATGCCTATGTCATTTCAGAATACTACCCAT 420 PbCtrNt_plas AAATTGAATATGATGATGAGTGATTTATCTATGCCTATGTCATTTCAGAATACTACCCAT 420 ************************************************************
PbCtrNt_vect ACTATTATTTTATTCAAATTTTGGGAA 447 PbCtrNt_plas ACTATTATTTTATTCAAATTTTGGGAA 447 ***************************
Figure 4.2 Alignment of the RT-PCR amplified putative P. berghei copper transporter's amino terminus coding sequence with the PlasmoDB sequence
The sequenced P. berghei copper transport protein amino-terminus (PbCtrNt_vect) and PlasmoDB sequence
[PbCtrNt_plas (PBANKA_130290)] were aligned with the ClustalW program
(http://www.ebi.ac.uk/Tools/msa/clustalw2/).
Using a similar approach, the putative P. falciparum copper transporter coding domain was amplified. Here, both the full length and amino terminus coding domains were targeted, whilst amplification of the LDH coding domain served as a positive control. A LDH product was
amplified from the P. falciparum genome that was approximately 820bp in size (Figure 4.3), which was similar to the results obtained for P. yoelii and P. berghei (Figure 4.1). Having identified that two potential copper transport proteins are expressed by P. falciparum (Section 3.2.2), it was necessary to probe for the presence of both coding domains (PF14_0211 and PF14_0369) in the parasite genome. Agarose gel analysis of the respective PCR reactions indicated amplification products of approximately 410bp for the full length PF14_0211 domain and 890bp for the full length PF14_0369 domain (Figure 4.3). The size of each PCR product corresponded well with the predicted sizes of each coding domain, which were 423bp for PF14_0211 and 920bp for PF14_0369. Since P. falciparum DNA was isolated from cultured parasites, the PCR control used in this instance was a reaction containing no template. These control reactions produce no amplicons (data not shown).
Figure 4.3 PCR amplification of the coding sequences for two putative P. falciparum copper transport proteins and their predicted amino termini
The full-length and amino terminus coding domains for PF14_0211 (lanes 3 and 4, respectively) and PF14_0369 (Lanes 1 and 2, respectively) were PCR amplified from P. falciparum genomic DNA and analysed on a 1% (w/v) agarose gel. Lane 5 is the P. falciparum LDH control. Molecular mass markers (M) are shown to the left of the gel (See materials and methods for details).
To confirm the identity of the P. falciparum amplicon, the amplicons corresponding to the amino termini of the putative P. falciparum copper transporters were cloned and sequenced. The PF14_0211 amplicon was approximately 150bp and the PF14_0369 amplicon 290bp (Figure 4.3), which was in agreement with the predicted sizes. Sequencing of each product identified that both amplicons corresponded well with the respective PlasmoDB sequences, with 98%
identity for PF14_0211 and 100% for PF14_0369 (Figure 4.4). In silico translation of these sequences yielded amino acid sequences with 97% and 100% identity to the PlasmoDB sequences for PF14_0211 and PF14_0369, respectively (data not shown).
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PfCtr211Nt_vect TTCACTTTCGATTTTGTGAGCAGCAGTTGTTGTCATTCAAAAAATGACGATGGAGTTATG 60 PfCtr211Nt_plas TTCACTTTCGATTTTGTGAGCAGCAGTTGTTGTCATTCAAAAAATGACGATGGAGTTATG 60 ************************************************************
PfCtr211Nt_vect TTACCTATGTATTTTTCAAATAATGGAAATATAAAAATGTTATTTGACATATTTCAAGTA 120 PfCtr211Nt_plas TTACCTATGTATTTTTCAAATAATGAAAATATAAAAATGTTATTTGACATATTTCAAGTA 120 ************************* **********************************
PfCtr211Nt_vect AAAAAT 126 PfCtr211Nt_plas AAGAAT 126 ** ***
B.
PfCtr369Nt_vect GACAAAAGCGACAATAGTATTTGTAAACCTTCTGTTAAAATATCATGTGCTCACAATTGT 60 PfCtr369Nt_plas GACAAAAGCGACAATAGTATTTGTAAACCTTCTGTTAAAATATCATGTGCTCACAATTGT 60 ************************************************************
PfCtr369Nt_vect TGTAAGAATAAATTATCTTTTATGTATAATTGTTGGAAACACTATGATAAATATAGTAAT 120 PfCtr369Nt_plas TGTAAGAATAAATTATCTTTTATGTATAATTGTTGGAAACACTATGATAAATATAGTAAT 120 ************************************************************
PfCtr369Nt_vect ATAATAAAAGATAATTTACAAAAAGAAGAAGATACAGTTGTTCAATTACAAGATCATGAT 180 PfCtr369Nt_plas ATAATAAAAGATAATTTACAAAAAGAAGAAGATACAGTTGTTCAATTACAAGATCATGAT 180 ************************************************************
PfCtr369Nt_vect AATATTGATATTGTCGAACATGTTGAAACTATGCCAATGTCCTTTCAGCTGACTACACAC 240 PfCtr369Nt_plas AATATTGATATTGTCGAACATGTTGAAACTATGCCAATGTCCTTTCAGCTGACTACACAC 240 ************************************************************
PfCtr369Nt_vect ACAATTATTTTATTTAACAAATGGGAAACCAAATCG 276 PfCtr369Nt_plas ACAATTATTTTATTTAACAAATGGGAAACCAAATCG 276 ************************************
Figure 4.4 Alignment of the sequenced amplicons for PF14_0211 and PF14_0369 putative copper transport protein amino terminus coding domains with the PlasmoDB sequences
A PF14_0211 (PfCtr211Nt_vect) and B PF14_0369 (PfCtr369Nt_vect) amino termini amplicons. PlasmoDB sequences are PfCtr211Nt_plas and PfCtr369Nt_plas, respectively. Sequences were aligned using the ClustalW program (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
4.2.2 Immunolocalisation of the murine and human malaria parasite putative copper