Download Supplementary material for "The Plasmodium HU homolog, which

Supplementary material for "The Plasmodium HU homolog, which binds the plastid
DNA sequence-independently, is essential for the parasite's survival" by Sasaki et al.
Contents
1. Materials and methods
1.1. Molecular cloning of the PfHU gene.
1.2. Antibody preparation.
1.3. Immunofluorescence microscopy.
1.4. Transfection of parasite and localization of expressed fluorescent proteins.
1.5. Production of recombinant PfHU.
1.6. Targeted disruption of P. berghei genes
2. References
3. Figure legends
1. Materials and methods
1.1. Molecular cloning of the PfHU gene
A pool of first-strand cDNA was prepared from the total RNA of P.
falciparum C10 using the FirstChoice RLM RACE Kit (Ambion), and cDNA
encoding the BHL domain-containing protein of the parasite (PfHU) was PCRamplified with set of synthetic primers hu-550 (5'tcagatctaaaATGTATGTAATATTATATTG-3'), hu-551 (5'ATCCTAGGCATTATCTTTTCAACAGTC-3'), hu-552 (5'AGTCTAGATGCAGCATAAATTAACTTTTC-3') and those supplied in the kit.
The genomic DNA sequence of the chromosomal region corresponding to the cDNA
sequence was also cloned to confirm that the cDNA sequence originated from the
genomic region. The genomic DNA sequence of the PfHU gene was deposited in the
EMBL database with accession numbers FM204793.
1.2. Antibody preparation
Part of cDNA encoding the predicted mature form of PfHU (PfHU53-189)
was recombined to pET28 (Novagen) and the 6 × His-tagged protein expressed in E.
coli BL21(DE3) CodonPlusTM (Novagen) was purified with Ni-column (HisTrap; GE)
to immunize a rabbit. Anti-PfHU antibodies were isolated from the serum using
MABTrap (GE).
1.3. Immunofluorescence microscopy
The red blood cells (RBCs) infected by P. falciparum transfectant with
pSSPF2/PfACP-GFP [1] were fixed in 4% paraformaldehyde containing 0.075%
glutaraldehyde followed by termination of fixation reaction with NaBH4 and
permeabilization of the membrane by incubating in PBS containing Triton X-100 at
0.1% as described elsewhere [2]. After blocking in PBS containing 3% bovine serum
albumin, the RBCs were incubated in PBS containing the anti-HU antibody prepared
as described in section 1.2 and then with the anti-rabbit IgG secondary antibody
conjugated with Alexa Fluor 594 (Invitrogen). After washes in PBS, the RBCs were
analysed by fluorescence microscopy using the DeltaVision microscopic system.
1.4. Transfection of parasite and localization of expressed fluorescent proteins
The bsd gene of the plasmids pEM7/Bsd (Invitrogen) was recombined to the
P. falciparum expression plasmids pSSPF2/PfHsp60-GFP [1] and
pSSPF2/PfACP-DsRed [3] replacing the hDHFR gene to generate
pSSPF3/PfHsp60-GFP and pSSPF3/PfACP-DsRed, respectively. The yfp genes of
pEYFP-N1 (Clontech) was recombined to the pSSPF3/PfHsp60-GFP replacing the
GFP coding sequence to make pSSPF3/PfHsp60-YFP.
cDNA of PfHU was cloned and the region encoding Met1-Met82 was
amplified by PCR and recombined to pSSPF3/PfHsp60-YFP as described in the
supplemental material. Sixty micrograms of the resulting plasmid
pSSPF3/PfHU-YFP and 50 µg of pSSPF3/PfACP-DsRed were introduced into P.
falciparum 3D7 simultaneously by electroporation [3] and dual-transfectant parasites
were selected with blasticidin S HCl (Invitrogen) at 1 µg/ml. Subcellular distribution
of fluorescent proteins expressed in the living parasite was analysed by fluorescence
microscopy using the DeltaVision system (Applied Precision), after the nucleus was
stained with Hoechst 33342 (Sigma).
1.5. Production of recombinant PfHU.
The Recombinant PfHUs, PfHU53-189 and PfHU53-148, were expressed as
fusion proteins with glutathione-S-transferase (GST) using vector pGEX6P-1 (GE) in
E.coli BL21(DE3) CodonPlusTM and purified using Hi-trap Q and SP column. After
further purification with glutathione-Sepharose 4B columns (GE), each protein was
processed with Prescision protease to remove the GST tag. The processed proteins,
which have only five excess amino acid residues (Gly-Pro-Leu-Gly-Ser) remaining at
the N-terminus, were used in the assay after dialysis against buffer (20 mM Tris-HCl,
pH 7.5, 1mM EDTA, 50 mM NaCl, 1mM DTT).
1.6. Targeted disruption of P. berghei genes
The 5’ and the 3’ non-coding regions of the PbHU gene were amplified from
the genomic DNA of P. berghei ANKA clone 2.34 with two sets of primers, PbHUF1-KpnI (5’-ggtaccGCACATCTTTTGATTTTTTAGCCT-3’) and PbHU-R1-HindIII
(5’-aagcttTTTGTTCTCTTGTATTTTAAGATA-3’), and PbHU-F2-EcoRV (5’gatatcTGCCTTTTATTGTAAAGAAAATAA-3’) and PbHU-R2-BamHI (5’ggatccTACCTTTAATACCCATTTGCATA-3’), respectively. The two fragments
were recombined to the plasmid pBS-DHFR [4] to obtain pPbHU-KO.
The pPbHU-KO plasmid and the control plasmid pPbGCS1-KO [4] were
linearised by restriction digestion with KpnI and BamHI and separately introduced
into P. berghei by electroporation as described by Janse et al. [5]. The electroporated
parasites were injected to mice and selected with pyrimethamine as a supplement to
the drinking water at 70ng/ml for one week. The parasite whose target gene is
successfully disrupted acquires resistance to pyrimethamine and reaches 0.5-2%
parasitaemia on day 7 after transfection.
2. References
[1]
Sato, S., Rangachari, K. and Wilson, R.J.M. (2003). Targeting GFP to the
malarial mitochondrion. Mol Biochem Parasitol 130, 155-8.
[2]
Tonkin, C.J., van Dooren, G.G., Spurck, T.P., Struck, N.S., Good, R.T.,
Handman, E., Cowman, A.F. and McFadden, G.I. (2004). Localization of
organellar proteins in Plasmodium falciparum using a novel set of transfection
vectors and a new immunofluorescence fixation method. Mol Biochem
Parasitol 137, 13-21.
[3]
Sato, S. and Wilson, R.J.M. (2004). The use of DsRED in single- and dualcolor fluorescence labeling of mitochondrial and plastid organelles in
Plasmodium falciparum. Mol Biochem Parasitol 134, 175-9.
[4]
Dessens, J.T., Beetsma, A.L., Dimopoulos, G., Wengelnik, K., Crisanti, A.,
Kafatos, F.C. and Sinden, R.E. (1999). CTRP is essential for mosquito
infection by malaria ookinetes. EMBO J 18, 6221-7.
[5]
Janse, C.J., Ramesar, J. and Waters, A.P. (2006). High-efficiency transfection
and drug selection of genetically transformed blood stages of the rodent
malaria parasite Plasmodium berghei. Nat Protoc 1, 346-56.
[6]
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G.
(1997). The CLUSTAL_X windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Res 25,
4876-82.
[7]
Carlton, J.M. et al. (2008). Comparative genomics of the neglected human
malaria parasite Plasmodium vivax. Nature 455, 757-63.
3. Figure legends
Fig. S1. PfHU is a plastid protein. The P. falciparum transfectant expressing the
plastid-localizing PfACP-GFP (the plastid targeting sequence of the P. falciparum
acyl carrier protein C-terminally tagged with GFP) was fixed and the presence of HU
was analyzed by immunofluorescence microscopy with the anti-HU antibody. The
parasites were either the young trophozoite (A) or the schizont (B) stage.
Microscopic observation was carried out using the DeltaVision system. The green
and red signals were merged (Merge) and superimposed on the differential
interference contrast image along with blue signals of Hoechst 33342 from the
parasite's nucleus (+DiC). Scale bar: 5m.
Fig. S2. The N terminal sequence of PfHU is a functional plastid targeting sequence.
Fluorescence microscopy analysis of the live dual-transfectant parasite expressing
both PfHU-YFP (Met1-Met82 of PfHU fused to the N terminus of YFP) and PfACPDsRed (the plastid targeting sequence of the P. falciparum acyl carrier protein Cterminally tagged with DsRed) confirmed that these two fluorescent proteins
exclusively co-localize in the same subcellular compartment in the parasite. This
suggests that the N terminal sequence of PfHU functioned as a strict plastid targeting
sequence. The parasites were either the young trophozoite (A) or the schizont (B)
stage. Microscopic observation was carried out using the DeltaVision system. The
green and red signals were merged (Merge) and superimposed on differential
interference contrast image along with blue signals of Hoechst 33342 from the
parasite's nucleus (+DiC). Scale bar: 5m.
Fig. S3. Western blotting of PfHU53-189 and PfHU53-148 used in the DNAmobility shift assay. A. The two truncated forms of the recombinant PfHUs affinitypurified from expressing E. coli. B. The apparent molecular mass of PfHU53-189
(~18 kDa) was slightly smaller than but almost the same as the authentic PfHU
present in the parasite (~19 kDa: the main band in "Total lysate").
Fig. S4. Sequence alignment of Plasmodium HUs
The amino acid sequences of the putative HUs of P. vivax (PvHU), P. knowlesi
(PkHU), P. berghei (PbHU) and P. yoelii (PyHU) were deduced from the nucleotide
sequence data downloaded from the databases summarized in Supplementary Table. 1
and aligned with the PfHU using Clustal X program [6]. The BHL domain was
highlighted and the cluster of hydrophobic amino acid residues and the following
basic sequence, which compose the plastid targeting sequence of Plasmodium spp.
[7], were indicated with a red and a cyan bars on the alignment, respectively.