Proc. Natl. Acad. Sci. USAVol. 92, pp. 973-977, February 1995Genetics

Transfection ofPlasmodium falciparum within human redblood cells

(malaria/chloramphenicol acetyltransferase/gene expression/codon usage)

YIMIN WU, C. DAVID SIFRI, HSIEN-HSIEN LEI, XIN-ZHUAN SU, AND THOMAS E. WELLEMSLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Communicated by Louis H. Miller, National Institutes of Health, Bethesda, MD, November 9, 1994 (received for review October 4, 1994)

ABSTRACT Plasmodium falciparum malaria parasiteswithin human red blood cells (RBCs) have been successfullytransfected to produce chloramphenicol acetyltransferase(CAT). Electroporation of parasitized RBCs was used tointroduce plasmids that have CAT-encoding DNA flanked by5' and 3' untranslated sequences of the P. falkiparum hsp86,hrp3, and hrp2 genes. These flanking sequences were requiredfor expression as their excision abolished CAT activity intransfected parasites. Transfection signals from native CAT-encodingDNA compared well with those from a synthetic DNAsequence adapted to the P. falciparum major codon bias,demonstrating effective expression of the bacterial sequencedespite its use of rare P. falciparum codons. Transfectedring-stage parasites produced CAT signals at least as strongas transfected schizont-stage parasites even though ringstages are surrounded by more RBC cytoplasm than schiz-onts. The transfection of erythrocyte-stage P. falciparumparasites advances our ability to pursue genetic analysis ofthis major pathogen.

Of the four Plasmodium parasites that usually infect man, P.falciparum is the major focus of research because this organismcauses almost all fatalities from malaria and is producing anincreasingly serious drug-resistance problem. P. falciparumresearch moved forward smartly when conditions were foundto continuously propagate the parasite in vitro in human redblood cells (RBCs) (1, 2). Methods of maintaining the com-plete parasite life cycle in the laboratory have since beendeveloped and have led to two P. falciparum crosses (3, 4).Progeny from these crosses have supported linkage studies andmapping of genetic loci-e.g., a chromosome segment gov-erning chloroquine resistance (5)-but techniques for intro-ducing and modifying genes are clearly needed to advancegenetic studies of P. falciparum parasites.Apicomplexans related to P. falciparum have been trans-

fected, including the extracellular stages of Toxoplasma gondiiand purified gametes of the chicken parasite Plasmodiumgallinaceum (6-8). Until now, transfection with P. falciparumremained elusive despite many attempts. One problem hasbeen the cloning of (A+T)-rich sequences flanking the codingregions of P. falciparum genes, as many recombinant DNAconstructs containing these sequences are unstable in Esche-richia coli (9). For example, the full P. falciparum dihydrofolatereductase-thymidylate synthase gene, an attractive selectablemarker because of the pyrimethamine resistance engenderedby its mutant forms (10, 11), rearranges frequently duringcloning (unpublished findings). Nevertheless, sequences flank-ing some P. falciparum coding regions have been stablyisolated in E. coli plasmids. These include the 5' and 3'sequences of hsp86, a P. falciparum gene of the heat-shockprotein 90 family (12), and flanking sequences of the hrp2 and

hrp3 (hrp3/sharp) genes that encode two related histidine-richproteins (ref. 13; unpublished results).Here we describe transfection of P. falciparum with expres-

sion plasmids that incorporate the 5' and 3' flanking sequencesof hsp86, hrp2, and hrp3. Chloramphenicol acetyltransferase(CAT) was chosen as a reporter in this work because of itssmall size, utility in assays of transient expression, and poten-tial for selection of stably transformed parasites.

MATERIALS AND METHODSPlasmid Constructs. The bacterial cat sequence (b-cat) was

amplified from pCAT-Basic DNA (Promega) with primers5'-AGC TAT GCA TGA GAA AAA AAT CAC T-3' and5'-AAC TGC CTT AAA AAG CTT ACG C-3' using routinepolymerase chain reaction (PCR) conditions (14). These prim-ers placed Nsi I and HindlIl sites at the 5' and 3' ends of theb-cat open reading frame and introduced an additional codon(CAT/His) in the second position of the open reading frame(see Fig. 2). The product was cloned into pCRII (Invitrogen),excised with Nsi I and HindIlI, and gel-purified.For construction of pHRPCAT (Fig. la), the 5'-hrp3 cas-

sette was obtained from a cloned 2.4-kb Hinfl fragment inpUC13 that contains 1.9 kb of the 5' flanking sequence fromP. falciparum clone 7G8 (unpublished). After amplificationfrom this plasmid using the primers 5'-TTT GAG AAG GAATGC ATT TTT TTA A-3' and 5'-GAG GTA CCC CCA GTCACG ACG TTG T-3' (complementing nucleotides 299-315 ofpUC13), the 5'-hrp3 sequence was cloned into pCRII, excisedwith Nsi I and Kpn I, and gel-purified. The purified b-cat and5'-hrp3 inserts were inserted together between the HindIII andKpn I sites of pBluescript (Stratagene) in a single ligation step.The 3'-hrp2 flanking sequence (0.6 kb) was amplified froma mung-bean nuclease insert in Agtll (13) using primers5'-CGC CAT TAA GCT TAT TTA ATA ATA GAT-3' and5'-TTT CTC TGC AGT TTA ATA AAT ATG TTC TT-3'.The amplified fragment was cloned into pCRII, excised withHindIII and Pst I, and inserted between the HindIlI and Pst Isites of the 5'-hrp3/b-cat/pBluescript construct to yieldpHRPCAT. Coding regions and junctions were confirmed byDNA sequencing.

Plasmid pHRPCAT has a single Pac I site 0.56 kb upstreamfrom the b-cat cassette (Fig. la). This site was replaced withNsi I andKpn I sites in separate plasmid derivatives by insertionof the annealed adapter sequences 5'-AGG TAC CTA T-3'and 5'-GAT GCATCA T-3'. The 0.56-kb segment was excisedby Nsi I from the first derivative to yield pHRPCATAN. The1.3-kb fragment distal to the destroyed Pac I site was excisedby Kpn I from the second derivative to yield pHRPCATAK.

Abbreviations: CAT, chloramphenicol acetyltransferase; b-cat, bacte-rial CAT-encoding DNA sequence; hrp2, gene encoding P. falciparumhistidine-rich protein 2; hrp3/sharp, gene encoding P. falciparumhistidine-rich protein 3; hsp86, gene encoding P. falciparum heat-shockprotein 86; RBC, red blood cell; s-cat, CAT-encoding DNA sequenceadapted to the P. falciparum major codon bias.

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aK A NBCH PM

pHRPSCATK N B H PM

q pHRPCAT

-)E. =.-

b-catN': B H .yN I I pHRPCATANb-cat

K N TNBCH N PM........................S'd 1 i i pSOCS251-hsP8 s-at T-

TNB CH N PMpSOCS3s-cat

NB CH N PMpsocs,&s

s-catK N TNB C M

s-cat

K N TNB CH N PMee,

b-cat

974 Genetics: Wu et al.

,pSOCSA3

-pSOCS2H1 kb

FIG. 1. Schematic depiction of the plasmid constructs used in transfection experiments. (a) Plasmid pHRPCAT and derivative constructs. (b)Plasmid pSOCS2 and derivative constructs. Vertical arrows indicate mapped positions of the hsp86 transcript start point and mRNA terminus (12).Restriction sites: A, Pac I; B, Bsm I; C, Nco 1; H, Hindlll; & Kpn I; M, BamHI; N, Nsi 1; P, Pst 1; T, BstBI.

The synthetic cat sequence (s-cat; Fig. 2a) was designed tocontain the synonymous codons used frequently by P. falcipa-rum (15-17). Six overlapping oligonucleotides (122-149 nt inlength) were synthesized and amplified by PCR. Amplifiedfragments were cloned into pCRII and those that were free ofnucleotide errors were identified by DNA sequencing.* Atwo-step strategy of amplification and overlap extensionserved to combine the cloned inserts ("a9._Ccf99 , Fig. 2b). VentDNA polymerase (New England Biolabs) was used to avoidartifact from unwanted nucleotides at the ends of amplifiedsubsections. The final s-cat product (GenBank accession no.

U17001) was restricted and placed between the Nsi I andHindIII sites of pHRPCAT to yield pHRPSCAT.

Plasmid pSOCS2 (Fig. lb) incorporates gene control elementsof hsp86. To construct this plasmid, we restricted pHRPSCATwithKpn I and Nsi I, thereby removing the 5'-hrp3 sequence. Thelinearized plasmid was then religated with an adaptor pair,.5'-CGA GAT TCG AAT AAA ATG CA-3' + 5'-TTT TAT

TCG AAT CrC GGT AC-3', that introduced the BstBI site and10 bp of the 5'-hsp86 sequence upstream ofs-cat start codon (12).The fufl 5'-hsp86 flanking sequence was then amplified from Dd2genomicDNA with primers 5'-TAAATG GTA CC7TGATATATT TTT AGA TAT ATG GAT-3' and 5'-TCC GTA TG-CATT TTA TTC GAA ATG TG-3', restricted with Kpn I andBstBI, and inserted between the Kpn I and BstBI sites of thisplasmid. In the final step, the 3'4zsp86 sequence was amplifiedfrom Dd2 genomicDNA with primers 5'-GACTAAAGC TTTTAT ATA ATA TAT TTA TGT AC7 C-3' and 5'-CAT AATGCT GCA GTA TTT GAT GAA TTA AC7 ACA C-3 t

9

restricted with HindIII and Pst I, and inserted between theHindIII and Pst I sites of the plasmid to yield pSOCS2. PlasmidpSOCS3 was generated by a similar strategy, except that primers5'-TATAAGGTACCGGATCCATATAATTATTAATAGGTA C-3' and 5'-TCC GTA TGC ATT TTA TTC GAAATG TG-3' were used to amplify a 1.35-kb 5'-hsp86 se-

quence (having a Nsi I site only at the 3' end), which was thenrestricted with Kpn I and Nsi I and placed into the plasmid.Plasmid pSOCSA5 was obtained from pSOCS2 by removingthe 5'-hsp86 sequence with Kpn I and BstBI, followed by

*We found 70-90% of the cloned inserts to have effors that most likelyarose in oligonucleotide synthesis (18).

M H E K K I T G Y T T V D I S Q W H R K E H F E A F 0 S V A 0 C T Y N 35s-cat acagaattcacagct..AAAAAAAAATTACTGGATATACAACAGTAGATATATCACAATGGCATAGAAAAGAACATTTTGAAGCATTTCAATCAGTAGCTCAATGTACATATAAT 104b-cat G-------- C----------- C--C--T -------- C --------- C-T -------------- G -------- G-----T ----------- C-----C

Q T V 0 L D I T A F L K T V K K N K H K F Y P A F I H I L A R L M N A H P E F R 75s-cat CAAACAGTACAATTAGATATTACAGCATTTTTAAAAACAGTAAAAAAAAATAAACATAAATTTTATCCAGCATTTATTCATATTTTAGCAAGATTAATEZZ=ATCCAGAATTTAGA 224b-cat --G--C--T--G=-------- G--C -------- G--C ----- G -------- G--C--G ------ IIIIIIIIII-C -------- C C-T-MIIIIIIIIIIIIIIII ------------=-----CC-T

M A M K D G E L V I W D S V H P C Y T V F H E 0 T E T F S S L W S E Y N D D F R 115s-cat ATGGCAATGAAAGATGGTGAATTAGTAATATGGGATAGTGTTCACCCATGTTATACAGTTTTTCATGAACAAACTGAAACATTTTCATCATTATGGAGTGAATATCACGATGATTTTAGA 344b-cat -------------- C -----GM--G --------------------T-----C--C-----C---_-G----------- G ------OW-C -----------C-----C-----C=

Q F L H I Y S 0 D V A C Y G E N L A Y F P K G F I E N M F F V S A N S F T 155s-cat CAATTTTTACACATATATTCACAAGATGTAGCATGTTATGGTGAAAATTTAGCATATTTTCCAAAAGGATTTATTGAAAATATGTTTTTTGTATCAGCAAATPMYMV.TAAGTTTTACA 464b-cat --G C -----------M--------GM-----C --------CM--C ----- C--T-----G-------- G -----------C-C----- C ----- C ----- G-----C--C

S F 0 L N V A N M D N F F A P V F T N G K Y Y T Q G D K V L M P L A I 0 V H H A 195s-cat AGTTTTGATTTAAATGTAGCAAATATGGATAATTTTTTTGCACCAGTTTTTACAATGGGAAAATATTATACACAAGGAGATAAAGTATTAATGCCATTAGCAATTCAAGTTCATCATGCA 584b-cat -------------- C--G--C -------- C--C--C--C--C--C-----C--C ----- C ----------- G-----C--C--G--G= G ----------- c

V C D G F H V G R M L N E L 0 0 Y C D E W Q G G A * 220s-cat GTATGTGATGGATTTCATGTAGGAAGAATGTTAAATGAATTACAACAATATTGTGATGAATGGCAAGGAGGTGCAT..actgcagaya 677b-cat --C -------- C--C-----C--C ------ C-T -------------- G--C--C -----G-----G--C--.-------- ttaaggc--t

C 344 565a ->. e

I's 816 b 2211 I'96 3119 d 468 d43 5a40 f 677

196 c+d 468

i_ a+b e+f 6717-115 2 2'I 443

Nfil -NC" Hiqdllls-cat

FIG. 2. Sequence and construction strategy of a synthetic CAT-encoding DNA (s-cat) adapted to the P. falciparum major codon bias. (a)Comparison of the s-cat sequence against the b-cat sequence amplified from the pCAT-Basic plasmid. The s-cat open reading frame containssynonymous codons that are used frequently by P. falciparum. Codons in b-cat that are seldom used by P. falciparum (i.e., those whose frequencyof use for a particular amino acid is <2%) are shaded. Boxed nucleotides identify the Nsi I, Bsm I, Nco I, and HindIII sites of s-cat. The secondcodon that was added to introduce the Nsi I site in b-cat and s-cat is indicated in italics. Numbering is set so that the first nucleotide of the startcodon is position 0. (b) Strategy of s-cat assembly by overlapping PCR amplification. Limits of the six-segment sequences 'W'---.'f'are given by thes-cat nucleotide numbers. Arrows indicate the 25-bp oligonucleotide primers used for production of the intermediate segments (a+b, c+d, e+f)and for final amplification of the full s-cat cassette.

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blunt-end repair and religation. Plasmid pSOCSA3 wasobtained from pSOCS2 by removing the 3'-hsp86 sequencewith HindIII and Pst I.A hybrid CAT-encoding cassette (h-cat) was made in which

codons of the central part of the s-cat sequence are replacedby b-cat codons (pSOCS2H, Fig. lb). For this purpose a b-catsegment was amplified from pCAT-Basic using primers 5'-CTG ATG AAT GCT CAT CCG G-3' and 5'-CTC ACC CATGGA TTG GCT G-3'. After restriction with Bsm I and Nco I,the amplified product was gel-purified and inserted in framebetween the unique Bsm I and Nco I sites of pSOCS2 to yieldpSOCS2H.

All plasmids expressed b-cat, h-cat, or s-cat in E. coli.Bacterial cultures were therefore routinely examined for CATactivity as a test of the expression plasmids. Plasmid DNAswere prepared from overnight E. coli cultures by SDS/alkalinelysis and CsCl gradient centrifugation or purification on acommercial resin (Plasmid Mega Kit, Qiagen, Chatsworth,CA).

Parasite Transfection. P. falciparum was cultivated in hu-man RBCs by standard methods (1, 2). All RBC stocks weremade leukocyte-free by passage through Sepacell R-500 sets(Baxter Health Care, Mundelein, IL). Synchronizations wereperformed by treating parasitized RBCs with 5% sorbitol for10 min (19). In some experiments, ring-infected RBCs werepurified by Percoll/sorbitol gradient centrifugation (20) toremove residual extracellular forms and mature-stage para-sites before and after electroporation.For electroporation, RBCs (usually containing 10% para-

sitized forms) were pelleted and resuspended in 800 ,ul ofincomplete Cytomix (120 mM KCl/0.15 mM CaCl/2 mMEGTA/5 mM MgCl2/10 mM K2HPO4/KH2PO4/25 mMHepes, pH 7.6) (21) containing plasmid DNA. Electropora-tions were performed using a Bio-Rad Gene Pulser and PulseController Unit set at 200 fQ. Routine electroporations wereperformed at settings of 2.5 kV and 25 ,F with 4-mm cuvettes.Time constants were 0.7-0.9 ms. Electroporated samples wereimmediately mixed with 10 ml of culture medium, placed into25-cm2 canted-neck culture flasks (Coming), and cultivatedwith daily medium replacement.CAT Assays. Samples (500 IlI) of E. coli overnight cultures

were pelleted at 15,000 x g and resuspended in 1 ml of TENbuffer (1 mM EDTA/150mM NaCl/40mM Tris HCl, pH 7.6).Infected RBCs were recovered from culture, washed once withphosphate-buffered saline (PBS; 1.7 mM KH2PO4/5 mMNa2HPO4/150 mM NaCl, pH 7.2) and incubated in twovolumes of 0.15% saponin/PBS at 37°C for 10 min. After thereleased parasites were packed by centrifugation (15,000 x g,10 min), the supematant and RBC ghosts were aspirated fromthe pellet and the parasites were resuspended in 1 ml of TEN.For CAT assays, the cells inTENwere pelleted, resuspended

in 120 ,ul of 0.25 M Tris-HCl (pH 7.6), and disrupted by threecycles of freezing on dry ice and thawing. Lysates wereincubated for 10 min at 65°C and cleared by centrifugation ina Microfuge (15,000 x g, 2 min). As a quantitative standard,CAT (10 units/,ul, Promega) was diluted in 250 mM Tris HCl(pH 7.6). One hundred microliters of lysate or CAT standardsolution was combined with 20 ,ul of 40 mM acetyl-coenzymeA (Calbiochem-Nova Biochem), 60 ,ul of 250 mM Tris-HCl(pH 7.6), and 1 ,ul (2.5 nCi) of [14C]chloramphenicol (110mCi/mmol; 1 Ci = 37 GBq; Moravek Biochemicals, La Brea,CA). Reaction mixes were incubated overnight at 37°C, ex-tracted with 400 ,ul of ethyl acetate, dried under vacuum, andredissolved in 10 ,lI of ethyl acetate. Samples were spotted ontoaluminum-backed, thin-layer silica gel 60A plates (Whatman)and developed for 45 min with a 20:1 chloroform/methanolsolvent in a glass chromatography chamber (PGC Scientific,Gaithersburg, MD). Signals were measured by a PhosphorIm-ager system (Molecular Dynamics) and exposed to KodakX-Omat scientific imaging film for photographic prints.

RESULTSTransfection by CAT Constructs (pSOCS2, pSOCS2H)

Containing Control Elements of the hsp86 Gene. Electropo-ration conditions were determined from the survival of par-asites subjected to different shocks. At a capacitance of 25 ,F,voltages of 2.0-2.5 kV with 4-mm cuvettes and 1.5-2.5 kV with2-mm cuvettes reduced parasitemia on the following day by48-78%, whereas, at 960 ,uF, voltages of 0.34-0.42 kV with2-mm cuvettes reduced parasitemia by 44-81%, relative tounshocked controls. We chose settings of 2.5 kV and 25 ,uFwith 4-mm cuvettes for routine electroporations.CAT signals were initially detected from parasites trans-

fected with pSOCS2. Fig. 3 shows time courses of expressionfrom ring- and schizont-stage parasites. Immediately aftertransfection (0 h) no cat expression was apparent. CAT signalsthen developed within 12 h and increased over the next 48 h.Signals from ring-forms (Fig. 3a) increased more rapidly thanthose from transfected schizonts (Fig. 3b), perhaps reflectingdifferential survival of rings and schizonts after electropora-tion. These observations were repeated in three separateexperiments. Similar results were obtained from plasmid DNApurified by Qiagen columns or CsCl gradient centrifugation.No cat expression was obtained when uninfected RBCs were

transfected or when parasitized RBCs were mixed with plas-mid DNA but not electroporated (Fig. 3). The possiblepresence of bacteria or yeast was tested by incubating samplesof transfected cultures in RPMI medium 1640, in LB broth,and on sheep blood agar plates for 48 h at 37°C. No bacterialor fungal growth was observed. Transfected parasites were alsotreated with 10 jig of cycloheximide per ml. This inhibitor ofeukaryotic protein synthesis completely blocked CAT signalsfrom P. falciparum but not from E. coli (Fig. 4a). These resultsdemonstrate that the CAT signals were produced by P. falci-parum and did not arise from the host cells themselves,

a 0.

3M-

IM-

..* * *4pjJ* .......... ......... .. ...... .. .........

b &= . = .c e z

FIG. 3. CAT signals from P. falciparum-infected RBCs. (a) Timecourse of signals from synchronized, ring-stage parasites transfectedwith pSOCS2. TIwenty samples from a single culture (each containing1 x 109 RBCs with 10% infected forms) were transfected separately,pooled, centrifuged, resuspended into culture medium, and distributedinto 10 culture flasks so that each contained 2 x 109 RBCs. Thesamples were harvested at selected time points, examined by lightmicroscopy, and assayed for CAT activity. Lanes show CAT signalsfrom the parasites at different times after transfection: 0 h (earlyrings), 12 h (rings/trophozoites), 24 h (schizonts), 36 h (segmenters/rings), 48 h (rings), 60 h (trophozoites), and 72 h (schizonts). Para-sitized RBCs mixed with 50 jig of pSOCS2 without electroporation("No Ep") and uninfected RBCs (2 x 109) transfected with 50 ,ug ofpSOCS2 ("RBC") were also included in the experiment and harvestedafter 36 h. CAT signal from an overnight culture of E. coli containingpSOCS2 is shown at the right ("Ec."). (b) Time course of signalsobtained from synchronized, schizont-stage parasites transfected withpSOCS2. Lanes show CAT signals from the parasites at 0 h (schizonts),12 h (segmenters), 24 h (rings), 36 h (rings), 48 h (trophozoites), and60 h (segmenters/rings). Microscopy identified pyknotic forms fromthe electroporated schizonts that may have resulted from damage tothe mature parasite stages by electroporation. Labels at the leftindicate signals from the unacetylated (N) and monoacetylated forms(1M, 3M) of [14C]chloramphenicol.

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a 1 2 3 4 bAS;

d

3M- 9W* 90i |i70

1M - % 501

30

10

N- *. 0.5 07 1.0 1.5 20 25RBC (10')

1 2 3 4 C

3M- * 0 * 7

IM - Ad %

FIG. 4. Effect of cycloheximide, parasite number, amount ofplasmid DNA, and Percoll/sorbitol purification on transfection sig-nals. (a) Cycloheximide abolishes CAT signals from P. falciparum butnot E. coli. Lanes: 1, transfected parasitized RBCs (50 ,ug of pSOCS2,2 x 109 RBCs at 10% parasitemia) without cycloheximide treatment;2, transfected parasitized RBCs with 10 ,g of cycloheximide per mlapplied immediately after transfection; 3, pSOCS2-transfected E. coliwithout cycloheximide treatment; 4, pSOCS2-transfected E. coli with10 jg of cycloheximide per ml applied immediately after transfection.(b) Effect of parasite number on CAT signals. Fifty micrograms ofpSOCS2 was transfected into separate samples containing the indi-cated number of RBCs at 10% parasitemia. Samples were harvestedafter 40 h. Acetylation percentages were determined from the sum ofthe two monoacetylated signals (IM + 3M) divided by the sum of thesignals from monoacetylated and nonacetylated forms (1M + 3M +N). (c) CAT signals from 1 x 109 RBCs at 10% parasitemia aftertransfection with indicated amounts of plasmid DNA. Samples wereharvested after 40 h. (d) CAT signals from transfected parasitespurified at ring stage by Percoll/sorbitol gradient centrifugation(PSGC). RBCs (1 x 1010) containing 10% synchronized ring-stageswere recovered from the 90%/80% Percoll layers after PSGC, dividedinto 10 cuvettes, transfected with pSOCS2 (50 ,ug each), and pooled.One-fourth of the pooled cells (2.5 x 109 RBCs) was then immediatelyrepurified by PSGC. The cells were washed and returned to culture.After 48 h (ring stages again) a second sample of 2.5 x 109 RBCs waspurified by PSGC. Portions of the samples representing 5 x 108 RBCsat 10% parasitemia were assayed for CAT activity at 72 h. Lanes: 1,uninfected RBC control; 2, transfected ring stages without subsequentPSGC; 3, transfected ring stages purified by PSGC immediately aftertransfection; 4, transfected ring stages purified by PSGC 48 h aftertransfection.

contaminating microorganisms, or artifactual introduction ofCAT protein.CAT standards showed that 0.005 unit of enzyme produced

25% acetylation of the [14C]chloramphenicol substrate in ourassays, a conversion percentage about equivalent to thatobtained from 5 x 108 RBCs at 10% parasitemia (5 x 107infected RBCs) 40 h after transfection with 50 ,ug of pSOCS2.Greater numbers of cells produced increases in CAT produc-tion until 2.5 x 109 cells (2.5 x 108 infected RBCs), themaximum number used in these experiments (Fig. 4b). Trans-fection of 1 x 109 RBCs (1 x 108 infected cells) with 1-100 jigof pSOCS2 showed that CAT signals increased with theamount of transfected DNA (Fig. 4c).To confirm that transfection signals were from parasitized

RBCs, we purified ring-infected RBCs by Percoll/sorbitolgradient centrifugation before and after transfection. Signalintensities from the purified cells were comparable to thosefrom control samples that had not been gradient-purified,indicating that the CAT signals indeed arose from the para-

sitized cells (Fig. 4d). Since gametocytes could be anothersource of transfection signals from the Dd2 P. falciparumclone, we checked transfection signals from the L.F4.F1 clonethat is defective in gametocytogenesis (22). The CAT signalsfrom Dd2 and L.F4.F1 were indistinguishable (data notshown).As a test of the effect of cat codon usage on expression we

modified pSOCS2 so that a 230-bp section in the middle ofs-cat was replaced by the corresponding section from b-cat(Fig. lb). The pSOCS2H plasmid containing this hybrid form(h-cat) has seven rare codons between the Bsm I and Nco Isites, including a CGG(Arg) codon that was not found at all inthree different codon-usage analyses of P. falciparum genes(15-17). Fig. 5 shows that the pSOCS2H hybrid form isefficiently expressed by P. falciparum. In three separate ex-periments, CAT signals from pSOCS2H were 30-80% of thosefrom pSOCS2 (Fig. 5; data not shown).

Transfection by CAT Constructs (pHRPCAT, pHRPSCAT)Containing Control Elements of the hrp2 and hrp3 Genes. P.falciparum parasites complete the malaria life cycle despitedeletion of the hrp2 and hrp3 genes (23). Plasmids incorpo-rating sequences of these genes are thus candidates for theintegration of selectable markers into the parasite genome.Two control elements from these genes were available in thiswork, a 5'-hrp3 flanking sequence and a 3'-hrp2 flankingsequence. These were incorporated into pHRPCAT (contain-ing b-cat) and pHRPSCAT (containing s-cat) and tested forexpression. In two separate experiments, the signal intensitiesfrom pHRPCAT were -80% of those from pHRPSCAT (Fig.5; data not shown), confirming efficient expression despiterare codons in the b-cat sequence.

Effects of Flanking Sequence Deletions on pSOCS2 andpHRPCAT Expression. Fig. S shows that pSOCSA5, fromwhich the 5'-hsp86 sequence is excised, produced no CATsignals from transfected parasites. The part of this 5' sequencenecessary for expression lies within 1.3 kb of s-cat, as pSOCS3,which lacks 0.5 kb distal to the Nsi I site, is expressed at thesame level as pSOCS2. CAT activity was not detected inparasites transfected by pSOCSA3, indicating that the 3'-hsp86flanking sequence is also essential to s-cat expression bypSOCS2.

Deletions from the 5'-hrp3 sequence were examined fortheir effects on b-cat expression by pHRPCAT. Fig. 5 showsthat excision of the 0.56-kb region adjacent to b-cat(pHRPCATAN) decreased but did not abolish the CAT signal.Upstream of b-cat, however, deletion of the 1.3-kb section

I-~~~~~~~~~~c0~~~ ~ ~~

0 ~ ~~ .w.w3M-

..... ......

e

N-

FIG. 5. CAT signals from P. falciparum-infected RBCs aftertransfection by selected expression constructs. In all experiments 2 x109 RBCs containing 10% infected forms (predominantly ring stages)were transfected with 50 ug of plasmid DNA and harvested after 40h. Control lanes show CAT assays from parasitized RBCs mixed with50 ,ug of pSOCS2 without electroporation ("No Ep") and uninfectedRBCs transfected with 50 ,ug of pSOCS2 ("RBC").

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distal to the Pac I site practically eliminated b-cat expressionby pHRPCATAK.

DISCUSSIONElectroporation of P. falciparum-infected RBCs is an effectiveprocedure for transfecting cultivated malaria parasites. Youngring-stage parasites, which take up a relatively small volume ofthe RBC cytoplasm, as well as mature schizonts, which fill thecell after digestion of most hemoglobin, yield transfectionsignals by this method. The parasites within RBCs are evi-dently the transfected forms, as CAT signals were found tosegregate with parasitized RBCs purified on Percoll/sorbitolgradients. The pathway by which the DNA enters the parasitesis unclear. Direct delivery through the RBC cytoplasm wouldrequire that the DNA cross multiple membrane layers, includ-ing those of the RBC, the parasitophorous vacuole, and theparasite itself. DNA may have had direct access to the intra-erythrocytic space around the parasite through ducts that areproposed to serve in molecular traffic to and from the RBCsurface (24).Codon usage in the cat cassettes was adjusted to test the

conjecture that expression may be affected by the P. falciparummajor codon bias. We compared expression from pSOCS2,containing the fully adjusted s-cat cassette, with that frompSOCS2H, containing the hybrid h-cat that has seven rarecodons in the middle part of the coding sequence, including theCGG(Arg) codon not found at all in three codon-usage tables(15-17). These comparisons showed that P. falciparum ex-presses h-cat efficiently despite its use of rare codons. Signalvariations that did occur may not have resulted from codonbias at all but, instead, may have arisen from other factors-e.g., differences in plasmid DNA preparations or effects ofplasmid structure on transcription factor loading. We note thata lack of effect by codon usage per se would be consistent withfindings from codon bias studies in other organisms (25).

Modifications of the 5' and 3' flanking sequences inpSOCS2 and pHRPCAT produced effects that reflect thespecific promoter and termination activity of P. falciparum. Inthe case of pSOCS2, expression was abolished by deletion ofthe 5'- or 3'-hsp86 sequences. These flanking sequences con-tain control regions of the hsp86 gene (12): an invertedCCAAT box and a (G+C)-rich sequence preceding a majortranscription start point at position -655, the 3'-poly(A)signal, and a downstream TATGT sequence similar to thatreported to affect transcript termination in yeast (26). In thecase of pHRPCAT, modifications of the 5' sequence localizethe hrp3 promoter >0.56 kb upstream of the start codon.These findings agree with a 5' untranslated region of >0.5 kbthat has been estimated for the hrp3 transcript (ref. 13;unpublished findings). Additional mapping experiments canbe expected to locate and characterize P. falciparum promoterregions in detail.

Stable transfection of P. falciparum may now be possibleusing CAT as a selectable marker.t Indeed, in the relatedapicomplexan T gondii, gene replacement has already beenachieved through homologous recombination of CAT con-structs under chloramphenicol selection (6). Similar success inthe stable transfection of P. falciparum would facilitate func-

tional studies of genes and experiments with genetically alteredmalaria parasites.

Note Added in Proof. In independent work with rodent malariaparasites, M. R. van Dijk, A. P. Waters, and C. J. Janse (personalcommunication) have used electroporation to transform Plasmodiumberghei blood stages with an episomally replicating plasmid thatencodes a pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase. We have obtained luminescence signals fromintraerythrocytic P. falciparum transfected with a pHRPCAT deriv-ative in which the b-cat cassette is replaced by luciferase-encodingDNA.

We thank Laura Kirkman for technical help and Janet Yee, FrankMaldarelli, Shyh-ing Jang, Michael Nerenberg, John Boothroyd, LouisMiller, and Steven Beverley for discussions and advice. C.D.S. wassupported by the Howard Hughes Medical Institute Research ScholarsProgram.

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Genetics: Wu et al.

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