Genetic Disruption of the Plasmodium falciparum DigestiveVacuole Plasmepsins Demonstrates Their Functional Redundancy*

Received for publication, August 20, 2004, and in revised form, October 13, 2004Published, JBC Papers in Press, October 18, 2004, DOI 10.1074/jbc.M409605200

A. Levi Omara-Opyene‡§¶, Pedro A. Moura§�, Carlos R. Sulsona‡, J. Alfredo Bonilla‡,Charles A. Yowell‡, Hisashi Fujioka**, David A. Fidock�, and John B. Dame‡ ‡‡

From the ‡Department of Pathobiology, University of Florida, Gainesville, Florida 32611-0880, the �Department ofMicrobiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, and**Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

The digestive vacuole plasmepsins PfPM1, PfPM2,PfPM4, and PfHAP (a histoaspartic proteinase) are 4aspartic proteinases among 10 encoded in the Plasmo-dium falciparum malarial genome. These have beenhypothesized to initiate and contribute significantly tohemoglobin degradation, a catabolic function essen-tial to the survival of this intraerythrocytic parasite.Because of their perceived significance, these plas-mepsins have been proposed as potential targets forantimalarial drug development. To test their essenti-ality, knockout constructs were prepared for each cor-responding gene such that homologous recombinationwould result in two partial, nonfunctional gene copies.Disruption of each gene was achieved, as confirmed byPCR, Southern, and Northern blot analyses. Westernand two-dimensional gel analyses revealed the ab-sence of mature or even truncated plasmepsins corre-sponding to the disrupted gene. Reduced growth rateswere observed with PfPM1 and PfPM4 knockouts, in-dicating that although these plasmepsins are not es-sential, they are important for parasite development.Abnormal mitochondrial morphology also appeared toaccompany loss of PfPM2, and an abundant accumula-tion of electron-dense vesicles in the digestive vacuolewas observed upon disruption of PfPM4; however,those phenotypes only manifested in about a third ofthe disrupted cells. The ability to compensate for lossof individual plasmepsin function may be explained byclose similarity in the structure and active site of thesefour vacuolar enzymes. Our data imply that drug dis-covery efforts focused on vacuolar plasmepsins mustincorporate measures to develop compounds that caninhibit two or more of this enzyme family.

Malaria remains a public health problem of enormous mag-nitude in the tropical and subtropical regions of the world,annually afflicting an estimated 500 million people and killingnearly 2 million, mostly children (1, 2). Of the four species ofmalaria parasites that infect humans, the most lethal one,Plasmodium falciparum, is becoming increasingly resistant to

the available drugs, making it essential to pursue the develop-ment of new antimalarial compounds that act on essentialparasite pathways unencumbered by current mechanisms ofdrug resistance (3). One validated pathway whose metabolicfunction is unique to the parasite is hemoglobin digestion,which results in the sequestration of free ferriprotoporphyrinIX as an inert, crystalline form (hemozoin) in the digestivevacuole (DV)1 of the parasite.

Previous studies by others suggest that hemoglobin digestionis a semiordered process initiated in the DV by two asparticproteinases named plasmepsins (plasmodium pepsins) I and II(PfPM1 and PfPM2, respectively). These enzymes can mediatethe first cleavage of the Phe33–Leu34 bond in the hinge regionof the � chain, causing the whole molecule to unravel in theacidic DV environment (4–7). Genome sequencing and subcel-lular localization studies have identified two additional DVplasmepsins, a histoaspartic proteinase (PfHAP) and plasmep-sin 4 (PfPM4) that are thought to be involved in hemoglobindegradation (8–11). These enzymes share �60% sequenceidentity with PfPM1 and PfPM2, including similar targetingproregions (11), and have been found localized to the DV byboth immunoelectron microscopy (10) and co-purification withthe DV (11). Their capacity to digest native hemoglobin issignificantly less than that of PfPM1 or PfPM2, but both ac-tively cleave globin as demonstrated in vitro (10). Cysteineproteases falcipain 2, 2*, and 3, plus the metalloprotease falci-lysin, are believed to be involved in further degrading largehemoglobin fragments into short peptides and free heme (12–14) and may themselves be able to initiate hemoglobin degra-dation (15–17). The released toxic heme product is neutralizedby the formation of coordinated hematin dimers that generatehemozoin, also known as malaria pigment (18–20). Hemoglo-bin degradation provides an abundant source of amino acidsthat can be used by the parasite during protein assembly. Inaddition, this process may provide the means for the parasite toregulate intracellular osmolarity during its maturation andreplication in the red blood cell (21–23). Plasmepsins have beenthought to play a critical role in hemoglobin degradation; fur-thermore, the finding that aspartic protease inhibitors can killmalaria parasites (16, 19, 24–27) has made them prime candi-dates for antimalarial drug development. However, these can-didate targets remain unvalidated. To address whether theseDV plasmepsins are critical, we performed gene disruptionexperiments and report our findings herein.

* This work was supported by National Institutes of Health GrantsAI39211 (to J. B. D.) and AI058186 (to H. F.). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.¶ Present address: Dept. of Pathology, Immunology, and Laboratory

Medicine, University of Florida College of Medicine, P. O. Box 100275,Gainesville, FL 32610-0275.

‡‡ To whom correspondence should be addressed. Tel.: 352-392-4700(ext. 5818); Fax: 352-392-9704; E-mail: damej@mail.vetmed.ufl.edu.

1 The abbreviations used are: DV, digestive vacuole; PfHAP, P. fal-ciparum histoaspartic protease; PfPM, P. falciparum plasmepsin;CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorp-tion ionization time-of-flight; IEF, isoelectric focusing.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 52, Issue of December 24, pp. 54088–54096, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org54088

EXPERIMENTAL PROCEDURES

Parasite Cultivation, Synchronization, and Release from Erythro-cytes—P. falciparum parasites were cultured as described (28, 29) at 4%hematocrit in RPMI 1640 (Invitrogen) medium supplemented with 0.5%Albumax (Invitrogen), 0.225% sodium bicarbonate, and 0.01 mg/mlgentamycin. Parasites were synchronized using 5% sorbitol when theywere mainly in the ring stage. The parasites used for RNA and proteinpreparations were collected at �12-h intervals. The parasitemia andthe synchronicity of each culture was determined by microscopic exam-ination of Giemsa or Protocol Hema (Fisher)-stained smears.

Knockout Constructs—Internal 0.7-kb fragments from PfPM1 (nu-cleotides 481–1195, GenBankTM accession number X75787), PfPM2(nucleotides 483–1200, GenBankTM accession number L10740), PfPM4(nucleotides 481–1195, PlasmoDB gene Pf14_0075), and PfHAP (nu-cleotides 481–1186 GenBankTM accession number AJ009990) were am-plified by PCR from parasite genomic DNA. JNB196 (5�-GCATGGAT-CCGCAAATTTATGGGTTCCAAG-3�) plus JNB195 (5�-GCATGCGGC-

CGCTTTATCATCATCATCTTTATAATCTGCTGCGTTGTAAGTAGT-ATTCTGGTTCTAAGG-3�) were used for PfPM1; JNB197 (5�-GCATG-GATCCGCTAATTTATGGGTCCCAAGTGTTA-3�) plus JNB199 (5�-G-CATGCGGCCGCTTTATCATCATCATCTTTATAATCTGCAGCGTGT-TGAAGGTAGTATTCAGGTTC-3�) were used for PfPM2; JNB200 (5�-GCATGGATCCTCTAATGTATATGGGTACCCAGTATA-3�) plus JNB-202 (5�-GCATGCGGCCGCTTTATCATCATCATCTTTATAATCTGCA-GCGGTTCAAGGTATTGTTTAGGTTC-3�) were used for PfHAP; andJNB203 (5�-GCATGGATCCGCTAATTTATGGGTTCCAAGTG-3�) plusJNB205 (5�-GCATGCGGCCGCTTTATCATCATCATCTTTATAATCT-GCAGCGGGTCCATATAGAATTCAGGTTC-3�) were used for PfPM4.5� primers introduced a BamHI restriction site, and 3� primers intro-duced a NotI restriction site. The resulting PCR products were clonedinto pCR2.1 (Invitrogen) and then subcloned into the P. falciparumtransfection vector pHD22Y, which uses human dihydrofolate reduc-tase as the selectable marker (30). This yielded the 6.6-kb constructspHD-�PM1, pHD-�PM2, pHD-�PM4, and pHD-�HAP (Fig. 1).

FIG. 1. Genetic strategy for disruption of the four DV plasmepsins. A, schematic of the constructs (pHD-�PM1, pHD-�PM2, pHD-�PM4,or pHD-�HAP) designed to integrate into the chromosomal copy of the targeted plasmepsin gene via a single crossover event. The genericplasmepsin coding region is represented by an open box with stippled bands. The arrow designates the direction of transcription. Integration of thepHD-�PM constructs produces two nonfunctional fragments of the targeted gene. The 5�-fragment lacks the gene region encoding Psi loop 2 at theC-terminal end of the mature enzyme, whereas the 3�-fragment lacks the 5�-end encoding the targeting peptide, the proregion, and the catalyticloop 1. Plasmid integration into the plasmepsin genes was detected by PCR using primers p1/p4 and p3/p2 (numbered arrows). PCR with primersp1/p2 detected the wild-type gene locus, whereas primers p3/p4 detected plasmid DNA. B–E, these indicate the sizes and origins of restrictionfragments containing each DV plasmepsin gene from the parental line (upper representation) as well as the predicted sizes for the knockout locus(lower representation). A, AccI; Bm, BsmI; Bt, BstB1; M, MfeI; P, PacI; S, StyI. B, AccI digestion of the PfPM1 locus yields a 1.5-kb fragmentcontaining the coding sequence for this gene. Gene disruption was predicted to produce 4.5- and 1.8-kb fragments. StyI was predicted to produce9.2- and 15.8-kb bands from the uninterrupted and disrupted PfPM1 loci, respectively. C, for PfPM2, AccI digestion was predicted to yield a 5.7-kbfragment for Dd2 versus 8.2- and 2.3-kb bands for the disrupted locus. Insertion of a single copy of the knockout plasmid into PfPM2 was predictedto increase the size of the BsmI fragment from 5.3 kb in Dd2 to 11.8 kb in the knockout line. D, for PfHAP, AccI was predicted to yield a 5.8-kbband for Dd2 versus 5.9- and 4.6-kb bands for the PfHAP knockout line. Insertion of a single plasmid copy was predicted to increase the size of theBstB1 fragment from 7.4 kb in Dd2 to 14.0 kb in the knockout line. E, for PfPM4, PacI was predicted to yield a 2.0-kb fragment for Dd2 versus 5.5-and 3.0-kb bands for the knockout, with insertion of a single plasmid copy resulting in an increase in size of the MfeI fragment from 11.3 kb in Dd2to 17.0 kb in the knockout line.

P. falciparum DV Plasmepsins Are Individually Nonessential 54089

Probes for Southern and Northern Blot Analysis—These were pre-pared from the 0.7-kb PCR product used to prepare each knockout con-struct by random primer labeling (Stratagene) with [�-32P]dATP. ThesePCR products were found not to cross-hybridize with one another whenused as probes under stringent hybridization and wash conditions.

Parasite Transfection and Cloning—P. falciparum transfectionswere performed by electroporating parasitized erythrocyte cultures asdescribed (31). Transfected parasites were selected in culture mediumcontaining 2.5 nM WR99210 (a P. falciparum-specific antifolate kindlyprovided by Dr. David Jacobus, Jacobus Pharmaceuticals, Princeton,NJ). Initially, plasmids replicated episomally until recombination oc-curred with homologous chromosomal sequences, leading to genomicintegration via single crossover (32). Integration of each knockout con-struct into the parasite genome was detected by PCR with the primercombinations p1/p4 and p3/p2, which were specific for integrationevents (Fig. 1A). The use of p1 with p2 detected the wild-type gene, andp3 with p4 detected episomal plasmids (Fig. 1A). Primers 1 and 2 forPfPM1 were JNB9 (5�-CCGGAATTCCGAGGATCCTCAAAACATGTA-ATAATTGGA-3�) and JNB10 (5�-CCGGAATTCGGATCCCGTGATTTA-CAATTTTTTTTTGGC-3�); for PfPM2 were JNB266 (5�-TCCGAACAT-TTAACTATTGGATTTAAAG-3�) and JNB267 (5�-GCAAGAGCAATAC-CAACACTG-3�); for PfHAP were JNB268 (5�-GACTTCAAAATATTCG-ACAGTAGGA-3�) and JNB269 (5�-GCTAAAGCAAATCCAACGGTG-3�); and for PfPM4 were JNB135 (5�-CCGGAATTCCGAGGATCCACA-AAACACAAACTATAGG-3�) and JNB137 (5�-CCGAATTCGGATCCTT-ATAAATTTTTAGCTACTGC-3�), respectively. Primers 3 and 4 wereJNB207 (5�-CCTAATCATGTAAATCTTAAATTTTTC-3�) and T3 (5�-GCAATTAACCCTCACTAAAGGG-3�), respectively. Mutant plasmep-sin-disrupted lines were cloned by limiting dilution in 96-well tissueculture plates (Costar, Corning, NY) and detected after 21 days usingthe Malstat assay that detects expression of parasite lactate dehydro-genase (33). Plasmid integration into the parasite genome was con-firmed by Southern blot analysis using the 32P-labeled DNA probesdescribed above.

Southern and Northern Blot Analysis—Parasites were prepared freeof excess erythrocytes by treatment with 0.05% saponin and washedtwice with phosphate-buffered saline. Genomic DNA was extractedusing TELT lysis buffer (50 mM Tris (pH 8.0), 62.5 mM EDTA (pH 8.0),2.5 M LiCl, 4% Triton X-100) (34). The DNA was further purified byphenol/chloroform/isoamyl alcohol (25:24:1, Fisher) deproteination, andisopropyl alcohol precipitation. Following restriction enzyme digestion,DNA samples were separated on 1% agarose gels and blotted ontoHybond N� membranes (Amersham Biosciences). Total parasite RNAwas extracted using Trizol (Invitrogen) and purified by isopropyl alco-hol precipitation. Quantification used both absorbance at 260 nM andagarose gel electrophoresis followed by ethidium bromide staining. ForNorthern blots, �6 �g of total RNA was loaded per well, resolved on 1%formaldehyde-agarose gels, blotted onto Hybond N� membranes, andhybridized with 32P-labeled DNA probes.

Isolation of DV—These were prepared from sorbitol-synchronizedcultures of P. falciparum trophozoites, using a method from Saliba et al.(35) with major modifications. Briefly, 100 ml of culture containingtrophozoites at 5–10% parasitema was treated with 0.05% saponin, andthe free parasites were immediately collected by centrifugation. Para-sites were washed twice by resuspension in ice-cold wash buffer (10 mM

Tris-HCl (pH 7.5), 250 mM sucrose) followed by centrifugation. Washedpellets were resuspended in 1 ml of ice-cold wash buffer, transferred to1.5-ml microcentrifuge tubes, and triturated four times through a 27-gauge needle (4 s per trituration). Samples were centrifuged at13,800 � g at 4 °C for 2 min. Supernatants were discarded, and pelletswere resuspended in 1 ml of uptake buffer (2 mM MgSO4, 100 mM NaCl,25 mM HEPES, 25 mM NaHCO3, 5 mM Na3PO4 (pH 7.4)) to which wasadded 50 �l of 1 unit/�l DNase. After 10 min of incubation at 37 °C,samples were centrifuged at 13,800 � g at 4 °C for 2 min. Supernatantswere discarded, and the pellets were resuspended in 0.1 ml of ice-coldwash buffer and then mixed with 1.3 ml of ice-cold 42% Percoll preparedin 0.25 M sucrose, 1.5 mM MgSO4 (pH 7.4). Mixtures were triturated twotimes through a 27-gauge needle (9 s per trituration) and centrifuged at13,800 � g at 4 °C for 10 min. The DV appeared as a dark band in thebottom 50 �l of the Percoll gradient. These were collected and trans-ferred to clean 1.5-ml microcentrifuge tubes and washed twice withice-cold wash buffer, involving centrifugation at 13,800 � g at 4 °C for2 min and removal of the supernatant each time. After the second wash,the pellet was resuspended in 100 �l of IEF sample buffer (9 M de-ionized urea, 4% CHAPS, 60 mM DTT, 0.8% ampholytes (pH 3–10)) and10 �l of 10� protease inhibitor mixture (Sigma). Samples were stored at�80 °C. Prior to use, samples were thawed, vortexed, and centrifuged at

16,000 � g at room temperature for 5 min. Supernatants were used intwo-dimensional gel electrophoresis as described below.

Two-dimensional PAGE—This was performed by using a MultiphorII Electrophoresis Unit equipped with a MultiDrive EPS 3501XL gra-dient power supply (Amersham Biosciences). Precast Immobiline Dry-strips (IEF strips, pH 4–7 linear, 180 � 3 � 0.5 mm) were rehydratedovernight (in a re-swelling cassette (Amersham Biosciences)) with 50-�lDV extracts and brought up to 125 �l with re-swelling buffer (8 M urea,2% (w/v) CHAPS, 2% (v/v) IPG buffer (Amersham Biosciences), and0.002% bromphenol blue). Just prior to use, DTT was added at a finalconcentration of 3 mg/ml. Rehydration buffer and stock DTT (62.5mg/ml) were stored at �20 °C. Rehydrated IEF strips were isoelectri-cally focused at 15 °C under low viscosity oil (Fisher) with a gradientvoltage of 0–200 V for 1 min, 200–3,500 V for 1.5 h, and a constantvoltage of 3,500 V for 1.5 h. The IEF strips were equilibrated for 15 minin 10 ml of SDS equilibration buffer (50 mM Tris-HCl (pH 8.8), 6 M urea,30% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromphenol blue),and DTT was added at a final concentration of 10 mg/ml immediatelybefore use. The second dimension was SDS-PAGE, performed accordingto the method of Laemmli (36) with precast 10% polyacrylamide gels(Bio-Rad). The equilibrated IEF strips were each placed on a prepara-tive well and sealed using 1% agarose plus 0.002% (w/v) bromphenol.After electrophoresis the two-dimensional gels were silver-stained byusing a MALDI-TOF-compatible silver-staining protocol (37). Spots cor-responding to each plasmepsin were excised and confirmed by MALDI-TOF analysis (38) (performed by Kendrick Laboratories Inc., Madison,WI). Gel images were documented with an AlphaImager IS-1000.

SDS-PAGE and Western Blot Analysis—Parasites were lysed in 1�RIPA Lysis buffer (Upstate Biotechnology Inc., Lake Placid, NY) con-taining the protease inhibitors pepstatin (2 �g/ml), leupeptin (1.5 �g/ml), and phenylmethylsulfonyl fluoride (1 mM). Lysates were thentreated with an equal volume of 2% SDS as described (39), and theirconcentrations were adjusted to ensure equal protein loading. Bio-Radsample buffer was added to the solubilized protein to a final concentra-tion of 1� prior to boiling for 5 min and loading onto 12% polyacryl-amide gels (39). After electrophoresis, proteins were transferred tonitrocellulose (Bio-Rad). Membranes were hybridized with primary an-tibodies as described (10), followed by horseradish peroxidase-conju-gated goat anti-mouse or anti-rabbit IgG (1:10,000 dilution, Pierce).Immunoreactivity was detected using the SuperSignal chemilumines-cent substrate (Pierce).

Electron Microscopy—For electron microscopy, infected red bloodcells or DV preparations were fixed with 2.5% glutaraldehyde in 0.05 M

phosphate buffer (pH 7.4), containing 4% sucrose, for 30 min at roomtemperature followed by 90 min at 4 °C. Cells were then postfixed in 1%osmium tetroxide for 1 h. After a 30-min en bloc stain with 1% aqueousuranyl acetate, cells were dehydrated in ascending concentrations ofethanol and embedded in Epon 812. Ultrathin sections were stainedwith 2% uranyl acetate in 50% methanol and lead citrate and examinedusing a Zeiss CEM902 electron microscope (Oberkochen, Germany).

RESULTS

Generation of PfPM1-, PfPM2-, PfPM4-, and PfHAP-dis-rupted Clones—P. falciparum Dd2 asexual stage parasiteswere transfected by electroporation using the plasmepsinknockout constructs pHD-�PM1, pHD-�PM2, pHD-�PM4, orpHD-�HAP. Episomally transfected parasites, selected withWR99210, were first visualized by day 14–16 post-electropora-tion, and integration was detected by PCR by day 104 post-electroporation for all lines. Plasmepsin knockout parasiteswere subsequently cloned by limiting dilution. The Tx1.A3 andTx2.B6 clones were isolated and characterized from the trans-fections with pHD-�PM1 and pHD2-�PM2, respectively.Transfection with pHD-�HAP gave rise to the clones Tx3.C3,Tx3.F11, and Tx3.G7, whereas the pHD-�PM4 transfectionresulted in the clones Tx4.F8, Tx4.H12, Tx4.B10. For all clonesdisruption of the intact parental plasmepsin gene was con-firmed by PCR (data not shown).

Southern Blot Analysis—To confirm the integration of theseparate constructs into the plasmepsin gene loci, we carriedout Southern blot analysis with gene-specific probes (Fig. 2).For every knockout clone, the banding patterns were consistentwith integration of a single plasmid copy leading to disruptionof the targeted plasmepsin. Details are provided in the Fig. 1

P. falciparum DV Plasmepsins Are Individually Nonessential54090

legend. In view of the identical results obtained between theindependent clones, for HAP and PfPM4, we chose to pursueour investigations using a single representative clone for eachlocus, namely Tx1.A3, Tx2.B6, Tx3.C3, and Tx4.F8. These arereferred to hereafter simply as Tx1, Tx2, Tx3, and Tx4.

Northern Blot Analysis—To determine the effect of genedisruption on the expression and stability of the alteredmRNAs of each DV plasmepsin in the respective knockoutclones, we performed Northern blot analyses by using gene-specific probes. For this, total RNA was collected at four timepoints from synchronized parasite clones. The normal size ofthe mRNA for PfPM1 is 4.0 kb, for PfPM2 is 2.4 kb, for PfHAPis 2.4 kb, and for PfPM4 is 4.0 kb (Fig. 3). None of the fourknockout mutants expressed a comparably sized transcriptfrom the disrupted gene locus in the asexual cycle. Instead, all

four expressed an abnormally large RNA present in nearly allstages of the asexual cycle (Fig. 3). This product also hybridizedto a probe prepared from the pHD22Y plasmid vector (withoutplasmepsin sequence; data not shown). Thus, the large mRNAtranscript had both plasmepsin and plasmid sequences. Thesignificance of the slight fluctuations in the intensity of thisband from different stages of various knockouts is uncertain.Two other transcripts also hybridized with the plasmepsinprobes, one larger and one slightly smaller than the 2.37-kbmarker. These are most prominent on the Tx3 RNA blot, whichhad been washed at moderate stringency. Corresponding bandsmay be seen at lower intensity on Tx1, Tx2, and Tx4 blotswashed at high stringency (Fig. 3, A, B, and D). All of theseabnormal RNAs that hybridized with the plasmepsin probealso hybridized to a probe prepared from the pHD22Y vector

FIG. 2. Southern blot analysis con-firms the successful interruption ofeach of the four plasmepsin genes byinsertion of a single copy of the ho-mologous pHD-�PM construct. Re-striction enzymes and sources of genomicDNA are indicated. Probes hybridized toeach blot are as follow: A, PfPM1; B,PfPM2; C and D, PfHAP; and E and F,PfPM4. Sizes of all resulting bands wereas predicted in Fig. 1, indicating integra-tion of a single plasmid copy into the ho-mologous gene via a single crossoverevent.

P. falciparum DV Plasmepsins Are Individually Nonessential 54091

alone, indicating transcription from the chromosome-inte-grated truncated plasmepsin plasmid sequences. For all knock-out clones, additional Northern blot analyses revealed thatindividual gene disruptions were not accompanied by changesin transcript sizes of the other DV plasmepsins. Representativedata are shown in Fig. 3C, where RNA from Tx2 was includedas a control to demonstrate the normal size of the PfHAPmRNA (which was the same for Tx2 and Dd2).

Two-dimensional PAGE and Western Blot Analysis—To com-pare the plasmepsins present in DV preparations from Dd2 andeach of the four knockout clones, we used two-dimensional gelelectrophoresis. The first dimension was isoelectric focusingperformed on an Immobiline gel strip (pH 4–7), and the seconddimension was electrophoresis on a 10% SDS-PAGE slab gel.As shown in Fig. 4, PfPM1, PfPM2, PfHAP, and PfPM4 were allidentified in Dd2 DV preparations. The identity of each of thesespots in Dd2 was confirmed by MALDI-TOF mass spectroscopicanalysis. For each knockout clone, the expected individual plas-mepsin spot was absent. We note that whereas these resultsdemonstrate the presence of the expected individual plas-mepsins in these DV preparations, the overall protein complex-ity is somewhat overestimated because of the co-purification ofsome free merozoites as detected by electron microscopy (datanot shown).

Because each knockout construct was designed to generatetwo truncated gene fragments, and thereby potentially resultin two partial protein fragments with differing molecularmasses and isoelectric points, whole parasite preparations ofeach knockout line were further examined by Western blotanalysis using antibodies specific for each plasmepsin. No formof PfPM1 was detected by this technique in ring, early tropho-zoite, late trophozoite, or schizont stages of Tx1, for whichproteins in the range of 14–200 kDa could be observed byCoomassie staining (Fig. 5A). Similarly, no forms of PfPM2,PfHAP, and PfPM4 were detected by the antibodies employedin blots of Tx2, Tx3, and Tx4, respectively, over this same sizerange (data not shown). These analyses demonstrated the re-

activity of each antibody against the corresponding plasmepsinin Dd2 (Fig. 5B). As a control, anti-PfPM2 antibodies werereacted with blots of the same extracts, demonstrating thepresence of PfPM2 in all lines except for Tx2. In this instance,anti-PfPM1 antisera was used as a control to demonstrate thepresence of PfPM1.

Comparative Growth in Culture—During the propagation ofthese recombinant lines, we observed a noticeable effect ofcertain plasmepsin gene disruptions on rates of growth. Tostudy this, we sorbitol-synchronized our knockout clones andthe parental line, initiated cultures at 0.5% parasitemia, andquantified the increase in parasitemia over 96 h. In similarexperiments, the parasite rate of multiplication was followedfor 2 weeks, adjusting the parasitemia every 48 h to begin at1%. Both types of assays revealed a 30–50% decrease in para-site multiplication in Tx1 and Tx4 relative to Dd2. Thisprompted us to analyze growth rates from parasitemia meas-urements recorded over an 18-month period, which shouldminimize any short term differences arising from sorbitol treat-ment or culture care. Data points were retained only whenparasitemias were calculated at the beginning and end of a48-h generation period, and measurements were only selectedfrom periods when parasites were growing robustly, typicallyin the range of 0.7–6.0% parasitemia. Measurements meetingthese criteria totaled 27–45 independent data points over 18months of culture. These revealed a statistically significant30–35% decrease in growth rates of the Tx1 and Tx4 linesrelative to Dd2 (Table I). A slight drop in growth rates was alsoobserved for Tx3, although this was not statistically signifi-cant, whereas Tx2 grew at least as well as Dd2 and over someperiods appeared to grow even faster.

Knockout Clone Morphology—Each knockout clone and Dd2was examined by electron microscopy and compared at variousstages of their asexual growth in erythrocytes, with particularattention paid to the morphology of the DV (Fig. 6). For Tx1,Tx2, and Tx3, no significant differences were noted in eitherthe size or organization of the DV or the arrays of hemozoincrystals when compared with Dd2. However, in numerous im-ages of Tx4, there was a notable accumulation in the DV ofsmaller more electron-dense, single-membrane vesicles (Fig.6E). Yet in other Tx4 images the DV and their arrays of hemo-zoin crystals appeared normal, indicating that the phenotypeshown was not observed for all cells. From examining all othersubcellular structures visible by electron microscopy, theknockout mutants appeared normal except for Tx2. For thisclone, the mitochondria were enlarged in approximately one-third of the mature-stage parasites (Fig. 6C).

DISCUSSION

Two early observations have assigned key roles to asparticproteinases in essential metabolic functions critical to the sur-vival of the malaria parasite (reviewed in Ref. 24). The first isthat pepstatin, a classical aspartic proteinase inhibitor, killsmalaria parasites in culture (25); and the second is that PfPM1,localized in the DV by immunolabeling (40), is efficient in vitroat initiating the degradation of native hemoglobin at pH 5,which corresponds to the estimated DV pH (6). These observa-tions stimulated the proposal that hemoglobin digestion oc-curred via a semi-ordered pathway initiated by PfPM1, whichcleaved hemoglobin in the hinge region of the � chain at Phe33–Leu34, thereby unraveling the molecule and rendering it acces-sible for further proteolysis in the acidic environment of theDV. Cysteine proteases and other plasmepsins (e.g. PfPM2)were hypothesized to contribute at later stages of the degrada-tion pathway. Support for this model came from studies withthe inhibitor SC-50083 (Roche Applied Science), which wascomparable in efficiency to pepstatin in blocking parasite

FIG. 3. Northern blot analysis of clones Tx1, Tx2, Tx3; and Tx4.Total RNA was isolated from Dd2 rings (A and D) and early trophozo-ites (B and C), as a positive control, as well as from each knockout lineharvested at four points in the asexual development cycle: rings (R),early trophozoites (ET), late trophozoites (LT), and schizont (S). RNAloadings were equal for each lane. Blots were hybridized with probes forPfPM1 (A), PfPM2 (B), PfHAP (C), and PfPM4 (D). Blots in A, B, and Dwere washed at 60 °C in 0.2� SSMC, and in C at 55 °C in 2� SSC.

P. falciparum DV Plasmepsins Are Individually Nonessential54092

growth in vitro (40). This compound, which also blocked themajority of hemoglobin digestion in DV preparations, was re-ported to be more efficient in inhibiting PfPM1 than PfPM2 (5).Based on these observations, several projects have been under-taken to identify specific inhibitors for PfPM1 and PfPM2 (41–46). A search for inhibitors has also been performed with thePlasmodium vivax DV plasmepsin, PvPM4, for which activerecombinant protein was available (47). Elucidation of theP. falciparum genome has now led to the discovery of 10 plas-mepsin genes (24), including the 4 investigated here that en-code the DV enzymes (10, 48). Our comparative analyses of theavailable genome data from other Plasmodium spp. indicatethat whereas all six of the plasmepsins located outside the DV(10) are present in the primate, rodent, and chicken malariaparasite species, only a single DV plasmepsin is present (11).Phylogenetic analysis clearly places this DV plasmepsin as

being an ortholog of PfPM4 (11). The only exception is Plasmo-dium reichnowi, which infects chimpanzees and gibbons andwhich is very closely related to P. falciparum. This speciesharbors all four DV plasmepsin paralogs.2

Our present studies enable us to ask whether any of thespecialized functions that may have been acquired for PfPM1,PfPM2, PfHAP, or PfPM4, during the course of their evolution(presumed to have begun with sequence duplication and diver-gence from PfPM4 (11)), are essential to the survival ofP. falciparum asexual blood stages. Identifying one or more asbeing essential would validate them as rational drug designtargets. The most important finding of this study is thatP. falciparum asexual parasites can propagate in vitro with

2 C. A. Yowell and J. B. Dame, unpublished observations.

FIG. 4. Two-dimensional gel analysis of DV proteins. Following two-dimensional separation, proteins were visualized by silver staining, andspots, tentatively identified as plasmepsins (based upon size and isoelectric point), were identified by MALDI-TOF mass spectroscopy. All of theDV plasmepsins (1, 2, HAP, and 4) were identified in DV preparations from the parental line Dd2. The proteins in the DV of Tx1, Tx2, Tx3, andTx4 each lacked the spot corresponding to the mature form of PfPM1, PfPM2, PfHAP, and PfPM4, respectively.

P. falciparum DV Plasmepsins Are Individually Nonessential 54093

any three of the four DV plasmepsins still functioning, withoutpresenting a highly deleterious phenotype. Our data establishthat no single DV plasmepsin is essential to the P. falciparumintracellular growth. This includes PfPM1, which had beenthought to play the key role in initiating hemoglobin digestion,yet cannot be so on the basis of our data (5). Our results clearlysuggest redundancy in the functions performed by the DVplasmepsins. The function of the inactivated gene product maybe performed by either one or more of the other three plas-mepsins, or possibly the cysteine proteases falcipain 2, 2*, and3 that are also present in the DV (17, 49, 50). Parasites inwhich falcipain 2 has been knocked out also lack any obviouslydeleterious phenotype, suggesting that the cysteine proteasesalong the hemoglobin digestion pathway also have redundantfunctions (51). The observation that the falcipain 2-knockoutparasites become highly sensitive to pepstatin (51) suggeststhat its loss of function was compensated for by an increaseddependence on plasmepsin activity and raises the possibility

that cooperative, partially redundant roles exist for these twodifferent mechanistic classes of DV proteases.

Data from Bozdech et al. (52) and Le Roch et al. (53) indicatethat PfPM1 and PfPM4 are transcribed early in the asexualcycle and the steady-state level of the mRNA falls during thelatter half of the cycle, whereas for PfPM2 and PfHAP, the

FIG. 5. Western blot analysis of whole parasite lysates of Dd2 and plasmepsin knockout clones. Total parasite protein extracts wereprepared from each knockout clone at four stages in the asexual development cycle: rings, early trophozoites, late trophozoites, and schizonts.Equal amounts of protein from each knockout line at each stage, and from Dd2 schizonts, were assayed. A, protein extracts of Tx1 and control, Dd2,probed with anti-PfPM1 antibody demonstrated the presence of the �37-kDa PfPM1 protein in Dd2; however, no protein or partial proteinfragment was detected in the Tx1 line at any developmental stage. Similar results were found for Tx2, Tx3, and Tx4 samples probed with antibodiesspecific for PfPM2, PfHAP, and PfPM4, respectively. B presents data from a restricted section of those blots probed with antibodies against twodifferent plasmepsins. In each case the antibodies specific for the plasmepsin targeted by the knockout strategy bound to an �37-kDa band in Dd2but failed to react with bands of any size in any stage of development of the knockout clones.

TABLE IMean multiplication rates of Dd2 and plasmepsin knockout clonesThe unpaired two-tailed t tests also showed a statistically signifi-

cant difference in the growth rates of Tx1 and Tx4 when comparedwith Dd2 (p � 0.01 in both cases). The growth rates were estimatedby tracking parasitemias and dilutions over 12–18 months and indicatethe mean fold increase in parasite numbers (propagation rates) per 48-hgeneration.

Dd2 Tx1 Tx2 Tx3 Tx4

Meana 5.42 3.84 5.66 4.44 3.6S.E. 0.6 0.24 1.04 0.29 0.16No. samples 34 38 31 27 45Mean as % Dd2 100.0% 70.9% 104.3% 81.8% 66.4%Mann-Whitney

p value0.001 0.35 0.08 p � 0.0001

a Mean indicates the geometric mean.

FIG. 6. Ultrastructure of plasmepsin knockout mutants. Ultra-thin sections of late trophozoites and schizonts of Dd2 and each knock-out clone were examined by electron microscopy as follows: A, Dd2; B,Tx1; C, Tx2; D, Tx3; and E, Tx4. The left image in each panel is arepresentative image of the entire parasite where the bar is 1 �m, andthe right image is an enlargement of the DV. C, the arrowheads indicatea swollen mitochondria seen in a proportion of the Tx2 knockout para-sites; E, small vesicles seen in the DV of the Tx4 knockout line.

P. falciparum DV Plasmepsins Are Individually Nonessential54094

amount of transcript present is low in the first half of the cycleand rises prominently in the second half. This suggests thatperhaps PfPM1 and PfPM4 could complement for each other,whereas PfPM2 and HAP could functionally complement as theother pair. Most interestingly, Tx4 displayed in some, althoughnot all, parasites a noticeable accumulation of multiple smallervesicles inside the DV, which suggests that perhaps PfPM4plays a role in vesicle-mediated transport of hemoglobin intothe DV and subsequent elimination of these discrete vesicles,either by their incorporation into the DV membrane or theirproteolytic degradation. Most interestingly, the loss of PfPM4in Tx4 did not eliminate the production of hemozoin. A subtlephenotype was also observed for the PfPM2 knockout, which inabout a third of the parasites displayed unusually enlargedmitochondria. We have yet to be able to define this phenotypefurther. A pronounced detrimental effect on growth rates wasalso observed for both the PfPM1 and PfPM4 knockout lines,which was statistically significant in comparison with the pa-rental Dd2 line. This defect therefore indicates that these twoplasmepsins, although not essential, nonetheless are requiredfor normal robust rates of parasite propagation in vitro. Ongo-ing studies of the molecular changes together with any subtlephenotypes connected with the loss of function of these DVplasmepsins should provide further insight into their functionsand how that relates to hemoglobin degradation and antima-larial drug susceptibility.

The implications of our work are clear: rational anti-plas-mepsin drug design strategies must be revised to include ap-proaches to identify compounds capable of inhibiting at leasttwo if not all four of these enzymes, without toxicity to the host.This will only be feasible if these enzymes share structurallyvery similar active sites that can be bound by pantothenate-specific inhibitors. Some evidence for this being the casecomes from structural studies that reveal close active sitesimilarity between PfPM2 and the P. vivax ortholog of PfPM4(54, 56–58).3 Most importantly, these DV plasmepsins differsufficiently from the most closely related human asparticproteinase, cathepsin D (59), making it potentially feasible todevelop selective anti-plasmodial inhibitors (41, 44). Indeed,the practicality of a strategy to design inhibitors capable ofinhibiting the whole family of DV plasmepsins has beenreported recently (55). These genetic, structural, and bio-chemical data therefore all point toward a close degree offunctional and structural conservation that could be ex-ploited by the identification of plasmepsin inhibitors effectiveagainst multiple DV plasmepsins, making this a key consid-eration for subsequent inhibitor screening against this familyof enzymes.

Acknowledgments—All the antibodies used in these experimentswere kindly provided by Dr. Daniel E. Goldberg, Howard Hughes Med-ical Institute and Department of Medicine and Molecular Microbiology,Washington University School of Medicine, St. Louis, MO. Western blotanalyses were carried out in the laboratory of Dr. Kenneth A.Iczkowski, Department of Pathology, Immunology and Laboratory Med-icine, University of Florida and Veterans Affairs Medical Center,Gainesville, FL.

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