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• W1972959613 abstract "Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions. Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions. Malaria kills 1–2 million people every year, most of whom are children under the age of 5. The economic burden caused by malaria in endemic countries is large, estimated to be approximately 1–2% of the gross domestic product in these countries (1World Health Organization (2004) WHO Fact Sheet No. 94—Malaria, GenevaGoogle Scholar). Nearly all malarial deaths are caused by Plasmodium falciparum, a protozoan parasite that lives in human red blood cells during its asexual life cycle. Drug resistance in P. falciparum is an enormous problem and new antimalarial agents are needed desperately. In the effort to develop new treatments for malaria, the study of hemoglobin degradation in the food vacuole of the parasite has generated substantial interest because it is a major metabolic pathway of the parasite and is believed to be essential for the survival of the parasite (2Banerjee R. Goldberg D.E. Rosenthal P.J. Antimalarial Chemotherapy: Mechanisms of Action, Resistance, and New Directions. Humana Press Inc., Totowa, NJ2001: 43-63Google Scholar).A family of P. falciparum aspartic proteases called plasmepsins has been found to be important for the initiation of hemoglobin degradation in vitro and in vivo (3Bray P.G. Janneh O. Raynes K.J. Mungthin M. Ginsburg H. Ward S.A. J. Cell Biol. 1999; 145: 363-376Crossref PubMed Scopus (146) Google Scholar, 4Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 990-995Crossref PubMed Scopus (373) Google Scholar), and considerable effort is being spent in developing potent plasmepsin inhibitors (5Boss C. Richard-Bildstein S. Weller T. Fischli W. Meyer S. Binkert C. Curr. Med. Chem. 2003; 10: 883-907Crossref PubMed Scopus (101) Google Scholar, 6Johansson P.O. Chen Y. Belfrage A.K. Blackman M.J. Kvarnstrom I. Jansson K. Vrang L. Hamelink E. Hallberg A. Rosenquist A. Samuelsson B. J. Med. Chem. 2004; 47: 3353-3366Crossref PubMed Scopus (52) Google Scholar, 7Ersmark K. Feierberg I. Bjelic S. Hamelink E. Hackett F. Blackman M.J. Hulten J. Samuelsson B. Aqvist J. Hallberg A. J. Med. Chem. 2004; 47: 110-122Crossref PubMed Scopus (111) Google Scholar, 8Jiang S. Prigge S.T. Wei L. Gao Y. Hudson T.H. Gerena L. Dame J.B. Kyle D.E. Antimicrob. Agents Chemother. 2001; 45: 2577-2584Crossref PubMed Scopus (90) Google Scholar, 9Nezami A. Kimura T. Hidaka K. Kiso A. Liu J. Kiso Y. Goldberg D.E. Freire E. Biochemistry. 2003; 42: 8459-8464Crossref PubMed Scopus (107) Google Scholar). Plasmepsin II (PM II), 1The abbreviations used are: PM, plasmepsin; KO, knockout; HAP, histoaspartic protease; PBS, phosphate-buffered saline; E64, trans-epoxysuccinyl-l-leucylamino(4-guanidine)-butane.1The abbreviations used are: PM, plasmepsin; KO, knockout; HAP, histoaspartic protease; PBS, phosphate-buffered saline; E64, trans-epoxysuccinyl-l-leucylamino(4-guanidine)-butane. for which the recombinant enzyme can be expressed readily and the crystal structure is available, has been the primary focus of drug design efforts. However, the in vivo potency of generated compounds has been limited. The malaria genome project has revealed that there are 10 plasmepsins in the P. falciparum genome (10Coombs G.H. Goldberg D.E. Klemba M. Berry C. Kay J. Mottram J.C. Trends Parasitol. 2001; 17: 532-537Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Four of them, PM I and II, histoaspartic protease (HAP, known previously as PM III), and PM IV, reside in the food vacuole and can degrade hemoglobin or globin (4Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 990-995Crossref PubMed Scopus (373) Google Scholar). The four plasmepsins are highly homologous to each other, sharing more than 60% amino acid identity. HAP is unique in that it has a histidine in place of the first canonical aspartic acid but is an active protease that may function by an aspartic or serine protease mechanism (4Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 990-995Crossref PubMed Scopus (373) Google Scholar, 11Andreeva N. Bogdanovich P. Kashparov I. Popov M. Stengach M. Proteins. 2004; 55: 705-710Crossref PubMed Scopus (15) Google Scholar). Because of the high degree of similarity, the specific roles of the food vacuole plasmepsins in vivo has been unclear, and which of these genes (if any) is essential to the parasite has been unknown.To address this question and to determine whether a particular plasmepsin should be considered as the primary drug target, we systematically disrupted all four plasmepsin genes. Our results suggest that the four plasmepsins have overlapping roles in the hemoglobin degradation pathway and that there is a selective advantage for P. falciparum to keep the multiple food vacuole plasmepsin genes in its genome.EXPERIMENTAL PROCEDURESParasite Culture, Transfection, and Selection—Asexual stage P. falciparum clone 3D7 was cultured in human O+ erythrocytes at 2% hematocrit under 5% CO2, 5% O2, and 90% N2 (12Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6077) Google Scholar). Except as stated, cultures were grown in rich medium (RPMI 1640 medium supplemented with 27 mm NaHCO3,11mm glucose, 0.37 mm hypoxanthine, 10 μg/ml gentamicin, and 5 g/liter Albumax (Invitrogen)). Serum medium (Albumax replaced by 10% heat-inactivated human O- serum (Interstate Blood Bank Inc., Memphis, TN)) was used for cultures being electroporated or under drug selection. For some experiments, an amino acid-limited medium was used, which was made by omitting all but five essential amino acids, cysteine, glutamic acid, glutamine, isoleucine, and methionine, from rich medium (13Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar). Parasite synchronization was performed by 5% d-sorbitol treatment (14Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2792) Google Scholar). Ring stage parasites at 5–8% parasitemia were transfected by electroporation with 100 μg of supercoiled transfection vectors (see below) using low voltage, high capacitance conditions (15Fidock D.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10931-10936Crossref PubMed Scopus (412) Google Scholar). Following transfection, parasites were maintained in drug-free medium for 48 h at which time positive selection was initiated by the addition of 10 nm WR99210 (Jacobus Pharmaceuticals, Princeton, NJ) to the medium. Drug-resistant parasites appeared in 3–6 weeks. When parasites reached a parasitemia of ∼ 2%, 4 or 10 μm ganciclovir (Roche Applied Science) was added to start negative selection. After 2–8 weeks, parasites appeared. The WR99210/ganciclovir doubly resistant parasites were cloned by limiting dilution, and their genotypes were analyzed by Southern blotting and PCR.Construction of Transfection Vectors—The parental plasmid pHHT-TK was obtained from Dr. Alan Cowman (16Duraisingh M.T. Triglia T. Cowman A.F. Int. J. Parasitol. 2002; 32: 81-89Crossref PubMed Scopus (146) Google Scholar). The PCR primers, restriction enzymes, and DNA fragments used to construct the transfection vectors are listed in Table I. All DNA fragments were amplified from 3D7 genomic DNA by PCR, digested with the indicated restriction enzymes, and cloned into the specific sites of pHHT-TK. The DNA sequences of all inserts were confirmed.Table IPCR primers for transfection vector constructionDNA fragmentPrimerRestriction sitePosition relative to start codonLengthbpPM I-5′aUsed as gene-specific probe for Southern blottingForwardggcttccgcggttaaagaagatttttcaagcgSacII14–521508ReversettgttgttaacccaatagtattacattgagcHpaIPM I-3′ForwardgccctaggtcgaacaagccgtttttaccAvrII815–1342528ReversegccctaggcaagggcgaaaccaacagtgtgatAvrIIPM II-5′ForwardtacagccgcggaacatgattttaaacatggcSacII20–540521ReversegaatcgttaacatgtttagttaaacatcctgcHpaIPM II-3′aUsed as gene-specific probe for Southern blottingForwardgatacccatggattcgaaccaacttatactgcNcoI702–1348647ReversetaaatggcgcctagcaagagcaataccaacacKasIHAP-5′aUsed as gene-specific probe for Southern blottingForwardtaaccccgcgggaagaagattttaccaacacSacII20–623604ReversecttagttaacattccacttattgtaccagcHpaIHAP-3′ForwardattctccatgggacgttgatggtgttttcggNcoI724–1344621ReversetataaggcgccggctaaagcaaatccaacggKasIPM IV-5′aUsed as gene-specific probe for Southern blottingForwardccatccgcggttacaaatggctcttaccgSacII–6–613619ReversectaagttaacctctaacagttcctgaaccgHpaIa Used as gene-specific probe for Southern blotting Open table in a new tab Southern Blotting—Two clones from each knock-out (KO) construct were analyzed. Genomic DNA was extracted from parasite-infected red blood cells using QIAamp DNA blood mini kit (Qiagen, Valencia CA). For each clone, 1.5 μg of DNA was digested using the following restriction enzymes: ScaI for the PM I KO1 clones A9 and E9, StuI for the PM II KO2 clones C5 and D2, SpeI-MspI for the HAP KO3 clones G2 and G9, and PacI for the PM IV/I KO4/1 clones A5 and E5. 3D7 DNA was used as a control for each digestion. The digested DNA was resolved on an 0.7% agarose gel for KO1, KO2, and KO3 clones and on an 0.8% agarose gel for KO4/1 clones. Gels were blotted onto Nytran SuPer-Charge membrane (Schleicher & Schüll), and a signal was generated using an AlkPhos direct labeling and detection kit (Amersham Biosciences) with the manufacturer's recommended hybridization and washing conditions. All blots were hybridized against human dihydrofolate reductase using a probe made by the digestion of pHHT-TK with HindIII-BamHI. After stripping with 0.5% SDS, the blots were reprobed with gene-specific probes (PM I-5′ for KO1, PM II-3′ for KO2, and HAP-5′ for KO3). In the case of KO4/1, an identically loaded blot was probed with PM IV-5′.Antibodies and Western Blotting—Rabbit polyclonal antibodies 574 (anti-PM I) and 737 (anti-PM II) were described previously (17Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and anti-BiP was described in Ref. 18Kumar N. Koski G. Harada M. Aikawa M. Zheng H. Mol. Biochem. Parasitol. 1991; 48: 47-58Crossref PubMed Scopus (142) Google Scholar. Mouse monoclonal antibodies 1C6–24 (anti-PM I) and 13.9.2 (anti-PM IV) were described previously (4Banerjee R. Liu J. Beatty W. Pelosof L. Klemba M. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 990-995Crossref PubMed Scopus (373) Google Scholar), and 3F10–6 (anti-HAP) was generated as described previously (19Niebuhr K. Lingnau A. Frank R. Wehland J. Celis J. Cell Biology: A Laboratory Handbook. Academic Press, NY1998: 398-403Google Scholar). Rat antibody (anti-falcipain-2) was described previously (20Sijwali P.S. Shenai B.R. Gut J. Singh A. Rosenthal P.J. Biochem. J. 2001; 360: 481-489Crossref PubMed Scopus (198) Google Scholar). For immunoblotting, parasites were separated from the erythrocyte cytosol by treatment with ice-cold 0.1% saponin in PBS for 10 min on ice followed by a cold PBS wash. Parasite pellets were stored at -80 °C until use. Parasites were lysed by boiling in SDS-PAGE sample loading buffer. The lysates were resolved by 12% SDS-PAGE, and transferred to a Protran nitrocellulose membrane (Schleicher & Schüll). Primary antibodies used were: 574 (1:1000) for KO1, 737 (1:5000) for KO2, 3F10-6 (1:5000) for KO3, and 1C6-24 (1:2000) and 13.9.2 (1:1000) for KO4/1. All blots were reprobed using anti-BiP to assess loading. The signal was developed with horseradish peroxidase-conjugated anti-rabbit IgG (1: 3000) or anti-mouse IgG (1:3000) secondary antibodies and ECL Western blotting detection reagents (Amersham Biosciences).Flow Cytometry—The flow cytometry protocol was adapted from Barkan et al. (21Barkan D. Ginsburg H. Golenser J. Int. J. Parasitol. 2000; 30: 649-653Crossref PubMed Scopus (71) Google Scholar). In brief, parasite cultures were fixed in 0.05% glutaraldehyde in PBS and stored at 4 °C. The cells were permeabilized with 0.25% Triton X-100 in PBS for 5 min at room temperature and stained with 5 μg/ml propidium iodide (Molecular Probes, Eugene, OR). The cells were then diluted 10-fold in PBS and analyzed using an Epics XL-MCL flow cytometer (Beckman). 1 × 105 erythrocytes were counted in triplicate for each sample. Data were analyzed using FlowJo software (Treestar Inc., Ashland, OR).Parasite Growth Rate Analysis—Asynchronous cultures were maintained at parasitemias between 0.5 and 4% by daily passage. Samples were taken approximately every 24 h over a period of 7 days, and parasitemia was measured by flow cytometry. The total number of parasites, y (parasitemia × dilution factor), was graphed against time (x) and fitted to the exponential growth curve using KaleidaGraph software (Synergy Software, Reading, PA). y=m0⋅e(ln2⋅x/τ) Here, τ is the intrinsic parasite doubling time, and m0 is the theoretical parasite number at time 0. To compare directly the growth rate of cultures with slightly different starting parasitemias, the -fold increase of the parasite number, normalized to have a single theoretical parasite for each culture at time 0, was graphed and is shown in Figs. 3 and 5.Fig. 5Growth rate of knock-out clones in amino acid-limited medium. Asynchronous cultures were grown in amino acid-limited medium and sampled daily. Parasitemias were quantified by flow cytometry and plotted against time for KO1 clones A9 and E9 (A), KO2 clones C5 and D2 (B), KO3 clones G2 and G9 (C), and KO4/1 clones A5 and E5 (D). Symbols and curves are as in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Parasite Life Cycle Analysis—Synchronous cultures of each clone with parasitemias between 1 and 2% were prepared by sorbitol treatment and dispensed in triplicate into 96-well microtiter plates in 100-μl aliquots. Every 2 h, a plate was removed from the incubator, and parasites were fixed by the addition of 100 μl of 0.1% glutaraldehyde. The distribution of stages and parasitemia of each sample were analyzed by flow cytometry. The high fluorescence region that corresponds to schizont stage parasites was gated, and the percent of parasites in this region was plotted against time. The points were fitted to an exponential sine wave function using KaleidaGraph software (Synergy Software). y=m1+m2⋅e[m3+m4⋅sin(2πx/tr+m5)] Here, y is the percentage of schizonts in the culture, x is the independent variable (time), tr is the period of the function in radians, which is converted to degrees by the formula td = (180/π) · tr, and m1–m5 are curve-fitting parameters, which are not related to the period of the function. In all cases, the R values of the curve fitting were greater than 0.99.Determination of EC50 Values for Pepstatin A and E64 —Inhibitor EC50 values were determined essentially as described previously (13Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar). 200-μl aliquots of late ring stage cultures at ∼0.5% parasitemia were incubated with various concentrations of either pepstatin A (Roche Applied Science) or trans-epoxysuccinyl-l-leucylamino(4-guanidine)-butane (E64) in hypoxanthine-free rich medium for 42 h. 0.5 μCi of [3H]hypoxanthine (178.7 Ci/mmol, PerkinElmer) was added to the culture, and the incubation continued for 24 h. Cultures were harvested on glass fiber paper, immersed in UltimaGold scintillation counting mixture (PerkinElmer), and counted in a scintillation counter. The percentage of the inhibition of [3H]hypoxanthine uptake was plotted against the drug concentration, and the curve was fitted using the modified dose-response logistic equation in KaleidaGraph software.Statistical Analysis—One-tailed t tests were employed to analyze the significance of the doubling times and cell cycle times of the knockouts as compared with 3D7. The p values from each test are listed in Table II.Table IIGrowth of knock-out cultures compared with 3D73D7KO1KO2KO3KO4/1abA9E9C5D2G2G9A5E5Rich medium Doubling time (h)aThe R value is >0.995 for all curve fitting parameters in the table12.312.012.412.312.712.912.512.413.313.1 Error±0.04±0.06±0.02±0.03±0.01±0.01±0.01±0.03±0.03±0.03 p valuebThe p values are from one-tailed t test versus 3D70.180.040.110.02 Cell cycle time (h)aThe R value is >0.995 for all curve fitting parameters in the table39.439.039.038.839.539.639.239.338.739.0 Error±0.5±0.5±0.3±0.4±0.3±0.3±0.2±0.3±0.2±0.3 p valuebThe p values are from one-tailed t test versus 3D70.160.120.420.15 Calculated multiplication ratecProductive progeny/cell, calculated from the number of doublings/cell cycle8.99.69.08.98.48.48.89.07.57.9Amino acid-limited medium Doubling time (h)aThe R value is >0.995 for all curve fitting parameters in the table19.019.119.819.519.919.819.820.121.821.2 Error±0.02±0.06±0.02±0.001±0.02±0.01±0.01±0.02±0.02 p valuebThe p values are from one-tailed t test versus 3D70.030.0070.010.008a The R value is >0.995 for all curve fitting parameters in the tableb The p values are from one-tailed t test versus 3D7c Productive progeny/cell, calculated from the number of doublings/cell cycle Open table in a new tab Reagents—All reagents were from either Sigma or Fisher, except as stated.RESULTSDisruption of Food Vacuole Plasmepsin Genes—Plasmepsins I, II, IV, and HAP are arranged in tandem on chromosome 14, spanning ∼16 kb (Fig. 1A). To disrupt the plasmepsin genes, a positive/negative selection strategy was employed (16Duraisingh M.T. Triglia T. Cowman A.F. Int. J. Parasitol. 2002; 32: 81-89Crossref PubMed Scopus (146) Google Scholar). Approximately 500–650 bp of homologous sequence from the 5′ and 3′ ends of each gene was amplified from 3D7 genomic DNA and cloned into plasmid pHHT-TK at sites flanking the positive selectable marker human dihydrofolate reductase (which confers resistance to the antifolate WR99210). The sequences were chosen so that those regions of each plasmepsin gene that were necessary for the formation of the active site would be deleted on double crossover recombination. Following transfection, parasites were first selected with 10 nm WR99210 and then with 4 or 10 μm ganciclovir to obtain parasites that had undergone double crossover recombination (16Duraisingh M.T. Triglia T. Cowman A.F. Int. J. Parasitol. 2002; 32: 81-89Crossref PubMed Scopus (146) Google Scholar).Fig. 1Targeted gene disruption of plasmepsin I, II, IV and HAP.A, schematic diagram is shown of plasmepsin I, II, IV, and HAP gene loci. The four genes cluster in tandem on chromosome 14 in the order shown. Each gene is approximately 1.4 kb, and the distances between neighboring genes are indicated (the diagram is not drawn to scale). B–E, confirmation of gene disruptions by Southern blotting is shown. Restriction enzymes used are diagramed (not to scale). Dashed lines correspond to the targeted DNA fragments before and after integration. DNA for each of two clones of KO1 (B), KO2 (C), KO3 (D), and KO4/1 (E) was analyzed. 3D7 DNA was used as control (left lanes), and probes used are denoted under each blot. Arrowheads mark the migration positions of the digested targeting plasmid. hDHFR, human dihydrofolate reductase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We were able to recover parasites with individual disruptions of PM I and II and HAP. This was confirmed by Southern blotting (Fig. 1, B–D) and by PCR (data not shown). Two clones from each targeting experiment were analyzed. Using probes corresponding to the targeted region, there was a 2.2-kb increase in migration of the restriction fragment compared with parental 3D7 DNA, as predicted (Fig. 1, B–D, left panels). When the human dihydrofolate reductase gene was used as the probe, no signal was detected in the 3D7 lanes, and bands of the sizes predicted for recombination were detected for the knockout clones (Fig. 1, B–D, right panels). We were not able to recover PM IV-disrupted parasites despite multiple attempts with several different targeting constructs. As an alternative strategy, we designed a double knock-out vector with targeting sequences homologous to the PM IV-5′ end and the PM I-3′ end (no gene is predicted for the PM IV/I intergenic region). Following transfection and selection of this construct, parasites with disruption of both PM IV and PM I were obtained successfully (Fig. 1E). The PM IV-5′ probe detected the predicted 2.1-kb band in the 3D7 lane and the 9.0-kb band in the KO4/1 lanes. Hybridization to human dihydrofolate reductase also detected a band of the same size in the KO4/1 lanes.We further confirmed the disruption of each gene by Western blotting using specific anti-PM antibodies (Fig. 2). Parasites were lysed, and equal amounts of protein were loaded in each lane for each experiment. As a loading control, blots were stripped and reprobed for BiP, an ER resident protein (Fig. 2, bottom rows). In agreement with the DNA analysis, no targeted protein bands were detected in the clone of either KO1, KO2, KO3, or KO4/1 (Fig. 2). All antibodies detected the corresponding plasmepsin band in the 3D7 lane on the same blot. No significant induction of untargeted plasmepsins or of falcipain-2 was seen for any of the knockouts, as judged by Western blotting with heterologous plasmepsin and falcipain-2 antibodies (data not shown).Fig. 2Western blotting analysis of knock-out clones. Total parasite lysates were analyzed by immunoblotting using anti-PM I antibody 574 (A), anti-PM II antibody 737 (B), anti-HAP antibody 3F10–6 (C), and anti-PM I antibody 1C6–24 and anti-PM IV antibody 13.9.2 (D). Blots were reprobed with anti-BiP antibody as a loading control (lower panels). The lower band in B was seen with secondary antibody alone (34Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Parasite Growth Analysis—Giemsa-stained knock-out clones appeared morphologically normal. To compare parental and knock-out growth rates, we monitored parasite proliferation by flow cytometry. Two clones from each knock-out line and two independently maintained 3D7 cultures were grown asynchronously in rich medium and sampled approximately every 24 h for 1 week. The samples were fixed and stained with the DNA dye propidium iodide, and parasitemia was counted by flow cytometry. After 7 days, the -fold increase of both KO2 clones was approximately 80% of 3D7 (Fig. 3B) and approximately 60% of 3D7 for the KO4/1 clones (Fig. 3D). The growth of the KO1 and KO3 clones relative to 3D7 was not significantly different (Fig. 3, A and C). We fitted these data to an exponential growth curve and calculated doubling times (see “Experimental Procedures”). Compared with 3D7, the KO2 doubling time was 5% longer, and KO4/1 doubling time was 9% longer (Table II).To test the possibility that the slow growth of KO2 and KO4/1 was caused by an extended cell cycle, we monitored the growth of synchronous parasites for 56 h, with frequent sampling (total of 26 samplings taken at ∼2-h intervals). Cultures were analyzed by flow cytometry, and the percentage of parasites at the schizont stage (post-S phase) was plotted against time (Fig. 4). As expected, the curves were periodic and fitted an exponential sine wave function (R > 0.99, see “Experimental Procedures”). A one-tailed Student's t test of the cell cycle times indicated that there was no significant difference between 3D7 and any of the knockouts (Table II). Similar results were obtained by plotting either the percentage of ring or percentage of trophozoite forms from this same experiment (data not shown).Fig. 4Cell cycle time of knock-out clones. Synchronous cultures were grown in rich medium for 56 h and sampled every 2 to 3 h. The parasite stage was analyzed by flow cytometry, and the percentage of parasites at the schizont stage was plotted against time. Two independently maintained 3D7 cultures (open and filled circles, respectively) and the two clones for each knockout, KO1, A9 and E9 (open and filled green triangles), KO2, C5 and D2 (open and filled blue squares), KO3, G2 and G9 (open and filled yellow inverted triangles), and KO4/1, A5 and E5 (open and filled red diamonds), were analyzed. Error bars indicate the standard deviation of triplicate samples. The data were fitted to an exponential sine wave equation, and the curves are shown. Dotted lines correspond to open symbols, and solid lines correspond to closed symbols.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Parasite Growth in Amino Acid-limited Medium—An amino acid-limited medium was used to test parasite growth. This medium contains the five amino acids that are lacking or limited in hemoglobin and forces the parasite to rely on hemoglobin degradation to obtain amino acids to sustain its growth (13Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar). A growth rate analysis similar to that in rich medium was performed. All cultures grew more slowly in amino acid-limited medium, with ∼5% of the overall growth in rich medium after 7 days (Fig. 5). The slow growth phenotype of the KO4/1 clones was the most obvious at approximately 60% of the 3D7 level in 1 week, and the growth of KO1, KO2, and KO3 clones was 70–80% of the 3D7 level. The doubling times were calculated and are listed in Table II. Doubling times for KO1, KO2, and KO3 clones were 4–5% longer than for 3D7, whereas those for KO4/1 clones were 13% longer, all of which represent significant differences.Sensitivities to Pepstatin A and E64 —Because both aspartic and cysteine protease activities participate in hemoglobin degradation in the food vacuole, we were interested to see if the susceptibility of knockouts to aspartic and cysteine protease inhibitors would change. The EC50 values of pepstatin A and E64 for each clone were determined and are reported as the percentage of inhibition relative to 3D7 (Fig. 6). All knock-out clones were slightly less sensitive (increased EC50) to pepstatin A, although they were slightly more sensitive to E64, except the KO3 clones that remained unchange" @default.
• W1972959613 created "2016-06-24" @default.
• W1972959613 creator A5013369376 @default.
• W1972959613 creator A5027835055 @default.
• W1972959613 creator A5029982946 @default.
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• W1972959613 date "2005-01-01" @default.
• W1972959613 modified "2023-10-17" @default.
• W1972959613 title "The Role of Plasmodium falciparum Food Vacuole Plasmepsins" @default.
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