Plasmepsin 4-Deficient Plasmodium berghei Are Virulence Attenuated and Induce Protective Immunity against Experimental Malaria
2009; Elsevier BV; Volume: 176; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090504
ISSN1525-2191
AutoresRoberta Spaccapelo, Chris J. Janse, Sara Caterbi, Blandine Franke‐Fayard, J. Alfredo Bonilla, Luke M. Syphard, Manlio Di Cristina, Tania Dottorini, Andrea Savarino, Antonio Cassone, Francesco Bistoni, Andrew P. Waters, John B. Dame, Andrea Crisanti,
Tópico(s)Complement system in diseases
ResumoPlasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Δpm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines. Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Δpm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines. The digested vacuole (DV) of malaria parasites performs hemoglobin degradation, which is a crucial process for parasite growth and survival within the host erythrocyte. In Plasmodium falciparum, the most important human malaria parasite, this is achieved with the contribution of several digestive vacuole proteases including three aspartic proteinases, the plasmepsins (PM) PfPM1, PfPM2, and PfPM4 and one histo-aspartic protease, PfHAP.1Coombs GH Goldberg DE Klemba M Berry C Kay J Mottram JC Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets.Trends Parasitol. 2001; 17: 532-537Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 2Banerjee R Liu J Beatty W Pelosof L Klemba M Goldberg DE Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.Proc Natl Acad Sci USA. 2002; 99: 990-995Crossref PubMed Scopus (353) Google Scholar, 3Dame JB Yowell CA Omara-Opyene L Carlton JM Cooper RA Li T Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.Mol Biochem Parasitol. 2003; 130: 1-12Crossref PubMed Scopus (63) Google Scholar, 4Bonilla JA Bonilla TD Yowell CA Fujioka H Dame JB Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function.Mol Microbiol. 2007; 65: 64-75Crossref PubMed Scopus (80) Google Scholar, 5Liu J Istvan ES Gluzman IY Gross J Goldberg DE Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems.Proc Natl Acad Sci USA. 2006; 103: 8840-8845Crossref PubMed Scopus (248) Google Scholar The plasmepsins have long been studied as potential drug targets and subjected to functional and biochemical studies with the hope that inhibiting them would halt hemoglobin digestion and result in parasite death. Surprisingly, the systematic disruption of either individual or different combinations of the plasmepsin genes did not result in any striking growth defect. Presumably, this is due to redundant enzyme systems for digesting hemoglobin, which involve cysteine proteases, metalloproteases, and aminopeptidases, and to the presence of multiple pathways for the uptake of extracellular amino acids.6Omara-Opyene AL Moura PA Sulsona CR Bonilla JA Yowell CA Fujioka H Fidock DA Dame JB Genetic disruption of the Plasmodium falciparum digestive vacuole plasmepsins demonstrates their functional redundancy.J Biol Chem. 2004; 279: 54088-54096Crossref PubMed Scopus (100) Google Scholar, 7Liu J Gluzman IY Drew ME Goldberg DE The role of Plasmodium falciparum food vacuole plasmepsins.J Biol Chem. 2005; 280: 1432-1437Crossref PubMed Scopus (152) Google Scholar, 8Bonilla JA Moura PA Bonilla TD Yowell CA Fidock DA Dame JB Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum.Int J Parasitol. 2007; 37: 317-327Crossref PubMed Scopus (43) Google Scholar The P. falciparum and Plasmodium reichnowi clades differ from other Plasmodium species in that they have four genes encoding DV plasmepsins. In P. falciparum only the disruption of all four plasmepsin genes, which eliminates all aspartic protease activity from the DV, resulted in delayed in vitro schizont maturation accompanied by reduced formation of hemozoin (an insoluble crystal produced during hemoglobin degradation) and less efficient processing of endosomal vesicles in the DV.4Bonilla JA Bonilla TD Yowell CA Fujioka H Dame JB Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function.Mol Microbiol. 2007; 65: 64-75Crossref PubMed Scopus (80) Google Scholar We here investigated the impact of the loss of the various functions of the DV plasmepsins on parasite virulence by disrupting the single gene encoding the DV plasmepsin 4 (pm4) in the rodent malaria parasite Plasmodium berghei. This parasite is a well established and tractable model to study the function of Plasmodium genes in vivo and replicates several key features of human cerebral malaria.9Lou J Lucas R Grau GE Pathogenesis of cerebral malaria: recent experimental data and possible applications for humans.Clin Microbiol Rev. 2001; 14: 810-820Crossref PubMed Scopus (190) Google Scholar, 10de Souza JB Riley EM Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis.Microbes Infect. 2002; 4: 291-300Crossref PubMed Scopus (171) Google Scholar The phenotypic analysis of loss-of-function mutants has been used to gain an insight into a variety of host-parasite interactions.11Carvalho TG Menard R Manipulating the Plasmodium genome.Curr Issues Mol Biol. 2005; 7: 39-55PubMed Google Scholar In this study, we confirm that the disruption of PM4, which results in loss of all aspartic proteinase activity targeted to its lysosomal compartments, has only a modest effect on the intraerythrocytic development of P. berghei parasites, but we observed dramatic differences in the virulence of these parasites compared with that of wild-type parasites. Specifically, we report the growth and multiplication characteristics of Δpm4 parasites in different mouse strains and demonstrate that these parasites neither induce experimental cerebral malaria (ECM) in ECM-susceptible mice nor kill the host by hemolytic anemia in ECM-resistant mice. In these latter mice, Δpm4 parasites induce a self-resolving infection, which generates spleen-dependent protective immune responses. This is the first report of a mutant P. berghei parasite that does not induce cerebral complications as the result of a single gene mutation. A number of mutant parasite lines carrying a disrupted P. berghei pm4 locus (PB000298.03.0) were independently generated in different laboratories. Additional parasite lines expressing the green fluorescent protein (GFP)-luciferase fusion protein were generated either on the wild-type or on the Δpm4 background. Parasites of the P. berghei ANKA clone 2.34 and clone cl15cy112Janse CJ Franke-Fayard B Mair GR Ramesar J Thiel C Engelmann S Matuschewski K van Gemert GJ Sauerwein RW Waters AP High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.Mol Biochem Parasitol. 2006; 145: 60-70Crossref PubMed Scopus (316) Google Scholar have been used as a control (wild-type) and for the generation of the Δpm4 mutant lines. In addition, these parasites have been used to generate the transgenic wild type (wt+) parasites (1037cl1 line) and wt++ parasites (gfp-luc/cl2 line) that express a fusion protein (GFP-Luc) encompassing the GFP (mutant3) and the luciferase (LUC-IAV) coding sequence. Δpm4cl1 and Δpm4cl6 are two Δpm4 parasite lines deficient in expressing PM4. The pm4 gene has been disrupted by introducing the construct pRSpm4 into the genome of the P. berghei ANKA clone 2.34 as described below. 688cl2 and 688cl3 are two Δpm4 parasite lines deficient in expressing PM4. The pm4 gene has been disrupted by introducing construct pL1095 into the genome of cl15cy1 parasites as described below. wt+ (or 1037cl1) is a reference transgenic parasite line that expresses GFP-Luc under the control of the schizont-specific ama-1 promoter. The gfp-luc is inserted into the p230p locus (PB000214.00.0) on chromosome 3 of parasites of cl15cy1. This line does not contain a drug-selectable marker and has been selected by flow-sorting of GFP-expressing parasites directly after transfection as described below. 1092cl4 and 1092cl6 are two Δpm4+ parasite lines deficient in expressing PM4. The pm4 gene has been disrupted by introducing construct pL1095 into the genome of transgenic parasites of line 1037cl1. Both lines express GFP-luciferase under the control of the schizont specific ama-1 promoter. wt++ is a reference transgenic parasite line that expresses GFP-Luc under the control of the schizont-specific ama-1 promoter. The transgene is inserted into the c-/d-rrna gene unit by single crossover recombination as described below. This line contains a tgdhfr/ts drug-selectable marker cassette. Swiss-OF1 mice (OF1 ico, construct 242, age 6 weeks, Charles River Laboratories, Inc., Wilmington, MA), C57BL/6 (age 6 weeks, Charles River Laboratories, Inc.), CBA/J (age 6 to 8 weeks, Charles River Laboratories, Inc.), Swiss-CD1 (age 6 to 8 weeks; Harlan Sprague-Dawley, Indianapolis, IN), BALB/c (age 6 to 8 weeks; Harlan Sprague-Dawley, Indianapolis, IN), and NIH Swiss (age 6 weeks; Harlan Sprague-Dawley, Indianapolis, IN) were used. All studies in which animals are involved have been performed according to the regulations of the Dutch “Animal On Experimentation Act” and guidelines 86/609/EEG, the Italian regulation D.L 27 January 1992, n. 116, and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, as approved by the Institutional Animal Care and Use Committee of the University of Florida. These parasite lines were generated using the DNA construct pRSpm4. This construct contains the following elements (Supplemental Figure S1A, see http://ajp.amjpathol.org): i) a5′-untranslated region (UTR) 954-bp PCR fragment of the pm4 (sense 5′-CCGGGCCCTACAAAATATTTTCATAAGTTGGC-3′ [ApaI site is underlined] and antisense 5′-CCATCGATTTCCATTTTGAACTAATTAAAG-3′ [ClaI site is underlined]); ii) a 3′-UTR 813-bp PCR fragment of the pm4 (sense 5′-GGGAATTCTTATATATGATATATTACACGTAC-3′ [EcoRI site is underlined] and antisense 5′-CGGGATCCATGGTTTTACGATTTAAACTTTC-3′ [BamHI site is underlined]); and iii) the tgdhfr/ts drug-selectable marker cassette (Supplemental Figure S1A, see http://ajp.amjpathol.org). The plasmid was linearized with SacII and PvuII and used for the generation of Δpm4 lines. For the generation of Δpm4cl1 and Δpm4cl6, blood stages of the P. berghei ANKA clone 2.34 parasites were transfected with 10 μg of DNA linear fragments of pRSpm4, and mutant parasites were obtained by the standard method of drug (pyrimethamine) selection in mice.13Janse CJ Ramesar J Waters AP High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.Nat Protoc. 2006; 1: 346-356Crossref PubMed Scopus (383) Google Scholar Pyrimethamine-resistant parasites were subsequently cloned by limiting dilution. The disruption of the pm4 locus was confirmed by diagnostic PCR (data not shown) and by Southern blot analysis of BamHI (B)- and AvaII (A)-digested genomic DNA (Supplemental Figure S1B, see http://ajp.amjpathol.org). Loss of expression of PM4 was confirmed by Western blot analysis as described below (data not shown). These parasite lines were generated using the DNA construct pL1095. To generate the construct, we first amplified by PCR ∼700-bp fragments from either end of the pm4 locus. The primers used to amplify the 5′-UTR 754-bp fragment encoding from −311 to 443 bp were the following: sense 5′-GCATGGTACCCCTTATTAAAGAGATTGGGAAGC-3′ and antisense 5′-GCATATCGATTTTCCTAATTCTGCAGTACC-3′, flanked with engineered KpnI and ClaI restriction sites (underlined). The 3′-UTR 680-bp fragment was amplified using the following primers: sense 5′-GCATGAATTCCAGGACAAATTGAAAATGCAG-3′ and antisense 5′-GCATCCGCGGATAAATTTCTTTAATCTTATGGC-3′. The PCR product contained 545 bp of coding sequence, extending 135 bp into the 3′-UTR and flanked by EcoRI and SacII restriction sites (underlined). These PCR products were directionally cloned into the pL0001 plasmid backbone using the restriction sites described above, placing the fragments into the construct as shown in Supplemental Figure S1C (see http://ajp.amjpathol.org), flanking the Toxoplasma gondii dihydrofolate reductase/thymidylate synthase (tgdhfr/ts) drug-selectable marker cassette. This plasmid was linearized by cleavage with ScaI, NaeI, and SapI, and the ∼6.5-kb fragment was purified by agarose gel electrophoresis. For the generation of mutant lines 688, blood-stage parasites of cl15cy1 were transfected with 5 to 10 μg of gel-purified DNA linear fragments of pL1095, and mutant parasites were obtained by the standard method of drug (pyrimethamine) selection in mice.13Janse CJ Ramesar J Waters AP High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.Nat Protoc. 2006; 1: 346-356Crossref PubMed Scopus (383) Google Scholar Pyrimethamine-resistant parasites were subsequently cloned by limiting dilution. The correct disruption of pm4 in two clones examined (688cl2 and 688cl3) was confirmed by the failure of PCR to amplify the expected 1122-bp from the central region of the pm4 locus using primer pair 1 + 2 (primer 1: 5′-TCCGAATATTTAACAATTCGTGC-3′ and primer 2: 5′-GTTTTTTGCAACTGCAAAACC-3′) under conditions for which this fragment was readily amplified from parental lines (Supplemental Figure S1D, see http://ajp.amjpathol.org). The expected integration into the pm4 locus was verified by successfully amplifying the predicted 5′ and 3′ boundary sequences using primer pairs 3 + 5 (primer 3: 5′-TTCCCTTGTGTCCTTTAAG-3′ and primer 5: 5′-CGCATTATAGAGTTCATTTTAC-3′) and 6 + 4 (primer 4: 5′-AAGCGGAGTTTATTGTCTGTC-3′ and primer 6: 5′-CACATAAAATGGCTAGTATGAATAG-3′) as shown in Supplemental Figure S1D (see http://ajp.amjpathol.org). Southern blot analysis of digested DNA from wild-type, 688cl2, and 688cl3 further confirmed the predicted integration event (data not shown). For the generation of mutant line Δpm4+ blood-stage parasites of line 1037cl1 (wt+ parasites) were transfected, selected, and analyzed as described for line 688, resulting in the selection of a transgenic parasite line that expresses GFP-Luc and is deficient in PM4 expression. Two mutant clones of this line, 1092cl2 and 1092cl4, have been selected for further analysis. The loss of PM4 expression was confirmed by Western analysis of total protein extracted from saponin-lysed blood-stage parasites. Samples were extracted in an equal volume of SDS sample buffer (100 mmol/L Tris-Cl, pH 6.8, 10% glycerol, 2% SDS, and 100 mmol/L 2-mercaptoethanol), boiled for 5 minutes, and separated by electrophoresis on 10% acrylamide gels (Bio-Rad Laboratories, Hercules, CA). After electrophoresis, proteins in the gel were transferred to a 0.2 μmol/L polyvinylidene difluoride membrane. The membrane was blocked for 24 hours (100 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween 20, and 5% powdered milk) before incubation with primary antibody. Anti-PM4 antibodies were raised in a rabbit immunized with the predicted N-terminal peptide of the enzyme (KYEDSIELDQSLGLSC) cross-linked to carrier protein (keyhole limpet hemocyanin) and used at a dilution of 1:7,500 in blocking solution. Antibody bound to the membrane was visualized on X-ray film after incubation with a secondary antibody, goat anti-rabbit (horseradish peroxidase-conjugated), and reaction with the SuperSignal West Dura Extended Duration Substrate (Pierce Chemical, Rockford, IL). Equal loading of parasite protein samples was confirmed using anti-BiP antisera (1:10,000 dilution) provided by J. B. Adams and obtained from the Malaria Research and Reference Reagent Resource Center (MR4) (Manassas, VA), followed by incubation with goat anti-rat (horseradish peroxidase-conjugated) and developed with the chemiluminescent substrate, as above (Supplemental Figure S1E, see http://ajp.amjpathol.org). The transgenic parasite line 1037cl1 (wt+ parasites) contains the gfp-luc integrated into the p230p locus (PB000214.00.0) under the control of the schizont-specific ama1 promoter. The construct pL1156 is integrated by double crossover recombination and does not contain a drug-selectable marker gene. The DNA construct for the generation of this reporter line (Supplemental Figure S2A, see http://ajp.amjpathol.org) was made by replacing the α-tubulin II promoter of pL002414Kooij TW Franke-Fayard B Renz J Kroeze H van Dooren MW Ramesar J Augustijn KD Janse CJ Waters AP Plasmodium berghei α-tubulin II: a role in both male gamete formation and asexual blood stages.Mol Biochem Parasitol. 2005; 144: 16-26Crossref PubMed Scopus (22) Google Scholar with the ama1 promoter (EcoRV/BamHI fragment of pL0010). The ama1-gfpm3 (EcoRV/KpnI) fragment of this plasmid was subcloned in plasmid BSSK to create plasmid BSSKama1-gfp-3′utr. Finally, the eef1aa-gfp-3′utr cassette of plasmid pL0023 (pbGFP-Lucko230p-SMCON12Janse CJ Franke-Fayard B Mair GR Ramesar J Thiel C Engelmann S Matuschewski K van Gemert GJ Sauerwein RW Waters AP High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.Mol Biochem Parasitol. 2006; 145: 60-70Crossref PubMed Scopus (316) Google Scholar) was replaced with the PstI/KpnI ama1-gfp-3′utr fragment of BSSKamaI-gfp-3′utr to obtain pL1141. Subsequently, the gfpm3-luc-3′utr (HpaI/KpnI) fragment of pL0028 (pPbgfp-lucSCH15Franke-Fayard B Janse CJ Cunha-Rodrigues M Ramesar J Buscher P Que I Lowik C Voshol PJ den Boer MA van Duinen SG Febbraio M Mota MM Waters AP Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.Proc Natl Acad Sci USA. 2005; 102: 11468-11473Crossref PubMed Scopus (225) Google Scholar) was introduced into pl1141 to create pL1156. The plasmids pL0024, pL0010, pL0023, and pL0028 can be obtained from MR4. Blood-stage parasites (cl15cy1) were transfected with 5 μg of linear fragments of KspI-digested plasmid pL1156,13Janse CJ Ramesar J Waters AP High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.Nat Protoc. 2006; 1: 346-356Crossref PubMed Scopus (383) Google Scholar and transgenic parasites were selected by flow-sorting of GFP-expressing parasites as described.16Janse CJ Franke-Fayard B Waters AP Ramesar J Tomas AM van der Wel AM Thomas AW Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei.Nat Protoc. 2006; 1: 614-623Crossref PubMed Scopus (75) Google Scholar Flow-sorted, GFP-Luc-expressing parasites were subsequently cloned by limiting dilution. Correct integration of the construct into the p230p locus was analyzed by PCR using the following primers 831, 5′-CTTTATTTTTCAATTACCGCC-3′, 1348,13Janse CJ Ramesar J Waters AP High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.Nat Protoc. 2006; 1: 346-356Crossref PubMed Scopus (383) Google Scholar and 824, 5′-CCAAGAAGGGCGGAAAGATC-3′, and by Southern analysis of restricted DNA (data not shown). Primers and sizes of the products are indicated in Supplemental Figure S2, A and B (see http://ajp.amjpathol.org). Correct timing of expression of GFP-Luc in schizonts was analyzed by determination of GFP expression during synchronized development of blood stages in live parasites using a fluorescence microscope (Leica DMRA HC “upright” microscope) as described.15Franke-Fayard B Janse CJ Cunha-Rodrigues M Ramesar J Buscher P Que I Lowik C Voshol PJ den Boer MA van Duinen SG Febbraio M Mota MM Waters AP Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.Proc Natl Acad Sci USA. 2005; 102: 11468-11473Crossref PubMed Scopus (225) Google Scholar The transgenic parasite wt++ line (gfp-luc/cl2) contains the gfp-luc integrated into the c-/d-rrna gene unit under the control of the schizont-specific ama1 promoter. These parasites were generated using plasmid pL0028 (MRA-797, MR4)15Franke-Fayard B Janse CJ Cunha-Rodrigues M Ramesar J Buscher P Que I Lowik C Voshol PJ den Boer MA van Duinen SG Febbraio M Mota MM Waters AP Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.Proc Natl Acad Sci USA. 2005; 102: 11468-11473Crossref PubMed Scopus (225) Google Scholar (Supplemental Figure S2C, see http://ajp.amjpathol.org). Correct integration of the construct was analyzed by diagnostic PCR as described previously17Franke-Fayard B Trueman H Ramesar J Mendoza J van der Keur M van der Linden R Sinden RE Waters AP Janse CJ A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.Mol Biochem Parasitol. 2004; 137: 23-33Crossref PubMed Scopus (370) Google Scholar (Supplemental Figure S2D, see http://ajp.amjpathol.org) and Southern analysis of restricted DNA (data not shown). The asexual multiplication rate in vivo, determined during the cloning procedure, is calculated as follows. The percentage of infected erythrocytes in mice injected with a single parasite is determined at days 8 to 11 by counting Giemsa-stained blood films. The mean asexual multiplication rate per 24 hours is then calculated, assuming a total of 1.2 × 1010 erythrocytes/mouse (2 ml of blood). The percentage of infected erythrocytes in mice infected with reference lines of the ANKA strain of P. berghei consistently ranges between 0.5 and 2% at day 8 after infection, resulting in a mean multiplication rate of 10 per 24 hours.18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Waters AP Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.Mol Microbiol. 2003; 50: 1539-1551Crossref PubMed Scopus (42) Google Scholar For determination of the number of merozoites per schizont, infected blood samples containing schizonts are stained with the DNA-specific, fluorescent dye Hoechst 33258 and analyzed by flow cytometry.18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Waters AP Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.Mol Microbiol. 2003; 50: 1539-1551Crossref PubMed Scopus (42) Google Scholar, 19Janse CJ Van Vianen PH Flow cytometry in malaria detection.Methods Cell Biol. 1994; 42: 295-318Crossref PubMed Scopus (50) Google Scholar Infected blood (1 to 3% parasitemia) is collected from Swiss-OF1 mice or Wistar rats by heart puncture and cultured overnight under standard culture conditions for collecting of P. berghei schizonts.20Janse CJ Waters AP Plasmodium berghei: the application of cultivation and purification techniques to molecular studies of malaria parasites.Parasitol Today. 1995; 11: 138-143Abstract Full Text PDF PubMed Scopus (120) Google Scholar After overnight culture the infected erythrocytes are separated from uninfected cells by Nycodenz density centrifugation as described13Janse CJ Ramesar J Waters AP High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.Nat Protoc. 2006; 1: 346-356Crossref PubMed Scopus (383) Google Scholar and fixed in 0.25 (v/v) glutaraldehyde solution in PBS. These samples are stained with Hoechst 33258 at a concentration of 2 μmol/L for 1 hour at 37°C and analyzed with a FACScan (LSR II, Becton Dickinson, San Jose, CA). UV excitation of the Hoechst 33258 dye is performed with an argon ion laser (450/50 nm). The fluorescence intensity of a total of 50,000 cells per sample is measured, and data analysis is performed using CellQuest software (Becton Dickinson). The mean fluorescence intensity of free merozoites and/or ring-infected erythrocytes (first peak [P1] in the fluorescence histograms) (Supplemental Figure S3, see http://ajp.amjpathol.org) is proportional to the haploid DNA value18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Waters AP Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.Mol Microbiol. 2003; 50: 1539-1551Crossref PubMed Scopus (42) Google Scholar and is set at 1. The number of merozoites per schizont is calculated by dividing the mean fluorescence intensity of mature schizonts (peaks P2 and P3 in the histograms) (Supplemental Figure S3, see http://ajp.amjpathol.org) by the mean fluorescence intensity of the merozoites/ring forms.18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Waters AP Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.Mol Microbiol. 2003; 50: 1539-1551Crossref PubMed Scopus (42) Google Scholar The length of the asexual blood stage is determined in standard short-term cultures of synchronized P. berghei blood stages.18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Waters AP Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.Mol Microbiol. 2003; 50: 1539-1551Crossref PubMed Scopus (42) Google Scholar In brief, cultured and purified schizonts, collected as described by Janse et al,13Janse CJ Ramesar J Waters AP High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.Nat Protoc. 2006; 1: 346-356Crossref PubMed Scopus (383) Google Scholar are injected i.v. into the tail veins of mice. In these animals, merozoites invade within 4 hours after injection of the schizonts, giving rise to synchronized in vivo infections with a parasitemia of 0.5 to 3%, containing mainly (>90%) ring form parasites. At 2 to 4 hours after injection of the schizonts, infected blood is collected from the mice by heart puncture and incubated at a 1% cell density in complete culture medium (RPMI 1640 with 20% fetal calf serum) for a period of 24 hours at 37°C. At fixed time points after the start of the cultures, 1-ml samples are collected for analysis of the cell cycle by flow cytometry.18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Waters AP Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.Mol Microbiol. 2003; 50: 1539-1551Crossref PubMed Scopus (42) Google Scholar Cells, fixed in 0.25 (v/v) glutaraldehyde solution in PBS, are stained with Hoechst 33258 at a concentration of 2 μmol/L for 1 hour at 37°C and analyzed with a FACScan (LSR II). UV excitation of Hoechst 33258 dye is performed with an argon ion laser (450/50 nm). The fluorescence intensity and size (forward/sideward scatter) of a total of 50,000 cells per sample are measured, and data analysis is performed using CellQuest software. No gate is set in the forward/sideward scatter for size selection of erythrocytes to include the small, free merozoites for analysis. The fluorescence intensity of infected erythrocytes is proportional to the DNA content of the parasites.19Janse CJ Van Vianen PH Flow cytometry in malaria detection.Methods Cell Biol. 1994; 42: 295-318Crossref PubMed Scopus (50) Google Scholar The start of schizogony (the length of the G1 phase of trophozoites) is defined as the time point (hours postinvasion) at which the percentage of cells with more than the haploid/diploid DNA content (percentage of schizonts; gate P5 in Supplemental Figure S4, see http://ajp.amjpathol.org) had increased >5% compared with that at the previous time point.18Janse CJ Haghparast A Speranca MA Ramesar J Kroeze H del Portillo HA Wa
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