Serine Repeat Antigen (SERA5) Is Predominantly Expressed among the SERA Multigene Family of Plasmodium falciparum, and the Acquired Antibody Titers Correlate with Serum Inhibition of the Parasite Growth
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m207145200
ISSN1083-351X
AutoresSayaka Aoki, Jie Li, Sawako Itagaki, Brenda Okech, Thomas G. Egwang, Hiroyuki Matsuoka, Nirianne Palacpac, T. Mitamura, Toshihiro Horii,
Tópico(s)Computational Drug Discovery Methods
ResumoThe Plasmodium falciparum serine repeat antigen (SERA) is one of the blood stage malaria vaccine candidates. The malaria genome project has revealed that SERA is a member of the SERA multigene family consisting of eight SERA homologues clustered on chromosome 2 and one SERA homologue on chromosome 9. Northern blotting and real time quantitative reverse transcription-PCR with five independent parasite strains, including three allelic representative forms of the SERA gene, have shown that all of the SERA homologues are transcribed most actively at trophozoite and schizont stages and that SERA5 (SERA/SERP) is transcribed predominantly among the family. Polyclonal antibodies were raised against recombinant proteins representing the N-terminal portions of four significantly transcribed SERA homologues (SERA3 to -6) in the center of the cluster on chromosome 2. Using these antibodies, indirect immunofluorescence microscopy detected the expression of SERA3 to -6, with similar localization, in all trophozoite- and schizont-infected erythrocytes. We have examined 40 sera from Ugandan adults for their antibody reactivity and found that enzyme-linked immunosorbent assay titer against SERA5 N-terminal domain, but not against other SERA proteins, is positively correlated with the inhibition of in vitro parasite growth by individual sera. Our data confirm the usefulness of the N-terminal domain of SERA5 as a promising malaria candidate vaccine. The Plasmodium falciparum serine repeat antigen (SERA) is one of the blood stage malaria vaccine candidates. The malaria genome project has revealed that SERA is a member of the SERA multigene family consisting of eight SERA homologues clustered on chromosome 2 and one SERA homologue on chromosome 9. Northern blotting and real time quantitative reverse transcription-PCR with five independent parasite strains, including three allelic representative forms of the SERA gene, have shown that all of the SERA homologues are transcribed most actively at trophozoite and schizont stages and that SERA5 (SERA/SERP) is transcribed predominantly among the family. Polyclonal antibodies were raised against recombinant proteins representing the N-terminal portions of four significantly transcribed SERA homologues (SERA3 to -6) in the center of the cluster on chromosome 2. Using these antibodies, indirect immunofluorescence microscopy detected the expression of SERA3 to -6, with similar localization, in all trophozoite- and schizont-infected erythrocytes. We have examined 40 sera from Ugandan adults for their antibody reactivity and found that enzyme-linked immunosorbent assay titer against SERA5 N-terminal domain, but not against other SERA proteins, is positively correlated with the inhibition of in vitro parasite growth by individual sera. Our data confirm the usefulness of the N-terminal domain of SERA5 as a promising malaria candidate vaccine. Malaria remains a devastating disease worldwide, especially in the tropics. Among four species of human malaria parasites,Plasmodium falciparum is responsible for more than a million deaths annually. The appearance of drug-resistant parasites and insecticide-refractory mosquito vectors has made its control more difficult. It is therefore of increasing importance to develop effective malaria vaccines. P. falciparum serine repeat antigen (SERA) 1The abbreviations used are: SERA, serine repeat antigen; gDNA, genomic DNA; RT, reverse transcription; MSP-1, merozoite surface protein-1; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline (1Bzik D.J. Li W.B. Horii T. Inselburg J. Mol. Biochem. Parasitol. 1988; 30: 279-288Google Scholar) is an asexual blood stage antigen produced in large amounts, specifically during late trophozoite and schizont stages (2Delplace P. Fortier B. Tronchin G. Dubremetz J.F. Vernes A. Mol. Biochem. Parasitol. 1987; 23: 193-203Google Scholar, 3Fox B.A. Bzik D.J. Mol. Biochem. Parasitol. 1994; 68: 133-144Google Scholar). SERA protein (also called SERP (4Knapp B. Hundt E. Nau U. Kupper H.A. Mol. Biochem. Parasitol. 1989; 32: 73-83Google Scholar) or p126 (5Delplace P. Dubremetz J.F. Fortier B. Vernes A. Mol. Biochem. Parasitol. 1985; 17: 239-251Google Scholar)) is secreted into the lumen of the parasitophorous vacuole after removal of the signal peptide (6Debrabant A. Maes P. Delplace P. Dubremetz J.F. Tartar A. Camus D. Mol. Biochem. Parasitol. 1992; 53: 89-95Google Scholar). Upon schizont rupture, SERA is processed into a 47-kDa N-terminal, a 50-kDa central, an 18-kDa C-terminal, and a 6-kDa domain (6Debrabant A. Maes P. Delplace P. Dubremetz J.F. Tartar A. Camus D. Mol. Biochem. Parasitol. 1992; 53: 89-95Google Scholar). The complex of 47- and 18-kDa peptides is associated with merozoite, and the 50-kDa fragment is shed into the culture medium (7Pang X.L. Mitamura T. Horii T. Infect. Immun. 1999; 67: 1821-1827Google Scholar, 8Li J. Mitamura T. Fox B.A. Bzik D.J. Horii T. Parasitol Int. 2002; 53: 89-95Google Scholar). The activity responsible for the primary processing step of SERA to P47 and P73 is sensitive to the serine protease inhibitor diisopropyl fluorophosphate, whereas the activity for the conversion of P56 into P50 is sensitive to cysteine protease inhibitors E-64, leupeptin, and iodoacetoamide (9Li J. Matsuoka H. Mitamura T. Horii T. Mol. Biochem. Parasitol. 2002; 120: 177-186Google Scholar). Mouse and rat antibodies against the N-terminal 47-kDa domain have been shown to inhibit the intraerythrocytic proliferation of parasitesin vitro (7Pang X.L. Mitamura T. Horii T. Infect. Immun. 1999; 67: 1821-1827Google Scholar, 10Sugiyama T. Suzue K. Okamoto M. Inselburg J. Tai K. Horii T. Vaccine. 1996; 14: 1069-1076Google Scholar, 11Fox B.A. Xing-Li P. Suzue K. Horii T. Bzik D.J. Exp. Parasitol. 1997; 85: 121-134Google Scholar, 12Pang X.L. Horii T. Vaccine. 1998; 16: 1299-1305Google Scholar), but rat antibodies against the central 50-kDa domain have little effect (10Sugiyama T. Suzue K. Okamoto M. Inselburg J. Tai K. Horii T. Vaccine. 1996; 14: 1069-1076Google Scholar). Recombinant proteins corresponding to the 47-kDa domain of SERA conferred protective immunity in Aotus and squirrel monkeys against the parasite challenges (13Inselburg J. Bzik D.J. Li W.B. Green K.M. Kansopon J. Hahm B.K. Bathurst I.C. Barr P.J. Rossan R.N. Infect. Immun. 1991; 59: 1247-1250Google Scholar, 14Inselburg J. Bathurst I.C. Kansopon J. Barchfeld G.L. Barr P.J. Rossan R.N. Infect. Immun. 1993; 61: 2041-2047Google Scholar, 15Inselburg J. Bathurst I.C. Kansopon J. Barr P.J. Rossan R. Infect. Immun. 1993; 61: 2048-2052Google Scholar, 16Suzue K. Ito M. Matsumoto Y. Tanioka Y. Horii T. Parasitol. Int. 1997; 46: 17-25Google Scholar). The epidemiological study in a holoendemic area of Uganda has revealed that increased level of IgG against 47-kDa peptide correlates with lower parasitemias in the peripheral blood and absence of fever in a group of children, but IgG level against 50-kDa peptide does not (17Okech B.A. Nalunkuma A. Okello D. Pang X.L. Suzue K. Li J. Horii T. Egwang T.G. Am. J. Trop. Med. Hyg. 2001; 65: 912-917Google Scholar). Thus, the N-terminal domain of SERA is a promising candidate for a malaria vaccine. It was previously reported that the N-terminal domain of SERA is polymorphic, and according to the amino acid sequences, all of the examined alleles can be grouped into three major allelic families, namely FCR3 type, K1 type, and Honduras-1 type in laboratory strains and field isolates (18Morimatsu K. Morikawa T. Tanabe K. Bzik D.J. Horii T. Mol. Biochem. Parasitol. 1997; 86: 249-254Google Scholar, 19Liu Q. Ferreira M.U. Ndawi B.T. Ohmae H. Adagu I.S. Morikawa T. Horii T. Isomura S. Kawamoto F. Southeast Asian J. Trop. Med. Public Health. 2000; 31: 808-817Google Scholar). Knapp et al. (20Knapp B. Nau U. Hundt E. Kupper H.A. Mol. Biochem. Parasitol. 1991; 44: 1-13Google Scholar) have reported that a SERA homologue gene (SERP-H) is located adjacent to the SERA gene, although it does not contain a serine stretch, and that a 130-kDa polypeptide is expressed from the SERP-H gene during schizont stage and localizes in the parasitophorous vacuole. Fox and Bzik (3Fox B.A. Bzik D.J. Mol. Biochem. Parasitol. 1994; 68: 133-144Google Scholar) have shown that another SERA homologue (designated as SERA3 in the original paper) is located 1.8 kb upstream of the SERA gene and is transcribed at the trophozoite and schizont stages. Recently, the malaria genome project has revealed that these genes belong to the SERA multigene family, consisting of eight open reading frames clustered in tandem on chromosome 2 (21Gardner M.J. Tettelin H. Carucci D.J. Cummings L.M. Aravind L. Koonin E.V. Shallom S. Mason T. Yu K. Fujii C. Pederson J. Shen K. Jing J. Aston C. Lai Z. Schwartz D.C. Pertea M. Salzberg S. Zhou L. Sutton G.G. Clayton R. White O. Smith H.O. Fraser C.M. Hoffman S.L. et al.Science. 1998; 282: 1126-1132Google Scholar). The eight open reading frames on chromosome 2 are designated as SERA1 to SERA8 in the direction from centromere to telomere. Previously described SERA (SERP or p126), SERP-H and SERA3, correspond to SERA5 (PFB0340c), SERA6 (PFB0335c), and SERA4 (PFB0345c), respectively. A serine repeat is found only at the N-terminal region of SERA5. All members in the SERA multigene family contain a papain protease-like motif, and SERA1 to -5 contain a serine residue instead of a cysteine residue at the putative active nucleophile position, suggesting that they are serine proteases with a typical structure of cysteine protease (3Fox B.A. Bzik D.J. Mol. Biochem. Parasitol. 1994; 68: 133-144Google Scholar, 22Higgins D.G. McConnell D.J. Sharp P.M. Nature. 1989; 340: 604Google Scholar, 23Eakin A.E. Higaki J.N. Mckerrow J.H. Craik C.S. Nature. 1989; 342: 132Google Scholar, 24Mottram D.G. Coombs G.H. North M.J. Nature. 1989; 342: 132Google Scholar). The evasion and/or prevention of the protective host immune responses are critical for the successful survival of Plasmodiumparasites. Genetic polymorphism in a single locus gene or multigene family, frequently found in malaria vaccine candidate genes, may represent genetic backgrounds that function for parasite immune evasion mechanism. To see whether the SERA multigene family, as a vaccine candidate antigen, exhibits antigenic variation, we characterized the expression profile of each member in this family. The data obtained demonstrate that the SERA5 gene is predominantly expressed and co-expressed with adjacent SERA homologue genes (SERA3, -4, and -6) in every single parasite cell with similar localization in trophozoites and schizonts. Moreover, antibody level in an individual human serum against SERA5 N-terminal domain, but not those for other homologues, is correlated with in vitro parasite growth inhibition. FCR3 (25Kubata V.K. Eguchi N. Urade Y. Yamashita K. Mitamura T. Tai K. Hayaishi O. Horii T. J. Exp. Med. 1998; 188: 1197-1202Google Scholar), Honduras-1 (26Inselburg J. J. Parasitol. 1983; 69: 592-597Google Scholar), K1 (generous gift from Dr. Masatsugu Kimura), 3D7 (27Hanada K. Palacpac N.M.Q. Magistrado P.A. Kurokawa K. Rai G. Sakata D. Hara T. Horii T. Nishijima M. Mitamura T. J. Exp. Med. 2002; 195: 23-34Google Scholar), and Dd2 (28Wellems T.E. Panton L.J. Gluzman I.Y. do Rosario V.E. Gwadz R.W. Walker-Jonah A. Krogstad D.J. Nature. 1990; 345: 253-255Google Scholar) strains of P. falciparum were maintained in culture according to the methods mainly by Trager and Jansen (29Trager W. Jensen J.B. Science. 1976; 193: 673-675Google Scholar) and modified by Mitamura and co-workers (30Hanada K. Mitamura T. Fukasawa M. Magistrado P.A. Horii T. Nishijima M. Biochem. J. 2000; 346: 671-677Google Scholar, 31Mitamura T. Hanada K. Ko-Mitamura E.P. Nishijima M. Horii T. Parasitol. Int. 2000; 49: 219-229Google Scholar). Cultures were maintained in 5% O2 and 5% CO2 atmosphere with 3% type O erythrocyte (v/v) in the culture medium containing 10% heat-inactivated human serum. For large scale culture and growth inhibition assay with human serum, 5 mg/ml AlbuMax (Invitrogen) was used in place of 10% human serum. For synchronization, schizont-rich parasites were purified by 63% (v/v) Percoll (Amersham Biosciences) density centrifugation (32Tosta C.E. Sedegah M. Henderson D.C. Wedderburn N. Exp. Parasitol. 1980; 50: 7-15Google Scholar) and incubated within 4 h in fresh medium with 3% erythrocyte prior to 5% sorbitol treatment. Total RNA was isolated from 0.075% saponin-treated (Sigma) synchronized parasite cells of Honduras-1, FCR3, K1, Dd2, and 3D7 with TRIZOLTMreagent (Invitrogen). First strand cDNA was synthesized with the SuperscriptTM First-strand Synthesis System for RT-PCR (Invitrogen) using 20 ng of each total RNA. Target cDNAs were amplified by the following primer sets: SERA1/for (5′-AAATTCAGCAATTTGTATGAAATATCC-3′) and SERA1/rev (5′-AGAAATAGCATGTGGTTCATAACCTT-3′), SERA2/for (5′-GAAAAACCTGACACCACTACTAGGAT-3′) and SERA2/rev (5′-GCAGGTGCTATAAAATCATATTCATC-3′), SERA3/for (5′-GATATGTTTAAAGCAAATGAACATGG-3′) and SERA3/rev (5′-AAACTTTTAATGGGTTTGAACCTTCT-3′), SERA4/for (5′-AACTTAAAGCAACCAATAACATCCAT-3′) and SERA4/rev (5′-AAATGATATTCGCTAGATTCCTCATC-3′), SERA5/for (5′-CTTAGATAATTATGGGATGGGAAATG-3′) and SERA5/rev (5′-GTTGTATCAACATGTACGACACCTTT-3′), SERA6/for (5′-TTGTTAAAATCTCATTCTGACGAAAA-3′) and SERA6/rev (5′-CATCAGAATTTTCTTTGTCATCATTT3′), SERA7/for (5′-TAATTGTTCGGATAGAGATTCTGATG-3′) and SERA7/rev (5′-TTTTGTAGTCATACGTTGTCTTGGAC-3′), SERA8/for (5′-TACCTGAGAGGAAAATATTCAAACCT-3′) and SERA8/rev (5′-GTAAGCTGCTATAACAACACTCGAAG-3′). As an internal control, a primer set (MSP1/for (5′-TTCGTGCAAATGAATTAGACGTAC-3′) and MSP1/rev (5′-GGATCAGTAAATAAACTATCAATGT-3′)) that annealed to the conserved blocks 3 and 5 of the merozoite surface protein-1 (MSP-1) gene (33Tanabe K. Mackay M. Goman M. Scaife J.G. J. Mol. Biol. 1987; 195: 273-287Google Scholar) was mixed together with one of the SERA primer sets. All RNA preparations gave no PCR product when reverse transcriptase was omitted from the RT-PCR. The efficacies of PCR primers were confirmed by using genomic DNA (gDNA) as a template. gDNAs were isolated from saponin-treated parasite cells of Honduras-1, FCR3, and K1 with DNAZOLTMreagent (Invitrogen). The PCR cycle used was as follows: 91 °C (1 min 30 s) followed by appropriate cycles of 91 °C (30 s), 50 °C (30 s), and 58 °C (3 min). Total RNA from Honduras-1, FCR3, and K1 strains were fractionated on a 1.2% agarose/formaldehyde gel (1 μg/lane) and transferred onto Nytran membrane (Schleicher & Schuell). The membrane was probed with PCR products, which had been amplified by the same set of primers for RT-PCR and labeled with deoxycytidine 5′-triphosphate α-32P (250 μCi/mmol) (PerkinElmer Life Sciences), exposed to a Fuji Film BAS imaging plate, and analyzed with MacBAS 1500 (Fuji Film Co.). Real time quantitative PCR (34Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Google Scholar,35Gibson U.E. Heid C.A. Williams P.M. Genome Res. 1996; 6: 995-1001Google Scholar) was performed using the ABI PRISM 7700 (PerkinElmer Life Sciences), and results were analyzed with the accompanying software. A 50-μl mixture was formulated with first strand cDNA prepared above using TaqManTM PCR Core Reagent Kit (PerkinElmer Life Sciences), the corresponding primer sets, and the appropriate TaqMan probe. Primers and probe sets were as follows: SERA1/for (5′-AGTTGATATGTATGGACCATCAACA-3′), SERA1/rev (5′-ATGGTTTACCTTATCTTCTTGGGA-3′), and SERA1/probe (5′-TGTTCATCAGACGCATTAACCAATTTCA-3′); SERA2/for (5′-CCGCATCTGAGGCAGGA-3′), SERA2/rev (5′-ATCGGTTGATACAGGTAATGCTACA-3′), and SERA2/probe (5′-TCCTTGTTTCGTAATTTTTCCACCCGT-3′); SERA3/for (5′-TCTTACCAACAGAAGGAGATTATTCA-3′), SERA3/rev (5′-ATTTTGTTCTAATAATTTTGCATTTGC-3′), and SERA3/probe (5′-CTGGGCATGTTTCACCAACTTTACTTTG-3′); SERA4/for (5′-CCTCATCAAGCGGACAACAA-3′), SERA4/rev (5′-CTTCTGCCGGTGATGCTTCT-3′), and SERA4/probe (5′-CAACACAAGGACTATCACCAGCAACTGGAG-3′); SERA5/for (5′-TATTCTCTGAAAAGGAAGATAATGAAAACA-3′), SERA5/rev (5′-TGAAGTTCCTGCAGATTCTAATGC-3′), and SERA5/probe (5′-CCTGATCCTGCCGTATCTTGACCGAAT-3′); SERA6/for (5′-TGTAGCTAATTGTTCTAAGAGAAAACCTAT-3′), SERA6/rev (5′-AGGACAAGAATTACCTGCACTTGTA-3′), and SERA6/probe (5′-AAATTCTAATGGATTCGATCCTTCTTCACA-3′); SERA7/probe (5′-TCGTCGGATCGAATCCAGTTGAATTTCTAG-3′); SERA8/for (5′-TCTGTATTTGTTTCTATGGAAGTAACAGA-3′), SERA8/rev (5′-AATACTAAGGCATGATCCGGACTAT-3′), and SERA8/probe (5′-TCACAACTCATCATAACTTTTGTCCCATCA-3′); SERA9/for (5′-ACTGTTCATGGACAAAGTGGAGAA-3′), SERA9/rev (5′-ACAGCTCCTCTGTTCGAATCTTG-3′), and SERA9/probe (5′-TTCAACCTTCACAACTTCGATCTACCGCT-3′); MSP-1/for (5′-ATCCAAATCCTACTTGTAACGAAAATA-3′), MSP-1/rev (5′-TTCTTTCTGCTGCTACCTGAATC-3′), and MSP-1/probe (5′-TGGCATCTGCATCACATCCACC-3′). The first strand cDNA was added to the reaction mixture just prior to thermal cycling. The PCR cycle used was as follows: 50 °C (2 min) and 95 °C (10 min), followed by 60 cycles of 95 ° (15 s) and 60 °C (1 min). RT-PCR was used to prepare cDNA encoding a part of the N-terminal region of SERA3,SERA4, and SERA6 as described in the legend to Fig. 1. NdeI site and BamHI sites were introduced at the end of forward and reverse primers, respectively (restriction enzyme sites are underlined): SERA3/for (5′-GGAATTCCATATGACAACAGTGGACGAGAGTACC-3′ and SERA3/rev (5′-CGGGATCCAAATTTAAATGTTTGGTTTTTTCCAG-3′; SERA4/for (5′-GGAATTCCATATGACAACCGCCAGTACTACTCA-3′) and SERA4/rev (5′-CGGGATCCGAAATCAAATTTTTTTGTGTCATC-3′); SERA6/for (5′-GGAATTCCATATGGAAGGAAATAAAGTGACTGTGA-3′) and SERA6/rev (5′-CGGGATCCTAGTTTAAAATGATATCCTTCAGA-3′). The amplified fragment was digested with BamHI andNdeI and ligated to a BamHI- andNdeI-digested pET15b plasmid vector (Novagen). The resultant plasmid, pET-SE3N, pET-SE4N, or pET-SE6N, encodes the His tag (6 histidine residues) fused to its N-terminal domain of SERA3 (Thr67–Phe570), SERA4 (Thr67–Phe552), or SERA6 (Glu97–Leu765), respectively. The encoded fusion proteins were designated as His-SE3N, His-SE4N, and His-SE6N. The freshly transformed Escherichia coliBL21(DE3) cells with RIG plasmid (36Baca A.M. Hol W.G.J. Int. J. Parasitol. 2000; 30: 113-118Google Scholar) and either pET-SE3N, pET-SE4N, or pET-SE6N were grown in LB to a cell density of 1.0 × 108 cells/ml at 37 °C, and then isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 50 μg/ml. After incubation for an additional 3 h, cells were harvested and stored at −80° until use. Subsequent operations were carried out at 4 °C or on ice. The frozen cells expressing His-SE3N, His-SE4N, or His-SE6N protein were thawed and suspended in 5 cell paste volumes of buffer A (20 mmTris-HCl, pH 8.0, 0.5 m NaCl, 20 mm imidazole). The cells were disrupted by freezing and thawing, followed by repeated treatments with an ultrasonic disrupter (Tomy Seiko model UR-200P). The sonicated mixture was centrifuged at 10,000 rpm for 10 min, and guanidine HCl powder was directly dissolved into the supernatant at a final concentration of 6 m. Purifications of all three His-tagged fusion proteins (His-SE3N, His-SE4N, and His-SE6N) were performed with the same procedure provided from the Hi-Trap chelating column (Amersham Biosciences). The column (1-ml bed volume) was preloaded with 0.5 ml of 0.1 mNiSO4 to bind nickel ion and then equilibrated with 5 ml of buffer B (6 m guanidine HCl in buffer A). The 5–15 ml of cell lysate prepared above was applied onto the column. The proteins bound to the resin were further washed with 5 ml of buffer C (6m urea in buffer A) and then refolded with 10 ml of buffer A. Bound proteins were eluted with buffer D (20 mmTris-HCl, pH 8.0, 0.5 m NaCl, 500 mmimidazole). The eluted sample was applied again onto the column, and the whole purification procedure described above was repeated. The eluted fractions from second column chromatography were dialyzed against PBS, prior to thrombin protease treatment (10 units/1 mg of protein). The treated sample was loaded onto the equilibrated Hi-Trap chelating column bound with nickel ion, and flow-through fractions were collected. After removal of the His tag, each recombinant protein was designated as SE3N, SE4N, or SE6N. Each recombinant protein gave a single band with an expected molecular mass as follows: SE3N, 28 kDa; SE4N, 30 kDa; SE6N, 33 kDa. After they were concentrated to 1 mg/ml by Centriprep YM-10 (Millipore Corp.), each purified recombinant protein was used for the custom antibody preparation (Asahi Techno Glass). Purification of total IgG from each serum was performed with a HiTrap Protein G column (Amersham Biosciences) according to the methods described previously (7Pang X.L. Mitamura T. Horii T. Infect. Immun. 1999; 67: 1821-1827Google Scholar). Preparation of recombinant SE47′ protein and affinity-purified mouse and rabbit anti-SE47′ antibodies was previously described (10Sugiyama T. Suzue K. Okamoto M. Inselburg J. Tai K. Horii T. Vaccine. 1996; 14: 1069-1076Google Scholar). Recombinant protein of block 17 in merozoite surface protein-1 (rMSP-119) was prepared in the silkworm,Bombyx mori, as follows. Genomic DNA of the P. falciparum MAD 20 strain was used for PCR to obtain DNA fragments encoding signal sequence (Met 1–Leu32) and block 17 (Pro1571–Gly1686) of MSP-1 (31Mitamura T. Hanada K. Ko-Mitamura E.P. Nishijima M. Horii T. Parasitol. Int. 2000; 49: 219-229Google Scholar). The obtained DNA fragments were connected with spacer nucleotides, GGAATT (encoding Gly-Ile), and then ligated to plasmid pBm030 (37Maeda S. Kawai T. Obinata M. Fujiwara H. Horiuchi T. Saeki Y. Sato Y. Furusawa M. Nature. 1985; 315: 592-594Google Scholar). The constructed plasmid was co-transfected with a wild type of B. mori nuclear polyhedrosis virus into an insect cell line, BmN4 (Funakoshi), and the recombinant virus was purified by plaque assay (37Maeda S. Kawai T. Obinata M. Fujiwara H. Horiuchi T. Saeki Y. Sato Y. Furusawa M. Nature. 1985; 315: 592-594Google Scholar). The purified recombinant virus was injected into silk worms on the first day of the fifth larval instar (5 × 104plaque-forming units/worm). Hemolymph was collected 4 days later. An affinity purification column was prepared with Affi-Gel 10 (Bio-Rad) and MSP-1 block 17 specific monoclonal antibody 5.2 purchased from the American Type Culture Collection (Manassas, VA). With the affinity column, recombinant MSP-1 was purified from the hemolymph according to the manufacturer's instructions. 10 μg of purified rMSP-1 was obtained from 1 ml of the hemolymph. 100 μl of 1 μg/ml each recombinant protein (SE3N, SE4N, SE47′, SE6N, or MSP-119) was used as antigens to coat each well of a 96-well microtiter plate. The second antibody used was biotinylated goat IgG specific to human IgG (γ chain) (Vector Laboratories), and color development was conducted using Vectastain ABC kit (Vector Laboratories) with 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as a substrate. ELISA titers were determined by a cut-off absorbance of 0.2 at 412 nm with a microtiter plate reader (Titertek Multiskan MCC/340 MKII). For Western blot, Percoll-purified trophozoite and schizont stage parasites were lysed in SDS buffer and loaded onto 8% SDS-polyacrylamide gel. Membrane transfer, primary antibody binding, and horseradish peroxidase-conjugated secondary antibody binding, followed by color development with the DAB substrate kit (Funakoshi, Japan), were according to the methods described (9Li J. Matsuoka H. Mitamura T. Horii T. Mol. Biochem. Parasitol. 2002; 120: 177-186Google Scholar). Percoll-purified trophozoite- and schizont-infected erythrocytes were fixed with 4% paraformaldehyde in PBS on ice for 30 min, spread onto slides, and air-dried. To permeabilize cells, samples were treated with PBS containing 1% Triton X-100 (7Pang X.L. Mitamura T. Horii T. Infect. Immun. 1999; 67: 1821-1827Google Scholar). Slides were blocked with PBS containing 3% BSA (buffer E) for 1 h and subsequently reacted with the affinity-purified mouse SE47′-specific IgG and either rabbit α-SE3N serum, rabbit α-SE4N serum, or rabbit α-SE6N serum. All of the rabbit antisera used were diluted at 1:1000, and the concentration of the purified mouse IgG used was 1 μg/ml in buffer E. The slides reacted with two primary antibodies were washed five times with PBS and then incubated in buffer E containing 1000-fold diluted Cy3-conjugated sheep anti-rabbit IgG (Sigma), 100-fold diluted fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Sigma), and 1 μg/ml 4′,6′-diamidino-2-phenylindole (Sigma). After five washes with PBS, the slides were mounted with PermaFluorTM Aqueous Mounting Medium (ImmunonTM). Fluorescence microscopy was performed by using an Axioskop fluorescence microscope (Carl Zeiss). Images were recorded by an AxioCam MRm CCD camera (Carl Zeiss). Individual sera from Ugandans were collected from 40 healthy adults (age >18 years) living in Atopi Parish, a malaria holoendemic area, located 5 km west of Apac Town, 300 km north of Kampala. Blood samples were obtained with informed consent (and approval by the Uganda National Council for Science and Technology) by venipuncture and collected in Vacutainers containing EDTA. Serum samples were separated into fresh serum vials and stored at −20 °C. The parasite growth inhibition assay was performed in a 96-well microtiter plate with FCR3, Honduras-1, and K1 parasite strains. Individual Ugandan serum samples were added at 5% (v/v) to the parasite culture containing 3% erythrocyte with 0.3–0.5% trophozoite- and schizont-rich cells and incubated for 24 h. Japanese malaria naive serum was used as control. Parasitized erythrocytes were counted in Giemsa-stained thin smears, and the parasitemia was scored by counting over 5000 erythrocytes in a slide. The growth inhibition (%) is calculated by (A −B)/A × 100, where A andB are control parasitemia (%) and parasitemia from sample (%), respectively. Correlation coefficients (r) were calculated using Pearson's test for pairs of logarithm of ELISA titers to base 2 and the parasite growth inhibition (%). The pvalues under 0.05 are considered significant. Transcriptional activity of each gene belonging to the SERA multigene family on chromosome 2 was examined by RT-PCR (Fig.1). Three parasite strains representative of typical SERA5 allelic forms, Honduras-1, K1, and FCR3, as well as two standard strains, Dd2 and 3D7, were used as total RNA templates for RT-PCR. When 20 ng of total RNA was used for the reverse transcriptase reaction, the 40 cycles of PCR could yield all of the expected products corresponding to SERA1–8 genes (data not shown), demonstrating that SERA1–8 genes are active in transcription. To estimate relative transcriptional activities ofSERA1–8 genes in each five parasite strains, PCR cycles were reduced to 20. As an internal control, the conserved region of MSP-1, block 3–5, was amplified with corresponding specific primers. As shown in Fig. 2, it appeared thatSERA5 was predominantly transcribed among SERA family genes and that the activities were followed by the adjacent SERA4, -3, -6, and -7 without any significant difference among five parasite strains examined. The observed transcription profiles were further confirmed by Northern blot analysis of total RNA prepared from three representative parasite strains (Fig.3). Northern blot indicates that theSERA5 transcription is severalfold higher than the internal control, MSP-1 gene.Figure 3Northern blot of SERA1–8genes in three parasite strains. Northern blot analysis ofSERA1–8 genes was carried out with total RNA (1 μg/lane). RNA was prepared from Percoll-purified trophozoite- and schizont-infected erythrocyte of parasite strains K1 (K), Honduras-1 (H), and FCR3 (F). The blotted membrane was probed with each of the radiolabeled PCR products ofSERA1–8 genes and MSP-1 that were radiolabeled in a single tube to keep a specific radioactivity of both probes constant.View Large Image Figure ViewerDownload (PPT) Since we have obtained consistent results with semiquantitative RT-PCR and Northern blot experiments, we carried out real time PCR for more quantitative comparison of the transcriptions among the SERA multigene family. In this experiment, the ninth SERA homologue, which was on chromosome 9 through the genome data base search (available on the World Wide Web at www.PlasmodDB.org), was also included (SERA9). To avoid possible inaccuracies caused by hybridization efficiency of primers used in above described RT-PCR, we prepared the new primer sets for each SERA gene except forSERA7. The new primer sets were designed to amplify the 3′-proximal region of each gene, because oligo(dT)-primed cDNA was used for the real time PCR. Based on the reproducible results from three independent experiments, the transcription profiles ofSERA1–8 genes are conserved in all of the parasite strains examined (Fig. 4). These results were consistent with the previous experiments described above. However,SERA9 and MSP-1 gene transcriptions varied from experiment to experiment and in parasite strains as well (Fig. 4). The stage specificity of each gene expression may largely affect the expression profile, especially SERA9 and MSP-1 genes; therefore, total RNA prepared from the tightly synchronized parasite cells were subjected to real time quantitative PCR. Ring, early trophozoite, late trophozoite, and schizont stage parasites were harvested, respectively, at 8, 15, 29, and 34 h after reinvasion. Fig. 5 showed the amount of each SERA gene transcript relative to that of the SERA5 gene detected at the late trophozoite stage as 100%. All of the SERA genes were transcribed at late trophozoite and schizont stages but not at ring and early trophozoite stages. The transcription of the MSP-1 gene was mainly at schizont stage. SERA3, SERA4, SERA5, and SERA6 genes were significantly transcribed in all of the parasite strains examined. To analyze the protei
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