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Analysis of the Plasmodium and Anopheles Transcriptional Repertoire during Ookinete Development and Midgut Invasion

2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês

10.1074/jbc.m307582200

1083-351X

Eappen G. Abraham, Shabana Islam, P. Srinivasan, Anil K. Ghosh, Jesús G. Valenzuela, José M. C. Ribeiro, Fotis C. Kafatos, George Dimopoulos, Marcelo Jacobs-­Lorena,

Insect Resistance and Genetics

Plasmodium, the causative agent of malaria, has to undergo sexual differentiation and development in anopheline mosquitoes for transmission to occur. To isolate genes specifically induced in both organisms during the early stages of Plasmodium differentiation in the mosquito, two cDNA libraries were constructed, one enriched for sequences expressed in differentiating Plasmodium berghei ookinetes and another enriched for sequences expressed in Anopheles stephensi guts containing invading ookinetes and early oocysts. Sequencing of 457 ookinete library clones and 652 early oocyst clones represented 175 and 346 unique expressed sequence tags, respectively. Nine of 13 Plasmodium and four of the five Anopheles novel expressed sequence tags analyzed on Northern blots were induced during ookinete differentiation and mosquito gut invasion. Ancaspase-7, an Anopheles effector caspase, is proteolytically activated during Plasmodium invasion of the midgut. WARP, a gene encoding a Plasmodium surface protein with a von Willebrand factor A-like adhesive domain, is expressed only in ookinetes and early oocysts. An anti-WARP polyclonal antibody strongly inhibits (70-92%) Plasmodium development in the mosquito, making it a candidate antigen for transmission blocking vaccines. The present results and those of an accompanying report (Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M. C., Dimopoulos G., Kafatos, F. C., Adams, J. H., and Jacobs-Lorena, M. (2004) J. Biol. Chem. 279, 5581-5587) provide the foundation for further analysis of Plasmodium differentiation in the mosquito and of mosquito responses to the parasite. Plasmodium, the causative agent of malaria, has to undergo sexual differentiation and development in anopheline mosquitoes for transmission to occur. To isolate genes specifically induced in both organisms during the early stages of Plasmodium differentiation in the mosquito, two cDNA libraries were constructed, one enriched for sequences expressed in differentiating Plasmodium berghei ookinetes and another enriched for sequences expressed in Anopheles stephensi guts containing invading ookinetes and early oocysts. Sequencing of 457 ookinete library clones and 652 early oocyst clones represented 175 and 346 unique expressed sequence tags, respectively. Nine of 13 Plasmodium and four of the five Anopheles novel expressed sequence tags analyzed on Northern blots were induced during ookinete differentiation and mosquito gut invasion. Ancaspase-7, an Anopheles effector caspase, is proteolytically activated during Plasmodium invasion of the midgut. WARP, a gene encoding a Plasmodium surface protein with a von Willebrand factor A-like adhesive domain, is expressed only in ookinetes and early oocysts. An anti-WARP polyclonal antibody strongly inhibits (70-92%) Plasmodium development in the mosquito, making it a candidate antigen for transmission blocking vaccines. The present results and those of an accompanying report (Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M. C., Dimopoulos G., Kafatos, F. C., Adams, J. H., and Jacobs-Lorena, M. (2004) J. Biol. Chem. 279, 5581-5587) provide the foundation for further analysis of Plasmodium differentiation in the mosquito and of mosquito responses to the parasite. Malaria, the deadliest of the human parasitic diseases, is transmitted exclusively by Anopheles mosquito vectors. Plasmodium, the causative agent of malaria, has to complete a complex developmental program in the mosquito for transmission to occur (2Ghosh A. Edwards M J. Jacobs-Lorena M. Parasitol. Today. 2000; 16: 196-201Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 3Sinden R.E. Sherman I.W. Malaria: Parasite Biology, Pathogenesis and Protection. American Society for Microbiology, Washington, D. C.1998: 25-49Google Scholar). Within minutes after a mosquito ingests an infected blood meal, gametocytes emerge from red blood cells and differentiate into male and female gametes. After fertilization, zygotes differentiate into ookinetes that move within the blood bolus. After crossing the peritrophic matrix and the midgut epithelium, the ookinetes lodge beneath the basal lamina, facing the hemocoel, and differentiate into oocysts. Each oocyst undergoes about 12 rounds of nuclear divisions to produce thousands of sporozoites that, upon oocyst maturation, are released into the hemocoel.Difficulties with the development of an effective malaria vaccine and the emergence of drug-resistant parasites make the search for alternative weapons to fight the disease a critical priority. Little is known about the molecular mechanisms that direct parasite development in its mosquito host. The molecular dissection of Plasmodium development in the mosquito may lead to new targets for malaria control. Of the ∼5000 predicted Plasmodium genes (4Hoffman S.L. Subramanian G.M. Collins F.H. Venter J.C. Nature. 2002; 415: 702-709Crossref PubMed Scopus (120) Google Scholar), only a few are known to be expressed specifically in ookinetes and oocysts (3Sinden R.E. Sherman I.W. Malaria: Parasite Biology, Pathogenesis and Protection. American Society for Microbiology, Washington, D. C.1998: 25-49Google Scholar). Proteome analysis of Plasmodium falciparum has identified a large number of proteins expressed in gametocytes and gametes (5Florens L. Washburn M.P. Raine J.D. Anthony R.M. Grainger M. Haynes J.D. Moch J.K. Muster N. Sacci J.B. Tabb D.L. Witney A.A. Wolters D. Wu Y. Gardner M.J. Holder A.A. Sinden R.E. Yates J.R. Carucci D.J. Nature. 2002; 419: 520-526Crossref PubMed Scopus (1086) Google Scholar, 6Lasonder E. Ishihama Y. Andersen J.S. Vermunt A.M. Pain A. Sauerwein R.W. Eling W.M. Hall N. Waters A.P. Stunnenberg H.G. Mann M. Nature. 2002; 419: 537-542Crossref PubMed Scopus (556) Google Scholar). Recent studies have begun to characterize the activation of mosquito innate immune responses during the course of Plasmodium development in the midgut (7Luckhart S. Vodovotz Y. Cui L. Rosenberg R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5700-5705Crossref PubMed Scopus (346) Google Scholar, 8Dimopoulos G. Richman A. Muller H.M. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11508-11513Crossref PubMed Scopus (321) Google Scholar, 9Dimopoulos G. Christophides G.K. Meister S. Schultz J. White K.P. Barillas-Mury C. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8814-8819Crossref PubMed Scopus (243) Google Scholar, 10Oduol F. Xu J. Niare O. Natarajan R. Vernick K.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11397-11402Crossref PubMed Scopus (101) Google Scholar, 11Tahar R. Boudin C. Thiery I. Bourgouin C. EMBO J. 2002; 21: 6673-6680Crossref PubMed Scopus (80) Google Scholar). With the long term goal of understanding the molecular mechanisms that drive Plasmodium differentiation, we constructed cDNA libraries enriched for Plasmodium and Anopheles transcripts induced during early parasite development in the mosquito. Numerous novel Plasmodium and mosquito transcripts were isolated from these libraries. This work led to initial insights as to how the mosquito uses induction of cell death genes as a possible defense mechanism and to the identification of a candidate parasite antigen for transmission blocking vaccine.EXPERIMENTAL PROCEDURESParasites and Mosquito Infection—See the accompanying report (1Srinivasan P. Abraham E.G. Ghosh A.K. Valenzuela J. Ribeiro J.M.C. Dimopoulos G. Kafatos F.C. Adams J.H. Jacobs-Lorena M. J. Biol. Chem. 2004; 279: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Ookinete Culture and Enrichment—Plasmodium berghei ookinetes were cultured in vitro and enriched as described (12Sinden R.E. Winger L. Carter E.H. Hartley R.H. Tirawanchai N. Davies C.S. Moore J. Sluiters J.F. Proc. R. Soc. Lond. B Biol. Sci. 1987; 230: 443-458Crossref PubMed Scopus (42) Google Scholar). A typical enrichment yielded 70-80% ookinetes containing 15-20% blood-stage parasites.Subtraction Library Construction—Total RNA was extracted using TRI reagent (Molecular Research Center, Inc.). Polyadenylated RNA was isolated using oligo(dT) (Stratagene) cellulose column chromatography. For enriched cDNA library construction, cDNA subtraction was carried out using the Clontech PCR select cDNA subtraction kit (catalog number K1804-1). Secondary PCR products were cloned into pGEMT-easy (Promega) and transformed into high efficiency DH5α Escherichia coli to yield the subtracted libraries. Two subtraction libraries, enriched ookinete and enriched early oocyst, were constructed (see "Results" for details).cDNA Cloning and Sequencing—Individual clone inserts from the subtracted libraries were amplified using T7 and SP6 primers and sequenced on a CEQ 2000 DNA sequencing instrument (Beckman Coulter, Inc.) (1Srinivasan P. Abraham E.G. Ghosh A.K. Valenzuela J. Ribeiro J.M.C. Dimopoulos G. Kafatos F.C. Adams J.H. Jacobs-Lorena M. J. Biol. Chem. 2004; 279: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The full-length cDNA for WARP 1The abbreviations used are: WARPvon Willebrand factor A (vWA) domain-related proteinTRAPthrombospondin-related adhesive proteinCTRPcircumsporozoite- and TRAP related protein. and ancaspase-7 were isolated by screening a separate cDNA library made with the λTriplEx™ vector (Clontech) using RNAs extracted from pooled midguts dissected 24, 36, and 52 h after an infectious blood meal.Clustering and Data Base Analysis—See the accompanying report (1Srinivasan P. Abraham E.G. Ghosh A.K. Valenzuela J. Ribeiro J.M.C. Dimopoulos G. Kafatos F.C. Adams J.H. Jacobs-Lorena M. J. Biol. Chem. 2004; 279: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Sequences with significant similarity on BlastX (BlastX, e ≥ 10-4) were grouped based on the function of the homologous protein. Sequences with no significant BlastX similarity were grouped based on their BlastN (BlastN e ≥ 10-10) similarity.Northern Analysis and Hybridization—Northern analysis of total RNA was done as previously reported (13Edwards M.J. Lemos F.J. Donnelly-Doman M. Jacobs-Lorena M. Insect Biochem. Mol. Biol. 1997; 27: 1063-1072Crossref PubMed Scopus (76) Google Scholar). A mosquito mitochondrial rRNA gene was used as a loading control (13Edwards M.J. Lemos F.J. Donnelly-Doman M. Jacobs-Lorena M. Insect Biochem. Mol. Biol. 1997; 27: 1063-1072Crossref PubMed Scopus (76) Google Scholar).Antibody Production, Immunoblotting, and Immunofluorescence— WARP was expressed as a glutathione S-transferase fusion protein (glutathione S-transferase fused to amino acids 45-303 of WARP) using the pGEX-4T1 expression vector (Amersham Biosciences) in E. coli strain BL21. The purified protein was used to raise anti-WARP antibody in a rabbit. Inhibition of oocyst formation and immunofluorescence assays were performed using anti-WARP IgG purified with the Immunopure A IgG purification kit (Pierce). Mosquito gut sheets were prepared by dissecting mosquito guts fed on infected or non-infected mice. Guts were opened longitudinally, and the resulting sheets were washed in PBS to remove blood. The sheets were fixed overnight in 4% paraformaldehyde at 4 °C, blocked for 2 h in PBS containing 4% bovine serum albumin, and incubated with WARP antibody (×1000 dilution). Antibody binding was detected with a fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody (Sigma).Full-length ancaspase-7-His6 was expressed using the pET-15b expression vector in E. coli (BL21). The purified protein was used to raise anti-ancaspase-7 antibody in a rabbit. Antibody was affinity-purified with immobilized ancaspase-7 protein using the AminoLink plus immobilization kit (Pierce, product #44894). For immunoblotting, 20 gut sheets (see above) from mosquitoes fed on infected or non-infected mice were suspended in 120 μlof1× Laemmli buffer containing 4 m urea and boiled for 10 min, and 12 μl were separated on 15% SDS-PAGE followed by a electrotransfer to a polyvinylidene fluoride membrane. A prestained protein ladder (Benchmark, Invitrogen) was used as molecular weight marker. The membrane was incubated with ancaspase-7 antibody (1:20,000), and the bound antibody was detected with a horseradish peroxidase-linked anti-rabbit immunoglobulin (Pierce, 1:30,000 dilution) by exposing the blots to x-ray films.Passive Immunization—To measure inhibition of oocyst formation by passive immunization, a batch of 30-40 female mosquitoes (control) were fed on a P. berghei-infected mouse for 20 min. Next, 1 mg of anti-WARP IgG (or pre-immune IgG as a control) was injected into the tail vein of the same mouse followed 20 min later by feeding of another batch of 30-40 mosquitoes (experimental). Fully engorged mosquitoes were selected, and the number of oocysts that formed in the gut of the surviving mosquitoes was counted 15 days later (14Ghosh A.K. Ribolla P E. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2001; 96: 13278-13281Crossref Scopus (151) Google Scholar).RESULTSSubtraction Libraries—To construct an enriched ookinete library, blood-stage parasite cDNAs were subtracted from a pool of ookinete cDNAs from four different developmental stages (6, 12, 18, and 24 h). The subtraction was expected to significantly reduce common cDNAs (e.g. housekeeping genes) and enrich for ookinete stage-specific cDNAs. Similarly, an enriched early oocyst library was constructed by subtracting midgut cDNAs from mosquitoes fed a non-infected blood meal plus blood-stage parasite cDNAs, from midgut cDNAs from mosquitoes fed a P. berghei-infected blood meal. This library was expected to be enriched for two classes of ESTs, ookinete- and early oocyst-specific ESTs and mosquito ESTs corresponding to genes induced by the parasite. To construct the enriched early oocyst library, guts were dissected at 24, 36, and 52 h after a non-infected or infected blood meal. Ookinete invasion of the midgut and the initial stages of ookinete differentiation into oocysts occurred during this period.To assess efficiency of the enrichment procedure, Southern blots of cDNAs before and after enrichment were hybridized with a mosquito actin probe. A ∼7-fold weaker signal was obtained with the subtracted cDNA compared with non-subtracted cDNA, indicating that the enrichment procedure was effective (see Supplemental Fig. 1). Enrichment was further confirmed by PCR analysis of the enriched cDNAs using circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP), an ookinete-specific gene (15Dessens J.T. Beetsma A.L. Dimopoulos G. Wengelnik K. Crisanti A. Kafatos F.C. Sinden R.E. EMBO J. 1999; 18: 6221-6227Crossref PubMed Scopus (230) Google Scholar). A ∼4-fold stronger signal was observed with the enriched cDNA template than with the non-enriched template (see Supplemental Fig. 1).Putative Ookinete ESTs—The 457 sequence reads from the enriched ookinete library could be grouped into 175 unique ESTs (Table I). Most of these (169 or 97%) had an (A+T) content higher than 55% (average 65%) and had homology with Plasmodium sequences in databases. This indicates that sequences with >55% (A+T) content have a high likelihood of being of Plasmodium origin (the library was constructed from in vitro transformed ookinetes, free of any mosquito tissues). Of the six (3%) ESTs with 55% (A+T) are likely to be of Plasmodium origin, whereas sequences with 55% (A+T)169 (97%)120 (35%) Unknown06 Plasmodium16988 Mosquito026Unique sequences with 55% A+T), 43 correspond to putative Plasmodium proteins with similarity to adhesive proteins, proteases and proteasomes, stress-related proteins, structural proteins, and proteins involved in transcription and translation (Fig. 1B; Supplemental Table 1B). Also, 12 ESTs corresponded to rRNA. An additional 19 ESTs had homology to Plasmodium EST data base (no presumed function) and are assumed to encode genes expressed in blood stages or mature sporozoites. Moreover, 10 showed homology only to Plasmodium genomic DNA but not to ESTs or other protein databases, suggesting that these are novel Plasmodium genes expressed during development in the mosquito. Finally, six sequences did not have any similarity upon BlastN (nucleotide-nucleotide) and BlastX (nucleotide query-protein) data base searches and were classified as unknown. We note that 21% (26/120) of the sequences with >55% (A+T) had homology to the Anopheles data base. Similarly, only 1% (3/226) of the sequences <55% (A+T) had homology to Plasmodium databases (Fig. 1B).Putative Mosquito ESTs—Of the 226 presumed mosquito unique ESTs ( 55% A+T) from the early oocyst subtraction library. E3108 had no significant matches to the Anopheles or Plasmodium databases, but further experiments indicated that it is of mosquito origin (E. G. Abraham and M. Jacobs-Lorena, unpublished observations). C, expression profile of putative mosquito clones ( 55% A+T) from the early oocyst library is shown in Fig. 2B. These ESTs were selected based on their similarity to Plasmodium genomic sequences except for E3108, which had no significant similarity with sequences of any data base. Each of the five randomly selected genes has a unique expression pattern. E125 may be expressed only in blood stage parasites. E351 appears to be induced early (2 h), whereas E182, E294, and E3108 are induced at progressively later times after infection. The absence of hybridization to RNA from non-infected guts is in agreement with the initial assignment of these clones as Plasmodium ESTs.Expression profiles of four putative mosquito genes ( 380 oocysts/gut). These results suggest that Ancaspase-7 activation in the mosquito gut is linked to ookinete invasion of the midgut and occurs at both transcriptional and post-transcriptional levels.The WARP Gene Plays an Essential Role in Early Parasite Development in the Mosquito—Subtraction library clones were analyzed with microarray experiments for P. berghei genes specific for mosquito stages of parasite development. 2Y. Dong, J. X. Xu, A. Kocan, E. G. Abraham, P. Srinivasan, S. Islam, B. Mimana, J. M.-C. Ribeiro, M. Jacobs-Lorena, F. C. Kafatos, and G. Dimopoulos, manuscript in preparation. One clone was further characterized. A full-length cDNA was isolated from a non-subtracted cDNA library prepared from mosquito guts dissected 24, 48, and 52 h after feeding with a P. berghei-infected blood meal. The cDNA was 1679 bp long and had a 303-amino acid-long open reading frame. While this work was in progress, the same gene was independently identified and named WARP (21Yuda M. Yano K. Tsuboi T. Torii M. Chinzei Y. Mol. Biochem. Parasitol. 2001; 116: 65-72Crossref PubMed Scopus (85) Google Scholar). The putative protein has an amino-terminal signal sequence and a vWA-like domain (Fig. 4A). This domain is found in Plasmodium proteins expressed in invasive stages, such as ookinete CTRP and sporozoite TRAP. A BlastX search identified CTRP and TRAP as the closest related proteins. However, WARP contains only one vWA domain (as opposed to six in CTRP) and does not have a predicted transmembrane domain (Fig. 4A).Fig. 4Structure and expression