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• W2022745094 abstract "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), two were of mouse and four were of Plasmodium origin. The ESTs were further grouped based on their similarity with sequences in the NR and PlasmoDB databases (Fig. 1A). A high proportion (29%) of the ESTs had no similarity to proteins in the NR data base but had significant similarity to sequences in the Plasmodium genomic databases, indicating that these are novel Plasmodium genes expressed during ookinete development. Moreover, 18% of the ESTs had similarity only to the Plasmodium EST data base, not to any known protein sequences. These are presumed to represent genes expressed in blood stages or mature sporozoites, since all ESTs deposited to date in databases are from blood-stage parasites or from sporozoites. The remaining ESTs belonged to various functional classes, as illustrated in Fig. 1A. A complete tabulation of all genes is provided in Supplemental Table 1A.Table ICharacteristics of the enriched libraries The name of the library heads each of the two right columns. All entries (except for insert size) represent the number of sequences. The percentage of the total is given in parenthesis. Unique sequences were classified into unknown, Plasmodium, mosquito, and mouse according to the results of BlastN searches (e × 10-10 cutoff value) for each of the sequences. Most sequences with >55% (A+T) are likely to be of Plasmodium origin, whereas sequences with <55% (A+T) are likely to be of mosquito origin.OokineteEarly oocystAverage size of inserts380 bp221 bpTotal number of sequences457652Number of unique sequences175 (38%)346 (53%)Unique sequences with >55% (A+T)169 (97%)120 (35%) Unknown06 Plasmodium16988 Mosquito026Unique sequences with <55% (A+T)6 (3%)226 (65%) Unknown029 Plasmodium43 Mosquito0194 Mouse20 Open table in a new tab Putative Early Oocyst ESTs—The 652 sequence reads from the enriched early oocyst library could be grouped into 346 unique ESTs (Table I). Of the 120 presumed Plasmodium ESTs (>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), 45 encode putative mosquito proteins with similarity to structural proteins, chitin-binding proteins, apoptosis-related proteins, detoxification and antioxidants, stress-related proteins, serpins, transmembrane proteins, proteins involved in carbohydrate and ornithine metabolism, proteases and proteasomes, and proteins involved in transcription and translation (Fig. 1B, Supplemental Table 1B). An additional 64 had homology to Anopheles gambiae genomic sequences encoding predicted proteins. Moreover, 71 had homology only to A. gambiae genomic sequences for which no open reading frame is predicted, suggesting that these are novel mosquito genes. These results should facilitate the ongoing annotation efforts. Although a number of the parasite ESTs had matches to Plasmodium rRNA genes (Fig. 1, A and B), no EST was identified with homology to mosquito rRNA. Possibly, Plasmodium rRNAs were primed by oligo(dT) relatively efficiently during reverse transcription because of its high (A+T) content.Expression Profiles—Seven randomly selected transcripts from the ookinete library having no significant similarity to the NR or Plasmodium EST databases were selected for expression analysis. As discussed below the presumed function of some of the gene products became known after the experiments were initiated. Fig. 2A shows the results obtained with five of the seven ESTs. The mRNA for Pbs21, an abundant ookinete surface protein, served as a Plasmodium sexual stage loading control (16Vervenne R.A. Dirks R.W. Ramesar J. Waters A.P. Janse C.J. Mol. Biochem. Parasitol. 1994; 68: 259-266Crossref PubMed Scopus (41) Google Scholar). As expected, none of the clones hybridized to RNAs obtained from the non-infected guts (Fig. 2A). O934 has similarity to an Arabidopsis thaliana putative non-clathrin coat protein (accession number NM_119610) and to a P. falciparum predicted protein. O934 transcript abundance increased 2 h after a blood meal, relative to Pbs21 (Fig. 2A). Expression of O1222, an EST with similarity to the human microtubule-organizing protein centrin 1 (17Lee V.D. Huang B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11039-11043Crossref PubMed Scopus (116) Google Scholar), was similar to O934. Expression of O2189 and O1198 also increased at the 2-h time point, whereas O1238 expression paralleled that of P. berghei ribosomal protein gene (Fig. 2, A and B). The expression profiles of O2091 and O1044 were similar to that of the ribosomal protein gene (data not shown), indicating that these may not have a specific function during ookinete differentiation. Thus, four of the seven ESTs from the ookinete library are up-regulated during ookinete differentiation.Fig. 2Temporal patterns of gene expression of selected sequences. Total RNA (5 μg/lane) from blood-stage (bl) parasites or from midguts dissected at different times (2 h to 6 days (d)) after an infected or non-infected blood meal as indicated was fractionated by electrophoresis on agarose gels and blotted onto nylon membranes. The blots were hybridized with the probes indicated to the left of each panel. An A. gambiae mitochondrial rRNA probe was used for the mosquito loading control. A, expression profile of clones isolated from the ookinete subtraction library. Pbs21, a sexual stage- and ookinete-specific protein, was used as a control. O1238 shows similarity to an uncharacterized Plasmodium yoelii EST 94471827. B, expression profile of putative Plasmodium clones (>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 subtraction library. All panels in this figure are from Northern blots prepared with RNA from the same infection (90% infection prevalence, mean 50-70 oocysts/gut). Similar results were obtained with RNAs from three separate infections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The analysis of five putative Plasmodium genes (>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 (<55% A+T) from the early oocyst library are shown in Fig. 2C. Consistent with the tentative mosquito assignment, no signal is detected for any of the four sequences with blood stage parasite RNA and all were hybridized with RNA from mosquito guts that had ingested a non-infected blood meal. Interestingly, expression of E151, E2864, and E2998 is induced, whereas E2954 is repressed by the parasite (compare infected with non-infected). E2954 is similar to A. gambiae G12 (accession number Z22925). E2864, which has similarity to 1-Cys peroxiredoxin-like sequences, may function as an antioxidant enzyme to protect cells against oxidant-induced membrane damage (18Manevich Y. Sweitzer T. Pak J.H. Feinstein S.I. Muzykantov V. Fisher A.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11599-11604Crossref PubMed Scopus (183) Google Scholar).An Anopheles Effector Caspase Gene Is Induced during Ookinete Invasion—Recent work suggested that apoptosis is triggered in midgut cells that are invaded by ookinetes (19Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (296) Google Scholar, 20Zieler H. Dvorak J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11516-11521Crossref PubMed Scopus (116) Google Scholar). In agreement with these observations, a group of genes showing similarity to apoptosis-related proteins was identified among early oocyst library sequences (Fig. 1B and Supplemental Table 1B). These included three caspases and one gene containing a death domain. One of the caspases was further characterized. The isolated cDNA has an open reading frame of 289 amino acids, and the putative protein shares 40% identity (60% similarity) with Drosophila DECAY (accession number NP_477462) and 39% identity (54% similarity) with mammalian caspase-7 (accession number NP_203124) (Fig. 3A). As is the case for DECAY and vertebrate effector caspases, the putative Anopheles caspase lacks a long amino-terminal pro-domain and carries a QARCG sequence encompassing the putative catalytic Cys167 residue (Fig. 3A).Fig. 3Analysis of the ancaspase-7 gene.A, ClustalW alignment of the deduced amino acid sequences of Ancaspase-7 with Drosophila DECAY-1 (NP_477462) and human caspase-7 (NP_203124). Identical amino acids are shaded dark gray, and conserved amino acids are shaded light gray. The conserved active site QARCG is underlined. B, Northern blot of total RNA (5 μg/lane) from A. stephensi fed on non-infected or P. berghei-infected blood. The probe was a full-length ancaspase-7 cDNA. bl, RNA from infected mouse blood; 0h, RNA from guts dissected before a blood meal. The time (in days) after a non-infected or infected blood meal at which guts were dissected is also indicated. The Ancaspase-7 mRNA was about 3-fold more abundant in the infected 1d sample than in the non-infected 1d sample. An A. gambiae mitochondrial rRNA probe was used for the loading control. C, immunoblot of A. stephensi gut sheets 30 h after a non-infected or P. berghei-infected blood meal (98% infection prevalence, mean oocyst intensity 404/gut) incubated with Ancaspase-7 antibody, washed, and detected using horseradish peroxidase-linked anti-rabbit antibody. A band of 19 kDa is detectable only in infected gut sheets, indicating that Ancaspase-7 activated during parasite invasion. Mobility of molecular mass markers (in kDa) is indicated on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Basal levels of the Ancaspase-7 transcript are detected in mosquito guts before a blood meal with a little or no further accumulation after a non-infected blood meal (Fig. 3B). However, after an infected blood meal, transcript abundance remains unchanged during the first 8 h (data not shown) and increases about 3-fold at 1 day compared with non-infected blood fed guts, suggesting that this gene is transcriptionally activated by the parasite (Fig. 3B). To further analyze the mechanism of gene regulation, an anti-Ancaspase-7 antibody was raised and used for immunoblot experiments. The purified antibody recognized a protein of ∼33 kDa in gut sheets from mosquitoes fed a non-infected or a P. berghei-infected blood meal (Fig. 3C). In addition, a ∼19-kDa band was detected only in gut sheets from P. berghei-infected mosquitoes (Fig. 3C). This apparent proteolytic cleavage product appears to be due to parasite invasion in that the ∼19-kDa band was not detected either in guts from mosquitoes fed a non-infected blood meal (Fig. 3C) or in guts from mosquitoes fed a P. berghei-infected meal, dissected before the start of parasite invasion (15 h) or after invasion was completed (2 days) (data not shown). Moreover, detection of the 19-kDa band required high infection rates (mean oocyst intensity >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" @default.
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• W2022745094 title "Analysis of the Plasmodium and Anopheles Transcriptional Repertoire during Ookinete Development and Midgut Invasion" @default.
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