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• W2212653570 abstract "Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission. Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission. Malaria remains a global public health crisis, and Anopheline mosquitoes transmit malaria parasites, of which Plasmodium falciparum is the most dangerous (1Christophides G.K. Crisanti A. Vector and vector-borne disease research: need for coherence, vision and strategic planning.Pathog. Glob. Health. 2013; 107: 385-386Crossref PubMed Scopus (1) Google Scholar). Female mosquitoes need to feed on blood for egg production (2Zhao B. Kokoza V.A. Saha T.T. Wang S. Roy S. Raikhel A.S. Regulation of the gut-specific carboxypeptidase: a study using the binary Gal4/UAS system in the mosquito Aedes aegypti.Insect Biochem. Mol. Biol. 2014; 54: 1-10Crossref PubMed Scopus (14) Google Scholar). Feeding on Plasmodium-infected blood can result in the ingestion of male and female haploid gametocytes that fuse to form diploid ookinetes, a process that initiates Plasmodium infection of the mosquito vector. Ookinetes start invading mosquito midgut epithelial cells between 12 and 24 h after a blood meal feeding (3Dinglasan R.R. Devenport M. Florens L. Johnson J.R. McHugh C.A. Donnelly-Doman M. Carucci D.J. Yates 3rd, J.R. Jacobs-Lorena M. The Anopheles gambiae adult midgut peritrophic matrix proteome.Insect Biochem. Mol. Biol. 2009; 39: 125-134Crossref PubMed Scopus (100) Google Scholar). Un-fused gametocytes and ookinetes located near the periphery of the blood bolus in the mosquito midgut are susceptible to attacks by diverse digestive proteases and bacteria (4Abraham E.G. Jacobs-Lorena M. Mosquito midgut barriers to malaria parasite development.Insect Biochem. Mol. Biol. 2004; 34: 667-671Crossref PubMed Scopus (84) Google Scholar, 5Pumpuni C.B. Demaio J. Kent M. Davis J.R. Beier J.C. Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development.Am. J. Trop. Med. Hyg. 1996; 54: 214-218Crossref PubMed Scopus (215) Google Scholar6Gonzalez-Ceron L. Santillan F. Rodriguez M.H. Mendez D. Hernandez-Avila J.E. Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vivax sporogonic development.J. Med. Entomol. 2003; 40: 371-374Crossref PubMed Scopus (188) Google Scholar), whereas gametocytes and ookinetes inside the blood bolus are protected by blood. However, mature ookinetes must cross and exit the blood bolus to initiate invasion of the midgut epithelium. Blood feeding regulates mosquito gene expression (7Mead E.A. Li M. Tu Z. Zhu J. Translational regulation of Anopheles gambiae mRNAs in the midgut during Plasmodium falciparum infection.BMC Genomics. 2012; 13: 366Crossref PubMed Scopus (27) Google Scholar, 8Christophides G.K. Vlachou D. Kafatos F.C. Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae.Immunol. Rev. 2004; 198: 127-148Crossref PubMed Scopus (206) Google Scholar) and stimulates the formation of the peritrophic matrix (PM) 3The abbreviations used are: PMperitrophic matrixNi-NTAnickel-nitrilotriacetic acidFBNfibrinogen-likeIHCimmunohistochemicalIFAimmunofluorescence assayPBSIPBS supplemented with protease inhibitors. within the midgut (9Billingsley P.F. The midgut ultrastructure of hematophagous insects.Annu. Rev. Entomol. 1990; 35: 219-248Crossref Scopus (152) Google Scholar). The newly formed PM completely surrounds the ingested blood, separating the blood bolus from secretory midgut epithelial cells, providing a second physical barrier that limits the infection by pathogens co-ingested with the blood meal (10Shao L. Devenport M. Jacobs-Lorena M. The peritrophic matrix of hematophagous insects.Arch. Insect Biochem. Physiol. 2001; 47: 119-125Crossref PubMed Scopus (122) Google Scholar). The PM is composed of 3–13% chitin microfibrils and is embedded with many known (3Dinglasan R.R. Devenport M. Florens L. Johnson J.R. McHugh C.A. Donnelly-Doman M. Carucci D.J. Yates 3rd, J.R. Jacobs-Lorena M. The Anopheles gambiae adult midgut peritrophic matrix proteome.Insect Biochem. Mol. Biol. 2009; 39: 125-134Crossref PubMed Scopus (100) Google Scholar) and unknown proteins (11Toprak U. Baldwin D. Erlandson M. Gillott C. Hegedus D.D. Insect intestinal mucins and serine proteases associated with the peritrophic matrix from feeding, starved and moulting Mamestra configurata larvae.Insect Mol. Biol. 2010; 19: 163-175Crossref PubMed Scopus (34) Google Scholar). Notably, when the ookinetes are mature 12 h after the blood meal (9Billingsley P.F. The midgut ultrastructure of hematophagous insects.Annu. Rev. Entomol. 1990; 35: 219-248Crossref Scopus (152) Google Scholar), the PM also becomes visible in the midgut lumen. To infect mosquitoes, the motile ookinetes must sequentially attach to and penetrate the PM and the midgut epithelium (12Sinden R.E. Billingsley P.F. Plasmodium invasion of mosquito cells: hawk or dove?.Trends Parasitol. 2001; 17: 209-212Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). At present, the detailed molecular mechanisms involved in ookinete attachment to and penetration of the PM and the subsequent midgut invasion are unclear. peritrophic matrix nickel-nitrilotriacetic acid fibrinogen-like immunohistochemical immunofluorescence assay PBS supplemented with protease inhibitors. We recently identified a mosquito gene, fibrinogen-related protein 1 (FREP1), that is implicated in Plasmodium infection in mosquitoes (13Li J. Wang X. Zhang G. Githure J.I. Yan G. James A.A. Genome-block expression-assisted association studies discover malaria resistance genes in Anopheles gambiae.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 20675-20680Crossref PubMed Scopus (28) Google Scholar). Specific genetic polymorphisms in FREP1 are significantly associated with P. falciparum infection intensity levels in wild Anopheles gambiae populations from Kenya. The FREP1 is a member of the fibrinogen-related protein family (FREPs or FBNs) that contains a highly conserved C-terminal interacting fibrinogen-like (FBN) domain. In vertebrates, fibrinogen molecules usually associate as hexamers and are comprised of two sets of disulfide-bridged α, β, and γ chains that participate as a principal component of both cellular and fluid coagulation (14Mosesson M.W. Fibrinogen and fibrin structure and functions.J. Thromb. Haemost. 2005; 3: 1894-1904Crossref PubMed Scopus (1249) Google Scholar). In invertebrates, FREPs/FBNs are common pattern recognition receptors (15Zhang H. Wang L. Song L. Song X. Wang B. Mu C. Zhang Y. A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor.Fish Shellfish Immunol. 2009; 26: 56-64Crossref PubMed Scopus (76) Google Scholar, 16Fan C. Zhang S. Li L. Chao Y. Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.Mol. Immunol. 2008; 45: 3338-3346Crossref PubMed Scopus (62) Google Scholar) responsible mainly for initiating innate immune responses (17Wang X. Zhao Q. Christensen B.M. Identification and characterization of the fibrinogen-like domain of fibrinogen-related proteins in the mosquito, Anopheles gambiae, and the fruitfly, Drosophila melanogaster, genomes.BMC Genomics. 2005; 6: 114Crossref PubMed Scopus (67) Google Scholar). For instance, tachylectin proteins in the horseshoe crab regulate host defense by recognizing bacterial lipopolysaccharides (18Kawabata S. Iwanaga S. Role of lectins in the innate immunity of horseshoe crab.Dev. Comp. Immunol. 1999; 23: 391-400Crossref PubMed Scopus (87) Google Scholar). Previous work examining the role and function of FREP/FBN family members in Anopheles mosquitoes has shown that two family members, FBN9 and FBN30, appear to restrict Plasmodium infection of midgut epithelial cells. Silencing the expression of either FBN9 or FBN30 in mosquitoes increased Plasmodium infection (13Li J. Wang X. Zhang G. Githure J.I. Yan G. James A.A. Genome-block expression-assisted association studies discover malaria resistance genes in Anopheles gambiae.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 20675-20680Crossref PubMed Scopus (28) Google Scholar, 19Dong Y. Dimopoulos G. Anopheles fibrinogen-related proteins provide expanded pattern recognition capacity against bacteria and malaria parasites.J. Biol. Chem. 2009; 284: 9835-9844Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Here, we report the role and function of a third FREP/FBN family member, FREP1, during P. falciparum infection of Anopheles mosquitoes. Our genetic and biochemical assays reveal that FREP1 functions as a critical molecular anchor in the PM that facilitates Plasmodium invasion and infection of mosquito midguts. In contrast to FBN9 and FBN30 that inhibit Plasmodium infection, our results show that FREP1 is an important host factor that promotes infection of mosquito midguts by the major human pathogen, P. falciparum. Collectively, our data reveal new insight into Plasmodium-Anopheles interactions and identify FREP1 as a promising transmission-blocking target. An. gambiae G3 strain was maintained at 27 °C, 80% humidity with a 12-h day/night cycle. Larvae were reared on ground KOI fish food supplements (∼0.1 mg/larvae per day). Adult mosquitoes were maintained with 8% sucrose and fed on ketamine/xylazine-anesthetized mice for egg production. FREP1 was cloned using PCR with primers shown in Table 1 from an An. gambiae mosquito cDNA library. The PCR product and pQE30 plasmid were digested with restriction enzymes XmaI and HindIII. Products were ligated into a His6 expression plasmid and transformed into Escherichia coli JM109. The sequence-verified construct was subsequently transformed into E. coli M15 strain. One mm isopropyl 1-thio-β-d-galactopyranoside was used to induce gene expression in E. coli M15 transformants. Cells were lysed in buffer B (8 m urea, 100 mm NaH2PO4, 10 mm Tris·Cl, pH 8.0). Recombinant FREP1 was purified by a Ni-NTA column using a standard protocol (20QIAGEN The QIAexpressionist: A handbood for high-level expression and purification of 6XHis-tagged proteins. QIAGEN, Hilden, Germany2003: 1-128Google Scholar). Four ml of E. coli lysate was collected from 200 ml of culture, and the amount of recombinant FREP1 in E. coli lysate was about 4.0 mg according to bands on the SDS-PAGE comparing to standard markers. In the end, about 0.85 mg of purified FREP1 was obtained with a recovery rate of 21%. SDS-PAGE and Coomassie Brilliant Blue R-250 staining confirmed the purity of recombinant FREP1. Purified recombinant FREP1 was then used as an antigen to generate customized polyclonal antibody against FREP1 in rabbits (Thermo Fisher Scientific, Rockford, IL). Anti-FREP1 antibody was purified from antiserum by protein A-agarose affinity chromatography and suspended in PBS.TABLE 1PCR primersPurposePrimer namePrimer sequenceIn vitro synthesis of FREP1 dsRNAForward5′-TAATACGACTCACTATAGGAGCTCGAGGTGAAGCAGAG-3′Reverse5′-TAATACGACTCACTATAGGTTCTCCAGCCGGTTGTGT-3′Verify FREP1 mRNA expressionForward5′-ACAGGGCAAGTTCGAGAAGA-3′Reverse5′-AAGTCAACCGTACCGTCCTG-3′AgS7 geneForward5′-GGCGATCATCATCTACGTGC-3′Reverse5′-GTAGCTGCTGCAAACTTCGG-3′In vitro synthesis of GFP dsRNAForward5′-TAATACGACTCACTATAGGCAAGTTTGAAGGTGATACCC-3′Reverse5′-TAATACGACTCACTATAGGCTTTTCGTTGGGATCTTTCG-3′Clone FREP1 into pQE30Forward5′-ACCCGGGCACTGCCCTGAACGGTGCAG-3′Reverse5′-GGCAAGCTTCGCGAACGTCGGCACAGTC-3′Clone FREP1 into pIB/V5-HisForward5′-TCAAAGCTTCACCATGGTGAATTCATTCGTGTCG-3′Reverse5′-ACTCTAGATTACGCGAACGTCGGCACAGTCGTG-3′Reverse with His tagACTCTAGAGCGAACGTCGGCACAGTCGTG Open table in a new tab The complete FREP1 coding sequence was PCR amplified using primers shown in Table 1 from an adult An. gambiae cDNA library. Products were cloned into plasmid pIB/V5-His (Life Technologies) to generate pIB-FREP1 (encoding FREP1) and pIB-FREP1-His (encoding FREP1 with a His6 tag) expression constructs. Constructs were amplified in E. coli DH5α and then purified with endotoxin-free plasmid preparation kits (Sigma). Cabbage looper ovarian cell-derived High Five cells (21Wickham T.J. Davis T. Granados R.R. Shuler M.L. Wood H.A. Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.Biotechnol. Prog. 1992; 8: 391-396Crossref PubMed Scopus (235) Google Scholar) were used to express recombinant FREP1 according to the manufacturer's instructions (22Invitrogen InsectSelect BSD system: for stable expression of heterologous proteins in lepidopteran insect cell lines using pIB/V5-His. Invitrogen, Grand Island, NY2008: 1-31Google Scholar). In brief, endotoxin-free plasmids were mixed with Cellfectin® Reagent (1 μl of Cellfectin/μg of plasmids, Invitrogen) in 5–6 ml of Express Five® SFM medium (Invitrogen). Cells were cultured in 25-cm2 cell culture flasks (Greiner Bio-One, Monroe, NC) for 48 h at 27 °C. Medium and cells were separated by centrifugation at 300 × g for 5 min. The proteins in the medium were concentrated using Amicon® ULTRA-4 Centrifugal Filter Devices (Milipore, Billerica, MA) by centrifugation at 5,000 × g for 10 min. The His6-tagged FREP1 was purified using a Ni-NTA column using a standard protocol (20QIAGEN The QIAexpressionist: A handbood for high-level expression and purification of 6XHis-tagged proteins. QIAGEN, Hilden, Germany2003: 1-128Google Scholar). In total, 0.3 mg of insect cell-expressed recombinant FREP1 was purified from 50 ml of culture medium with an initial amount of ∼1.0 mg of FREP1 in culture supernatant (estimated based on SDS-PAGE). The yield was ∼30%. Using standard protocols (23Duong-Ly K.C. Gabelli S.B. Gel filtration chromatography (size exclusion chromatography) of proteins.Methods Enzymol. 2014; 541: 105-114Crossref PubMed Scopus (26) Google Scholar), 0.03–1 mg of purified High Five-expressed recombinant FREP1 in 0.1 ml of PBS with or without 0.1% Triton X-100 was subjected to fast protein liquid chromatography gel filtration (Bio-Rad). Sephadex G-200 columns (30 cm in length, 1.0 cm in diameter, 5 to 600 kDa resolution) were used with flow rates regulated to 0.2 ml/min. The void volume (V0) of this column is about 8.0 ml, and the total elution volume (Vt) is about 24.0 ml. Fractions of ∼0.1 ml were collected. Absorbance (A280) was monitored constantly, and ELISA was used to detect recombinant FREP1 in each fraction. Briefly, 50 μl of sample from each fraction was coated per well onto an immunoGrade microplate (Brand, Wertheim, Germany) and incubated overnight at 4 °C. Wells were blocked for 1.5 h with 100 μl of 1.0% BSA in PBS, followed by sequential incubation for 1 h with 100 μl of anti-FREP1 antibody (diluted 5,000-fold in PBS-0.2% BSA to a concentration of 0.1 μg/ml) and then 1 h in 100 μl of alkaline phosphatase-conjugated anti-rabbit IgG (Sigma, 1:20,000 dilution with PBS, 0.2% BSA). Wells were washed three times with PBST (0.2% Tween 20 in PBS) between incubations. Wells were developed with 100 μl of p-nitrophenyl phosphate solution (Sigma). When colors in wells were visible, the optical density absorbance at 405 nm was measured. A set of molecular weight standards was used to establish standard curves of molecular masses for the gel filtration columns (aprotinin (6.5 kDa), ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), canalbumin (75 kDa), and ferritin (440 kDa)). The elution (retention) volumes were measured to be 21.05, 18.34, 16.91, 13.65, and 9.38 ml, respectively. The linear regression equation of standard curve based on these standards was y = −6.32x + 44.86, where x is log10 transformed molecular mass in daltons and y is elution volume in milliliters. The correlation coefficient between x and y was 0.99. Midguts from 3–5-day-old naive and blood-fed female mosquitoes were dissected in PBS supplemented with protease inhibitors (Thermo Scientific, Rockford, IL). Tissues were embedded in optimal cutting temperature compound and immediately frozen in liquid nitrogen. Frozen midguts were sectioned (8–10 μm), mounted on super frost plus slides (positively charged), air dried for 30 min at room temperature, fixed with 4% paraformaldehyde in PBS for 20 min, and stored at −20 °C until use. Prior to staining, sections were re-hydrated in Tris-buffered saline (50 mm Tris, 150 mm NaCl, pH 7.6) with 0.05% Tween 20 (TBST) for 10 min. Sections were blocked with 5% dry milk in TBST for 30 min, and then incubated for 2 h with 2.5 μg/ml of purified rabbit anti-FREP1 antibodies diluted in blocking solution. Control sections were incubated with blocking solution containing preimmune rabbit antibodies. Samples were washed 3 times for 5 min with TBST and then incubated for 30 min with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Sigma) diluted 1:20,000 in blocking solution. Slides were then washed 3 times in TBST and developed with BCIP/NBT Chromogenic solution (Sigma) for 10–20 min. Sections were examined under a microscope at ×4 and 40 magnification. Photoshop (version CC 2014) software was used to measure the “gray values” of an area of interest. The pixel intensity was obtained by subtracting the gray value from a constant value of 255. For IHC staining images of mosquito midguts, three different rectangles (0.01 square inches each, chosen randomly) within PM were selected with the “Rectangular Marquee Tool.” After clicking “Record Measurements,” the mean gray value of the region of interest was obtained. The average of three measured gray values (mean) from three selected areas was used to represent a value of an experimental image. The same procedure was conducted outside of the PM to establish gray values for background signals. The pixel intensity value of an area was obtained by subtracting the measured gray values of the area within PM from the background gray values. The measurements from three slices were used to calculate the mean ± S.D. The same procedure was conducted to obtain the pixel intensity of control groups. To measure the average pixel intensity on a Western blot, we conducted the same procedure as described above except selecting target bands from experimental groups and control groups, and the areas from experimental groups were the same as the areas from control groups. The pixel intensity of the background was calculated from regions adjacent to the bands. Three replicates of the Western blot that were conducted exactly the same were used to calculate the mean pixel intensity and standard deviations. Quantification of pixel intensity (gray values) was independently validated by both measuring pixel intensity as a function of exposure time and by using other software (such as Microsoft PowerPoint) to change the brightness and/or contrast of an image. Calculations were consistent across multiple software applications. P. falciparum parasites (NF54 strain from MR4) were propagated in O+ fresh human blood (4% red blood cells (RBC), 0.25–0.5% parasitemia). Cultures were maintained in 6-well plates (Corning Inc., Costar) with 5.0 ml of complete RPMI 1640 medium supplemented with 10% heat-inactivated human AB-type serum (Interstate blood bank, Memphis, TN) and 12.5 μg/ml of hypoxanthine. The plates were maintained under 37 °C in a candle jar (24Jensen J.B. Trager W. Plasmodium falciparum in culture: use of outdated erthrocytes and description of the candle jar method.J. Parasitol. 1977; 63: 883-886Crossref PubMed Scopus (258) Google Scholar) and the medium was replaced daily until days 15–17. The parasitemia or gametocytemia was checked every other day by Giemsa staining of thin blood smears. For ookinete enrichment, P. falciparum cultures harboring stage V gametocytes were diluted 10-fold in complete RPMI 1640 without sodium bicarbonate and incubated at room temperature for 24 h as described (25Beetsma A.L. van de Wiel T.J. Sauerwein R.W. Eling W.M. Plasmodium berghei ANKA: purification of large numbers of infectious gametocytes.Exp. Parasitol. 1998; 88: 69-72Crossref PubMed Scopus (67) Google Scholar). P. falciparum culturing and infection experiments were conducted in the biosafety level 2 (BSL2) laboratory at the University of Oklahoma. Standard IFA was performed as described previously (26Korochkina S. Barreau C. Pradel G. Jeffery E. Li J. Natarajan R. Shabanowitz J. Hunt D. Frevert U. Vernick K.D. A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands.Cell. Microbiol. 2006; 8: 163-175Crossref PubMed Scopus (69) Google Scholar). In brief, P. falciparum cultures were deposited on coverslips (Fisher Scientific). Cells and parasites were immediately fixed in 4% paraformaldehyde in PBS at room temperature for 30 min, which preserved intact (non-permeabilized) cell membranes. Cells were then sequentially incubated with 100 mm glycine in PBS for 20 min, 0.2% bovine serum albumin (BSA) in PBS for 90 min, High Five cell-expressed FREP1 (10 μg/ml) in 0.2% BSA-PBS for 2 h, enhancer (Alexa Fluor® 594 goat anti-mouse SFX kit, Invitrogen) for 30 min, 5 μg/ml of anti-FREP1 antibody in PBS containing 0.2% BSA for 1 h, and 2 μg/ml of secondary antibody (Alexa Fluor® 594 goat anti-rabbit antibody, in PBS containing 0.2% BSA, Invitrogen) for 30 min in the dark. Between each incubation, the cells were washed 3 times for 3 min in PBS containing 0.2% BSA. Coverslips were rinsed in distilled water for 20 s and mounted on glass slides using 50 μl of Vectashield mounting media (Vector Laboratories, Burlingame, CA). Cell staining was examined using a Nikon Eclipse Ti-S fluorescence microscope. The fluoresence (pixel) intensity was measured as described above. The fluoresence intensity of a target region of an image was calculated by substracting the mean background fluoresence intensity (gray values) from the mean target fluoresence intensity (gray values). The fluoresence intensity values from three independent experiments or three controls were measured to calculate the mean ± S.D. Three to 5-day-old mosquitoes were fed on anesthetized mice and then maintained with 8% sugar. About 18 h after the blood meal, the engorged mosquitoes were dissected in 1× PBS supplemented with protease inhibitors (PBSI) (Pierce Protease Inhibitor Mini Tablets, EDTA-free, Thermo Scientific, Rockford, IL). The blood bolus in each midgut was removed manually. The dissected midguts were washed three times with PBSI. Each replicate contained at least 10 midguts. The same number of unfed (control) mosquito midguts was examined in parallel. Insect cell-expressed recombinant FREP1 (0.75 μg in 100 μl of PBSI) was incubated with experimental and control midguts for 1.5 h. An equivalent amount (0.75 μg in 100 μl of PBSI) of BSA was used as an additional control. The midguts were then washed three times with PBSI, and shredded with a micro-pestle in PBSI containing 0.5% Tween 20 in 1.5-ml tubes. The insoluble materials were removed by centrifugation at 8,000 × g for 2 min. Supernatants (100 μl) were added into immunoGrade microplates and the plates were incubated overnight at 4 °C. ELISA was used to quantify the relative amount of FREP1 in each reaction. FREP1 was cloned from an An. gambiae mosquito cDNA library as described in our previous work (13Li J. Wang X. Zhang G. Githure J.I. Yan G. James A.A. Genome-block expression-assisted association studies discover malaria resistance genes in Anopheles gambiae.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 20675-20680Crossref PubMed Scopus (28) Google Scholar). Briefly, nested PCR using the primers listed in Table 1 was used to generate a DNA template for synthesis of double-stranded RNA (dsRNA) using the in vitro transcription system T7 Megascript (Amibon, TX). The non-mosquito, negative control sequence was amplified from the Aequoria green fluorescent protein (GFP) gene using the primers listed in Table 1. dsRNA was synthesized from these gene fragments using the in vitro transcription system T7 Megascript (Amibon, TX). The FREP1 targeting construct spans a unique sequence from nucleotide 791 to 1,268, corresponding to amino acids 263 to 423 of FREP1. Blast analyses confirmed that no other genes in An. gambiae share significant DNA sequence identity with the targeted sequence of FREP1. The dsRNA was purified with a Qiagen RNA purification kit. Approximately 207 ng of dsRNA in a 69-nl solution was injected into the hemocoel of each cold-anesthetized, 1-day-old An. gambiae G3 female mosquitoes. Approximately 100 mosquitoes per treatment were used for the RNAi knockdown experiments. Thirty-six hours after dsRNA injection, the treated mosquitoes were fed with P. falciparum-infected blood containing 0.2% gametocytes in standard membrane feeding assays (27Goodman A.L. Blagborough A.M. Biswas S. Wu Y. Hill A.V. Sinden R.E. Draper S.J. A viral vectored prime-boost immunization regime targeting the malaria Pfs25 antigen induces transmission-blocking activity.PloS One. 2011; 6: e29428Crossref PubMed Scopus (47) Google Scholar). Seven days post-infection and feeding, treated mosquitoes were dissected in PBS and oocysts numbers were counted using light microscopy after staining with 0.1% mercury dibromofluorescein disodium salt in PBS. In the negative control groups, ∼100 mosquitoes injected with dsRNA targeting GFP were exposed to the same parasite cultures, and were processed identically to the experimental groups. The transcript knockdown efficiency was confirmed by the quantitative RT-PCR in five treated mosquitoes that were taken randomly 12 h after infection. In parallel, reductions in FREP1 in mosquito midguts were confirmed using IHC assays as described above, except replacing secondary reagents with Alexa Fluor® 594-conjugated goat anti-rabbit antibody. Fluorescent staining intensity was quantified as described above. P. falciparum-infected blood cultures containing mature stage V gametocytes were diluted with the fresh O+ type human blood to get the 0.2% final concentration of stage V gametocytes. An equal volume of heat-inactivated (65 °C for 15 min) AB-type human serum was added. Identical volumes of PBS (1/10 volume of blood) containing different concentrations of rabbit polyclonal anti-FREP1 antibody (5, 4, 2, 1, and 0.5 mg/ml) were added to the gametocyte cultures. Artificial membrane feeding was conducted using 3-day-old female naive mosquitoes. After feeding for 15 min, engorged mosquitoes were separated and maintained with 8% sugar in a BSL2 insectary (28 °C, 12-h light/dark cycle, 80% humidity) at the University of Oklahoma. Seven days after infection, midguts were dissected, stained with 0.1% mercurochrome, and examined using light microscopy to count the number of oocysts. Equivalent amounts of purified preimmune rabbit antibody (Thermo Scientific) were used as controls. To begin to understand the basic biochemical characteristics of FREP1, we first examined its functional domains. According to our previous genome annotation (28Li J. Ribeiro J.M. Yan G. Allelic gene structure variations in Anopheles gambiae mosquitoes.PLoS One. 2010; 5: e10699Crossref PubMed Scopus (7) Google Scholar), full-length FREP1 comprises 738 amino acids, including a 22-amino acid signal peptide at the N terminus, three coiled-coil regions, and a conserved ∼200-amino acid FBN domain at the C terminus (Fig. 1A). All six cysteine amino acid residues are within the C-terminal FBN domain. To generate anti-FRE" @default.
• W2212653570 created "2016-06-24" @default.
• W2212653570 creator A5015206671 @default.
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• W2212653570 date "2015-07-01" @default.
• W2212653570 modified "2023-10-15" @default.
• W2212653570 title "Anopheles Midgut FREP1 Mediates Plasmodium Invasion" @default.
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