Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
PLoS Pathog
2007 Dec 01;312:e192. doi: 10.1371/journal.ppat.0030192.
Show Gene links
Show Anatomy links
Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development.
Yoshida S
,
Shimada Y
,
Kondoh D
,
Kouzuma Y
,
Ghosh AK
,
Jacobs-Lorena M
,
Sinden RE
.
???displayArticle.abstract???
The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.
Figure 1. Hemolytic and Hemagglutination Activities of CEL-III toward Human, Mouse, and Rat Erythrocytes(A) Serial 2-fold dilutions of CEL-III were mixed with human, mouse, or rat erythrocytes in V-shaped microtiter plate wells. Samples were incubated in the absence (−) or presence (+) of 5% FBS. Hemolysis was examined visually after incubation for 1 h at room temperature.(B) Hemolytic activity toward human, mouse, and rat erythrocytes was expressed as the absorbance at 550 nm resulting from release of hemoglobin.(C) Serial 2-fold dilutions of CEL-III were mixed with human, mouse, or rat erythrocytes in V-shaped microtiter plate wells. Samples were incubated in the presence of 10% Dextran 4. Agglutination was examined visually after incubation for 1 h at room temperature.(D) CEL-III was added to mouse (panels 1 and 2) or rat erythrocytes (panels 3 and 4), and bound CEL-III was detected with FITC-labeled anti-mouse IgG following mouse anti-CEL-III antibody by fluorescence microscopy. Panels 1 and 3, phase contrast; panels 2 and 4, FITC. Scale bars are 10 μm.
Figure 2. Binding of CEL-III to Ookinetes Inhibits Parasite Development In Vitro(A) Binding of CEL-III to ookinetes. CEL-III was added to cultured ookinetes purified from P. berghei–infected mouse blood. Bound CEL-III was detected with FITC-labeled anti-mouse IgG following mouse anti-CEL-III antibody by fluorescence microscopy (panels 1 and 2) (CEL-III (+)). As a negative control, ookinetes were incubated with FITC-labeled anti-mouse IgG following mouse anti-CEL-III antibody without CEL-III (panels 3 and 4) (CEL-III (−)). Panels 1 and 3, phase contrast; panels 2 and 4, FITC. Arrows indicate cultured ookinetes. Scale bars are 10 μm.(B) Effect of CEL-III on ookinete development in vitro. P. berghei–infected mouse blood was cultured for ookinetes for 24 h at 19 °C. CEL-III was added at initiation of the culture at various concentrations. Data are expressed as number of ookinetes relative to medium alone (100%). Results are the mean of two independent experiments, and bars represent standard errors of the mean.
Figure 3. Structure of the CEL-III Gene and Its Expression in Transgenic Mosquitoes(A) Schematic diagram of the pMinos-EGFP-carboxypeptidaseP-CELIII-antryp1T construct used for A. stephensi germ line transformation. The construct consists of the D. melanogaster actin5c promoter (actinP), egfp selectable marker (egfp), and D. melanogaster hsp70 terminator sequence (hspT), the A. gambiae carboxypeptidase promoter sequence (agcpP) plus its signal sequence (SP), fused in-frame to the coding sequence of CEL-III without its signal sequence followed by the A. gambiae trypsin terminator sequence (trypT). The left (ML) and right (MR) arms of Minos are indicated by triangles.(B) Induction of CEL-III mRNA by a blood meal. Transgenic mosquitoes were allowed to feed on a non-infected mouse and 6 h later total RNA was extracted from midguts of engorged mosquitoes (Blood-fed). As a control, total RNA was extracted from midguts of sugar-fed mosquitoes (Sugar-fed). CEL-III mRNA level was examined using RT-PCR. PCR products of the endogenous carboxypeptidase gene and the S7 gene were used as inducible positive controls and quantitative controls of the different mRNA preparations, respectively. These PCR products were fractionated by electrophoresis then stained with ethidium bromide.(C) Hemolytic activity of midgut contents of transgenic mosquitoes. Transgenic (CEL-III) and non-transgenic (WT) mosquitoes were offered a serum meal by membrane feeding. Six h after the meal, the supernatants of midgut lysates were added to human erythrocytes. Hemolytic activity was determined by visual examination of lysis of erythrocytes as described in Figure 1A.
Figure 4. CEL-III Expression in Midguts of Transgenic MosquitoesTransgenic (CEL-III) and non-transgenic (WT) mosquitoes were allowed to feed on naïve mice. After 6 or 24 h, midguts of engorged mosquitoes were dissected and lysed, then electrophoresed on 8% SDS-PAGE. As a control, midguts of sugar-fed mosquitoes (S) were dissected and lysed. CEL-III expression level was examined by western blotting using mouse anti-CEL-III antiserum. Each lane contains protein lysates equivalent to two midguts. The source of protein is indicated at the top of each lane (6, 24 h). For quantitative estimation of CEL-III per midgut, native CEL-III isolated from C. echinata body fluid was analyzed by western blotting. The amount of native CEL-III (5, 25, 100 ng) is indicated at the top of each lane. Arrows indicate the positions of monomeric and oligomeric forms of CEL-III.
Figure 5. Hemolysis of Human Blood in Mosquito MidgutMosquitoes were allowed to feed on a human volunteer. Representative photomicrographs of engorged mosquito gut sections 24 h after a blood meal are shown (HE staining, ×40 magnification for [A and B], and ×1,000 magnification for [C and D]). Midgut of non-transgenic mosquitoes was filled with intact erythrocytes (A and C), with many spaces between erythrocytes. In contrast, no space is observed in the midgut of transgenic mosquitoes (B and D). Erythrocytes appear to be completely hemolyzed, and HE-stained lymphocytes cells are detectable (arrows). Scale bars in (A and B) and (C and D) are 500 μm and 10 μm, respectively.
Abraham,
Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements.
2005, Pubmed
Abraham,
Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements.
2005,
Pubmed
Arrighi,
Design and activity of antimicrobial peptides against sporogonic-stage parasites causing murine malarias.
2002,
Pubmed
Billker,
Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito.
1998,
Pubmed
Billker,
Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite.
2004,
Pubmed
Bremer,
Targeted induction of apoptosis for cancer therapy: current progress and prospects.
2006,
Pubmed
Collins,
Infectivity of the Santa Lucia (El Salvador) strain of Plasmodium falciparum to different anophelines.
1977,
Pubmed
Curtis,
Possible use of translocations to fix desirable genes in insect pest populations.
1968,
Pubmed
Edwards,
Rapid induction by a blood meal of a carboxypeptidase gene in the gut of the mosquito Anopheles gambiae.
1997,
Pubmed
Ghosh,
The journey of the malaria parasite in the mosquito: hopes for the new century.
2000,
Pubmed
Ghosh,
Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library.
2001,
Pubmed
Hatakeyama,
Purification and characterization of four Ca(2+)-dependent lectins from the marine invertebrate, Cucumaria echinata.
1994,
Pubmed
,
Echinobase
Hatakeyama,
Interaction of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata with the erythrocyte membrane.
1995,
Pubmed
,
Echinobase
Hatakeyama,
Oligomerization of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata induced by the binding of carbohydrate ligands.
1996,
Pubmed
,
Echinobase
Hatakeyama,
Carbohydrate-binding properties of the hemolytic lectin CEL-III from the holothuroidea Cucumaria echinata as analyzed using carbohydrate-coated microplate.
1997,
Pubmed
,
Echinobase
Hatakeyama,
Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata.
2004,
Pubmed
,
Echinobase
Ito,
Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite.
2002,
Pubmed
Kokoza,
Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti.
2000,
Pubmed
Kouzuma,
Characterization of functional domains of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.
2003,
Pubmed
,
Echinobase
Lensen,
Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes.
1997,
Pubmed
Lobo,
Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies.
1999,
Pubmed
Moreira,
Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development.
2004,
Pubmed
Moreira,
Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes.
2002,
Pubmed
Moreira,
Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes.
2000,
Pubmed
Nakano,
Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin.
1999,
Pubmed
,
Echinobase
Nirmala,
Engineering Plasmodium-refractory phenotypes in mosquitoes.
2003,
Pubmed
Oda,
Temperature- and pH-dependent cytotoxic effect of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata on various cell lines.
1997,
Pubmed
,
Echinobase
Ponnudurai,
Synchronization of Plasmodium falciparum gametocytes using an automated suspension culture system.
1986,
Pubmed
Ranawaka,
Characterization of the effector mechanisms of a transmission-blocking antibody upon differentiation of Plasmodium berghei gametocytes into ookinetes in vitro.
1994,
Pubmed
Riehle,
Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges.
2003,
Pubmed
Rodriguez,
Effect of a cecropin-like synthetic peptide (Shiva-3) on the sporogonic development of Plasmodium berghei.
1995,
Pubmed
Shahabuddin,
Plasmodium gallinaceum: differential killing of some mosquito stages of the parasite by insect defensin.
1998,
Pubmed
Sinden,
Gametocytes of Plasmodium falciparum: phagocytosis by leucocytes in vivo and in vitro.
1976,
Pubmed
Skavdis,
Conserved function of anopheles gambiae midgut-specific promoters in the fruitfly.
1996,
Pubmed
Uchida,
Crystal structure of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata: implications of domain structure for its membrane pore-formation mechanism.
2004,
Pubmed
,
Echinobase
Vaughan,
Sporogonic development of cultured Plasmodium falciparum in six species of laboratory-reared Anopheles mosquitoes.
1994,
Pubmed
Walliker,
Genetic analysis of the human malaria parasite Plasmodium falciparum.
1987,
Pubmed
Yoshida,
Robust salivary gland-specific transgene expression in Anopheles stephensi mosquito.
2006,
Pubmed
Yoshida,
A single-chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers transmission blockade in the mosquito midgut.
1999,
Pubmed
Yoshida,
Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes.
2001,
Pubmed
Zieler,
A snake venom phospholipase A(2) blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface.
2001,
Pubmed
de Lara Capurro,
Virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glands in Plasmodium gallinaceum-infected Aedes aegypti.
2000,
Pubmed
