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 Mosquitoes
Transgenic (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 Midgut
Mosquitoes 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.
Table 1.
Rat–P. berghei Experiments
Table 2.
Mouse–P. berghei Experiments
Table 3.
Vectorial Competence of Transgenic Mosquitoes
Table 4.
Sporozoite Infectivity of Salivary Glands of Transgenic Mosquitoes
Table 5.
P. falciparum Gametocyte Membrane Feeding Experiments