Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites

Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines

Relative luciferase activity in <i>TEP1</i> and <i>Lp</i> knockdown mosquitoes infected with <i>glyc::GFP-LUC</i>.

<p>Mosquitoes were injected with dsRNA prior to infection with <i>glyc::GFP-LUC</i>. Surviving mosquitoes 18–21 dpi were freeze dried, ground and the luciferase activity was measured. The values are normalised to the values in control treatment (<i>dsLacZ</i>). Shown are the results of 3 independent experiments for each treatment expressed as means of duplicate or triplicate measurements; error bars represent the standard error of the mean. The dotted line marks the value 1, corresponding to the respective controls. Sample sizes in experiment 1: <i>dsLacZ</i> (n = 21), <i>dsTEP1</i> (n<b> = </b>17); experiment 2: <i>dsLacZ</i> (n = 19), <i>dsTEP1</i> (n<b> = </b>17); experiment 3: <i>dsLacZ</i> (n = 26), <i>dsTEP1</i> (n<b> = </b>11); experiment 4: <i>dsLacZ</i> (n = 21), <i>dsLp</i> (n<i> = </i>20); experiment 5: <i>dsLacZ</i> (n = 26), <i>dsLp</i> (n = 8); experiment 6: <i>dsLacZ</i> (n = 44), <i>dsLp</i> (n = 39).</p

Generation of transgenic parasites expressing <i>gfp-luciferase</i> under salivary gland-specific promoters.

<p>(A) Schematic representation of a strategy used to obtain transgenic parasites. The top panel shows the wild type locus of <i>p230p</i>; arrows indicate the primers used to identify an intact <i>p230p</i> locus. The middle panel shows the linearised plasmid containing the 5′ and 3′ <i>p230p</i> fragments used for recombination (orange), <i>tgdhfr/ts</i> flanked by the 5′ UTR and 3′ UTR of <i>pbdhfr</i> (<i>tgdhfr/ts</i> cassette, yellow), the salivary gland-specific promoter (magenta), the <i>GFP-luciferase</i> fusion gene (green and blue) and a second <i>pbdhfr</i> 3′ UTR (dark blue). Crossing-over event is illustrated by the two crosses. The bottom panel shows the resulting disrupted, transgenic locus of <i>p230p</i>. Arrows represent the primers used for the identification of the disrupted <i>p230p</i> locus or of <i>luciferase</i>. Blue blocks illustrate the probes used for the FIGE analysis. (B) Southern analysis of FIGE separated chromosome of cloned transgenic lines confirms correct integration of the construct into the <i>230p</i> locus on chromosome 3. Hybridisation with the 3′UTR <i>dhfr/ts</i> probe recognises the integrated construct in chromosome 3 and the endogenous <i>P. berghei dhfr/ts</i> gene on chromosome 7. Note the more intense signal of chromosome 3 in the transgenic lines resulting from the two 3′UTR <i>dhfr/ts</i> regions in the construct. (C) Diagnostic PCR analyses of cloned transgenic parasites, confirming the correct integration of the constructs.</p

Correlation between sporozoite numbers and luciferase activity in whole mosquitoes, and dissected midgut and salivary gland sporozoites of <i>uis3::GFP-LUC</i> and <i>glyc::GFP-LUC</i>.

<p>Mosquitoes were infected with either <i>uis3::GFP-LUC</i> (A and C) or <i>glyc::GFP-LUC</i> (B and D) and sporozoites extracted from salivary glands (sgs), midguts (mgs) or whole mosquitoes were collected 18–19 dpi. The sporozoites and whole mosquitoes were lysed and dilution series of cell extracts were generated and used to perform luciferase assays. Luciferase activity was measured and plotted as the value after subtraction of the baseline against the sporozoite number (RLU). Goodness of the linear curve fit is given as r². Graphs representing two independent experiments are shown. 10⁴ sgs correspond to 1.1 mosquito equivalent in (C) and 1.2 in (D). Outliers have been omitted from the curve fit (i.e. highest sgs concentrations for isolated sgs for (C) and whole mosquitoes (D)).</p

GFP detection in blood and mosquito stages by fluorescence analysis.

<p>(A) For blood stage images, blood of infected mice was diluted and stained with the blue nuclear dye Hoechst 33258. Ookinetes were cultured, pelleted and stained with Hoechst 33258 before imaging. To image oocysts and salivary gland sporozoites, mosquitoes were infected with the transgenic parasites. Midguts containing oocysts were dissected directly into PBS/Hoechst 33258 11–21 dpi; infected salivary glands were dissected into PBS/Hoechst 33258 16–21 dpi. Oocysts and sporozoites were stained with the nuclear dye for 15–30 min. The GFP was visualized using the GFP fluorescence channel, the nuclei using the UV channel. The scale bars represent 5 µm for all the blood stages, ookinetes and sporozoites and 10 µm for oocysts.</p

Luciferase detection in mosquito stages by immuno-fluorescence assay (IFA).

<p>Luciferase expression in mgs and sgs was detected by immunofluorescence in dissected fixed midguts and salivary glands (17–21 days post infection) using anti-firefly luciferase antibody. Nuclei were visualized with DAPI. The scale bars represent 20 µm.</p