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

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

Validation of extracellular ligand–receptor interactions by Flow-TriCEPS

Abstract Objective The advent of ligand-based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time-consuming. Here, we present a methodology that provides a fast and cost-efficient alternative for candidate target verification on living cells. Results In the described methodology a ligand of interest (e.g. transferrin, epidermal growth factor or insulin) was conjugated to a linker (TriCEPS) that carries a biotin. To confirm ligand/receptor interactions, the ligand–TriCEPS conjugates were first added onto living cells and cells were subsequently labeled with a streptavidin-fluorophore and analyzed by flow cytometry (thus referred as Flow-TriCEPS). Flow-TriCEPS was also used to validate identified receptor candidates when combined with a siRNA knock down approach (i.e. reduction of expression levels). This approach is versatile as it can be applied for different classes of ligands (proteins, peptides, antibodies) and different cell lines. Moreover, the method is time-efficient since it takes advantage of the large variety of commercially available (and certified) siRNAs

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk

ARTD1 in myeloid cells controls the IL-12/18–IFN-γ axis in a model of sterile sepsis, chronic bacterial infection, and cancer

Mice deficient for ADP-ribosyltransferase diphteria toxin-like 1 (ARTD1) are protected against microbially induced inflammation. To address the contribution of ARTD1 to inflammation specifically in myeloid cells, we generated an Artd1ΔMyel mouse strain with conditional ARTD1 deficiency in myeloid lineages and examined the strain in three disease models. We found that ARTD1, but not its enzymatic activity, enhanced the transcriptional activation of distinct LPS-induced genes that included IL-12, TNF-α, and IL-6 in primary bone marrow-derived macrophages and LPS-induced IL-12/18-IFN-γ signaling in Artd1ΔMyel mice. The loss of Artd1 in myeloid cells also reduced the TH1 response to Helicobacter pylori and impaired immune control of the bacteria. Furthermore, Artd1ΔMyel mice failed to control tumor growth in a s.c. MC-38 model of colon cancer, which could be attributed to reduced TH1 and CD8 responses. Together, these data provide strong evidence for a cell-intrinsic role of ARTD1 in myeloid cells that is independent of its enzymatic activity and promotes type I immunity by promoting IL-12/18 expression

Synthetic TALE transcription factors targeting the <i>TEP1</i> promoter.

<p><b>a</b>. Schematic representation of the ~3Kb <i>TEP1</i> promoter (not to scale). Shown are potential NF-κB sites (GGGRNNYYCC) (light grey boxes) and potential TATA boxes (dark grey boxes). The minimal promoter (MP) shown to be active in cells is indicated by a black line. Numbers on the promoter indicate the distance from the ATG. White boxes represent the binding position of each designed TALE. <b>b</b>. Luciferase assay in S2 cells showing the effect of TALEs on the minimal <i>TEP1</i> promoter. Different TALE constructs all carrying a VP16 activation domain and placed under the <i>Drosophila actin5c</i> promoter were co-transfected into S2 cells together with a firefly luciferase reporter gene under control of the minimal <i>TEP1</i> promoter. A plasmid carrying <i>GFP</i> placed under the same promoter as TALEs was used as reference for basal promoter activity. The <i>TEP1</i> promoter was further induced by addition of heat killed <i>E</i>. <i>coli</i> to the cells. Luciferase activity was normalized to the basal promoter activity without TALEs. <b>c</b>. qRT-PCR to assess the level of <i>TALE</i> and <i>TEP1</i> mRNA in transgenic mosquitoes expressing <i>Vg-TAL</i>_<i>6</i> comparing functional TALE with VP16 activation domain (<i>Vg-TAL</i>_<i>6</i>^<i>vp16</i>) and control line expressing the same TALE without activation domain (<i>Vg-TAL</i>_<i>6</i>^<i>ΔAD</i>), with or without induction of the <i>Vg</i> promoter by a blood meal 24h prior to mRNA extraction. <b>d.</b> qRT-PCR of transgenic mosquitoes constitutively expressing <i>Lp-TAL</i>_<i>6</i> or <i>Lp-TAL</i>_<i>0</i> or both, showing the level of <i>TEP1</i> mRNA. Levels of mRNA in each sample are normalized to <i>wt</i> and to the housekeeping reference gene <i>RPL19</i>. <b>e</b> Western blot analysis of transgenic mosquitoes constitutively expressing <i>Lp-TAL</i>_<i>6</i> or <i>Lp-TAL</i>_<i>0</i> or both. Wild type mosquitoes are used as controls. Shown are the bands for the full length and the cleaved forms of TEP1 as well as PPO2 as loading control. 2 samples for each mosquito line are presented to show sample variation. <b>f</b>. Quantification by densitometry of the TEP1 bands in (<b>e</b>). <b>g</b>. <i>P</i>. <i>berghei</i> infection of <i>Vg-TAL</i>^<i>vp16</i> transgenic mosquitoes. <b>h.</b> <i>P</i>. <i>berghei</i> Infection of transgenic mosquitoes constitutively expressing <i>TAL</i>_<i>0</i>, <i>TAL</i>_<i>6</i> or both under the <i>Lp</i> promoter. In all infections, the empty docking line (wt^X1) serves as control. Live oocysts (green circles) in each midgut are counted 7 days after infection. Number of oocysts and infection data are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006113#ppat.1006113.s003" target="_blank">S3 Table</a>.</p

Transgenic expression of <i>TEP1r</i> under the <i>Vg</i> promoter.

<p><b>a</b>. Western blot analysis of hemolymph from <i>Plasmodium</i> infected wild type (<i>TEP1s</i>), mutant (<i>TEP1</i>^<i>Δct2</i>) and mutant mosquitoes ectopically expressing <i>TEP1r</i> (<i>TEP1</i>^<i>Δct2</i><i>;TEP1r;</i>) showing full (TEP1-full) and cleaved (TEP1-cut) TEP1 forms, LRIM1 and APL1C protein, 25 and 48h after infectious blood feeding. PPO2 is used as a loading control <b>b</b>. Western blot analysis of hemolymph from <i>Plasmodium</i> infected mosquitoes of the <i>TEP1</i>^<i>ΔT</i> mutant background (<i>TEP1</i>^<i>ΔT</i>), ectopically expressing <i>TEP1r</i> (<i>TEP1</i>^<i>ΔT</i><i>; TEP1r</i>) and wild type L3-5 mosquitoes expressing only the endogenous <i>TEP1r</i> allele (<i>TEP1r</i>). 3–4 day old mosquitoes were blood fed to induce the <i>Vg</i> promoter. Samples were collected 24h after blood meal. The membrane was probed with the indicated antibodies, including anti-Vg to confirm proper induction of the <i>Vg</i> promoter. Two independent samples were loaded for each condition to account for variability. <b>c.</b> Western blot analysis of hemolymph from control and <i>TEP1</i> mutant mosquitoes showing the levels of APL1C and LRIM1. PPO2 is used as a loading control. Note the specific reduction of the two LRR proteins only in <i>TEP1ΔT</i> mosquitoes in which TEP1 lacks a single threonin. <i>TEP1Δct1</i> and <i>TEP1Δct2</i> are two different mutants lacking the entire C-terminus [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006113#ppat.1006113.ref027" target="_blank">27</a>]. <b>d.</b> Western blot analysis of carcasses from mosquitoes 48h after blood feeding showing the levels of TEP1-full and -cut in the indicated genotypes. Two independent samples of each genotype are shown to represent variability. Actin (upper band of the doublet) used as loading control. <b>e</b>. Western blot analysis of hemolymph from mosquitoes injected with heat-killed <i>E</i>. <i>coli</i> 24h after blood feeding comparing TEP1 levels in injected and non-injected mosquitoes of the indicated genotypes. Note the absence of transgenic TEP1r cleavage and reduced LRIM1 in the <i>TEP1ΔT</i> mutation background regardless of bacterial injection. <i>PPO2</i> was used as a loading control.</p

Expression patterns of transgenic reporter lines.

<p>The <i>3xP3</i> promoter drives expression of transgenesis markers in eyes and nerve cells. <b>a</b>. Control <i>3xP3-RFP</i> transgenic <i>A</i>. <i>gambiae</i> larva, image is a merge of green and red channels. Red arrows point to RFP expression in nerve cells, used as transgenesis marker (tm). <b>b</b>. <i>TEP1-GFP</i>, <i>3xP3-RFP</i> larva. Arrows point to some of the lobes of the fat body expressing GFP. Note the fine architecture of thoracic fat body revealed by GFP expression. <b>c</b>. control wild type pupa, showing little auto fluorescence in the green channel. <b>d</b>. <i>TEP1-GFP</i>, <i>3xP3-RFP</i> pupa showing GFP expression in fat body (fb, white arrows) and RFP expression in the eye (red arrows, tm). <b>e-g.</b> Larva carrying both <i>ppo6-RFP</i>, <i>3xP3-YFP</i> and <i>TEP1-GFP</i>, <i>3xP3-RFP</i>. RFP is expressed in hemocytes (white arrows) while GFP is expressed in fat body cells. YFP and RFP transgenic markers (red arrows, tm) are expressed in the nervous system. Scale bar = 200 μm <b>h.</b> RFP and YFP expression in <i>ppo6-RFP</i>, <i>3xP3-YFP</i> pupa, showing RFP expression in hemocytes (hc). White arrows point to some RFP expressing hemocytes. Transgenesis YFP marker is expressed in the eye (red arrow, tm). <b>i</b>. Midgut of <i>TEP1-GFP</i>,<i>3xP3-RFP</i> female 24h after blood feeding, showing distinct GFP expression in proventriculus. (pv) <b>j</b>. Close-up on a proventriculus expressing GFP in TEP1-GFP gut. <b>k</b>. <i>LRIM1-GFP</i>,<i>OpIE2-pac</i> larva showing GFP expression in the fat body, alongside a control (wt) larva. <b>l</b>. Larva expressing <i>APL1C-GFP</i>, <i>3xP3-CFP</i> showing GFP expression in the fat body (white arrows) and CFP transgenesis marker expression (red arrows, tm) in the nervous system (delineated with a thin line) <b>m</b>. <i>APL1C-GFP</i>,<i>3xP3-CFP</i> pupa expressing GFP in the fat body (white arrows) and CFP transgenesis marker in the eyes (tm, red arrow). <b>n.</b> <i>TEP1-GFP</i> (1) and <i>APL1C-GFP</i> (3) adult females with intense reporter expression in the fat body, visible through the cuticle, alongside a control female (2). Images <b>a</b>-<b>h</b>, <b>j</b>-<b>m</b> were acquired on a Zeiss Axiovert 200M microscope using a 5x or 10x objective. Images <b>i</b> and <b>n</b> were acquired on a Nikon SMZ18 binocular fluorescence microscope. Scale bar = 500μm except for <b>d</b>-<b>h</b>, <b>l</b>-<b>m</b>: 200 μm.</p

TEP1 expression in fat body cells.

<p><b>a.</b> Dissected carcass of <i>TEP1-GFP</i>,<i>3xP3-RFP</i> and <i>Lp-RFP</i>, <i>3xP3-CFP</i> mosquito exposing the fat body that shows GFP and RFP expression. Merged image shows co-expression of <i>Lp</i> and <i>TEP1</i> reporters in the fat body. Scale bar = 200 μm. <b>b-c.</b> Stained carcasses of wild type (<b>b</b>) and mosquitoes treated with dsTEP1 (<i>TEP1KD</i> (<b>c</b>)) showing TEP1 antibody staining (green) in Lp-expressing fat body cells. Note reduction in TEP1 staining in <i>TEP1KD</i> carcasses. Dapi stained cell nuclei (blue). Shown are z-projections of confocal images, scale bar = 10μm. Images were collected on a Zeiss LSM 710 confocal microscope.</p

Binding of transgenically expressed TEP1 to ookinetes.

<p>TEP1 antibody staining (red) of dissected midguts 24 h after infection with GFP-expressing <i>P</i>. <i>berghei</i>. Shown are wild type mosquitoes expressing the <i>TEP1s</i> allele <i>(TEP1s)</i>, wild type L3-5 mosquitoes expressing the <i>TEP1r</i> allele (<i>TEP1r</i>), mosquitoes transgenically expressing <i>TEP1r</i> in a wild type <i>TEP1s</i> background (<i>TEP1s;Vg-TEP1r</i>), mutant <i>TEP1</i> mosquitoes (<i>TEP1^ΔT</i>), and mutant mosquitoes transgenically expressing <i>TEP1r</i> (<i>TEP1^ΔT;Vg-TEP1r</i>). Ookinetes express GFP (green) and nuclei are stained with Dapi (blue). Images are stacks of confocal z-sections, scale bar = 20 μm</p