Use of a Drosophila Model to Identify Genes Regulating Plasmodium Growth in the Mosquito

Department of Microbiology and Immunology, Stanford University, Stanford, California 94305, USA.
Genetics (Impact Factor: 5.96). 10/2008; 180(3):1671-8. DOI: 10.1534/genetics.108.089748
Source: PubMed


We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.

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Available from: Carolina Barillas-Mury, Mar 11, 2014
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    • "For example, the development of Plasmodium gallinaceum, a close relative of the human malarial parasite, has been modeled in Drosophila melanogaster by directly injecting Plasmodium ookinetes into the insect’s hemocoel [48]. Use of D. melanogaster genetic knock-outs has led to the discovery of genes in Anopheles gambiae that reduce Plasmodium growth [49]. This was possible despite the fact that Plasmodium does not stably infect the intestines of D. melanogaster [48], and no parasites of the phylum Apicomplexa, of which Plasmodium is a member, have ever been found in Drosophila. "
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    ABSTRACT: Microbial parasites of animals include bacteria, viruses, and various unicellular eukaryotes. Because of the difficulty in studying these microorganisms in both humans and disease vectors, laboratory models are commonly used for experimental analysis of host-parasite interactions. Drosophila is one such model that has made significant contributions to our knowledge of bacterial, fungal, and viral infections. Despite this, less is known about other potential parasites associated with natural Drosophila populations. Here, we surveyed sixteen Drosophila populations comprising thirteen species from four continents and Hawaii and found that they are associated with an extensive diversity of trypanosomatids (Euglenozoa, Kinetoplastea). Phylogenetic analysis finds that Drosophila-associated trypanosomatids are closely related to taxa that are responsible for various types of leishmaniases and more distantly related to the taxa responsible for human African trypanosomiasis and Chagas disease. We suggest that Drosophila may provide a powerful system for studying the interactions between trypanosomatids and their hosts.
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    • "Genetic and molecular approaches have shown that Drosophila is an effective model system for studying innate immunity, the mechanisms of which seem to be remarkably conserved from flies to mammals (Brandt et al., 2008; Hoffmann and Reichhart, 2002; Hultmark, 2003; Sibley et al., 2008; Spresser et al., 2008). The immune response in flies can be stimulated by injury when an insect is pricked in the thorax (Haghayeghi et al., 2010). "
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    ABSTRACT: Drosophila melanogaster is one of the most important genetic models and techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) are being employed extensively for deciphering the genetics basis of physiological functions. In RT-qPCR, the expression levels of target genes are estimated on the basis of endogenous controls. The purpose of these reference genes is to control for variations in RNA quantity and quality. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in D. melanogaster studies are lacking. We analyzed the expression levels of seven candidate reference genes (Actin, EF1, Mnf, Rps20, Rpl32, Tubulin and 18S) in flies that were injured, heat-stressed, or fed different diets. Statistical analyses of variation were determined using three established software programs for reference gene selection, geNorm, NormFinder and BestKeeper. Best-ranked references genes differed across the treatments. Normalization candidacy of the selected candidate reference genes was supported by an analysis of gene expression values obtained from microarray datasets available online. The differences between the experimental treatments suggest that assessing the stability of reference gene expression patterns, determining candidates and testing their suitability is required for each experimental investigation.
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    • "OXR1 was also identified in a recent genetic screen for genes that affect the survival of Plasmodium gallinaceum parasites injected into the Drosophila hemocele [17]. Furthermore, OXR1 silencing reduces Plasmodium berghei infection in the mosquito A. gambiae [17]. In this study, we examined the participation of A. gambiae OXR1 in protecting mosquitoes from oxidative stress and on the survival of early stages of P. berghei parasites in the mosquito. "
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    ABSTRACT: OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage. OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response. The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.
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