Progeny from this cross were separated by fluorescent markers into two groups: the gRNA-RFP +/Cas9-GFP + transheterozygotes and the gRNA-RFP −/Cas9-GFP + siblings (referred to as siblings Fig. Heterozygous females of the gRNA-expressing line were crossed with homozygous males of the vasa-Cas9 strain ( Gantz et al., 2015 Hammond et al., 2016). 1 a) based on an established CRISPR/Cas9 system in A. To generate mosGILT-deficient mosquitoes, we first created a transgenic line expressing three guide RNAs (gRNAs) targeting the mosGILT gene ( Fig. Therefore, mosGILT is a mosquito factor that plays an important role in ovarian development and indirectly protects both human and rodent Plasmodium species from mosquito immunity. The refractory phenotype of mosGILT mutant mosquitoes is strongly associated with their reduced expression of Vg, which leads to a more efficient TEP1-mediated killing of ookinetes. This leads to a dramatic decrease in sporozoite load and infection of the SGs. These mutant mosquitoes have markedly lower numbers of oocysts, following infection by either rodent or human malaria parasites ( P. gambiae mosquitoes carrying mosaic mutations in the mosGILT gene show defects in ovarian development. To determine whether mosGILT influences Plasmodium infection in mosquitoes, we generated mosaic mosGILT-mutant mosquitoes using CRISPR/CRISPR–associated protein 9 (Cas9), a powerful technique that enables the study of mosquito gene function through efficient genome editing ( Dong et al., 2018). The inhibition of these motility components impairs the ability of the sporozoites to migrate to the liver and establish a normal hepatic infection. We showed that mosGILT can partially reduce the speed and cell traversal activity of both human and rodent Plasmodium sporozoites. Previously, we identified a protein, mosquito IFN-γ–inducible lysosomal thiol reductase (mosGILT), that binds to sporozoites in the saliva of infected Anopheles mosquitoes ( Schleicher et al., 2018). These mosquito immune factors constitute defense mechanisms that restrict or eliminate Plasmodium development.
In addition, positive regulators of this complement-like immunity have been identified, including two members of the leucine-rich repeat immune protein (LRIM) family, LRIM1 ( Fraiture et al., 2009 Osta et al., 2004 Povelones et al., 2009) and APL1C ( Fraiture et al., 2009 Povelones et al., 2009 Riehle et al., 2006, 2008), and the CLIP-domain serine protease homologue SPCLIP1 ( Dong et al., 2006 Povelones et al., 2013). Silencing of TEP1 enhances Plasmodium berghei oocyst numbers and abolishes parasite melanization in both the susceptible and refractory strains of Anopheles gambiae ( Blandin et al., 2004 Collins et al., 1986 Povelones et al., 2011).
The direct binding of TEP1 to the surface of ookinetes targets the parasites for destruction by lysis or melanization ( Blandin et al., 2004). Thioester-containing protein 1 (TEP1), a complement-like glycoprotein, is one of the key antiparasitic immune effectors in mosquitoes ( Blandin et al., 2004 Levashina et al., 2001). Mosquitoes mount potent immune responses against Plasmodium development between the ookinete and oocyst stages, causing substantial losses in parasite numbers ( Blandin and Levashina, 2004 Christophides et al., 2002 Dimopoulos et al., 1998, 2002 Sinden, 2002). MosGILT is a mosquito factor that is essential for ovarian development and indirectly protects both human and rodent Plasmodium species from mosquito immunity. Expression of vitellogenin (Vg), the major yolk protein that can reduce the parasite-killing efficiency of TEP1, is severely impaired in mutant mosquitoes. Following infection by either Plasmodium berghei or Plasmodium falciparum, mutant mosquitoes have significantly reduced oocyst numbers as a result of increased thioester-containing protein 1 (TEP1)–dependent parasite killing. Here, we show that female mosaic mosGILT mutant mosquitoes display defects in ovarian development and refractoriness to Plasmodium. To determine whether mosGILT influences Plasmodium within the mosquito, we generated Anopheles gambiae mosquitoes carrying mosaic mutations in the mosGILT gene using CRISPR/CRISPR associated protein 9 (Cas9). We previously showed that a protein in saliva from infected Anopheles, mosquito gamma-interferon–inducible lysosomal thiol reductase (mosGILT), inhibits the ability of sporozoites to traverse cells and readily establish infection of the vertebrate host. Plasmodium infection in Anopheles is influenced by mosquito-derived factors.
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