The role of NF-κB factor REL2 in the Aedes aegypti immune response

Abstract

Mosquitoes transmit numerous diseases that continue to be an enormous burden on public health worldwide. Transgenic mosquitoes impervious to vector-borne pathogens, in concert with vector control and drug and vaccine development, comprise an arsenal of means anticipated to defeat mosquito-spread diseases in the future. Mosquito transgenesis allows tissue-specific manipulation of their major immune pathways and enhances the ability to study mosquito-pathogen interactions. Here, we report the generation of two independent transgenic strains of Aedes aegypti overexpressing the NF-κB transcriptional factor REL2, a homologue of Drosophila Relish, which is shown to be under the control of the vitellogenin promoter in the mosquito fat body after a blood meal. We show that this REL2 overexpression in the fat body results in transcriptional activation of Defensins A, C, and D, and Cecropins A and N, as well as translation and secretion of Defensin A protein into the hemolymph. We also demonstrate that induction of REL2 results in the increased resistance of the mosquito to tested Gram-negative and Gram-positive bacteria. Importantly, induction of transgenic REL2 leads to the significant decrease in susceptibility of Ae. aegypti to Plasmodium gallinaceum infection. Consistently, RNAi knockdown of REL2 in wild-type mosquitoes results in a delay in Defensin A and Cecropin A expression in response to infection and in increased susceptibility to both bacteria and P. gallinaceum. Moreover, our transgenic assays demonstrate that the N-terminus of the mosquito REL2, which includes the His/Gln-rich and Serine-rich regions, plays a role in its transactivation properties.

The bacteriumWolbachiaexploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquitoAedes aegypti

“…To validate the activation of the transgenes in these lines, we measured the expression of REL1 and REL2 in mosquito fat bodies 7 days post eclosion and 24 h post blood meal (PBM). Consistent with previous reports Antonova et al, 2009), the expression of the transgenes did not change before a blood meal, but were induced markedly at 24 h post blood meal (Supplementary Figure S5), indicating that Wolbachia did not change the regulation patterns of transgene expression. We then compared wAlbB load in both the ovaries and the remaining carcass tissue of these mosquitoes at day 15 post blood meal.…”

“…To further test the effect of boosting the Toll and IMD pathways on Wolbachia in A. aegypti, we took advantage of transgenic lines that overexpress REL1 (REL1+) and REL2 (REL2+), and in which these immune regulatory factors are activated via the blood-meal inducible Vitellogenin (Vg) promoter Antonova et al, 2009). wAlbB was introduced into REL1+ and REL2+ mosquito lines through repeated crosses of wAlbBinfected females with REL1+ and REL2+ transgenic males for seven generations Antonova et al, 2009). These crosses resulted in wAlbB-infected transgenic mosquitoes, referred to as W+/REL1+ and W+/REL2+.…”

“…WBT is an A. aegypti mosquito line, in which the wAlbB infection was cleared by tetracycline treatment of WB1, and has subsequently been maintained for 410 generations. Vg-ΔREL1-A (REL1+) and Vg-REL2 (REL2+) are transgenic A. aegypti mosquito lines, in which the Rel1 and Rel2 genes, respectively, are overexpressed Antonova et al, 2009). The Rockefeller/UGAL is a wild-type A. aegypti line used as a control for the transgenic lines.…”

Blocking of Plasmodium transmission by cooperative action of Cecropin A and Defensin A in transgenic Aedes aegypti mosquitoes

“…Genetic modification of mosquitoes by means of transposable elements provides an outstanding tool for experimental testing of concepts of vector biology, including pathogen-vector interactions. Utilization of transgenesis to generate mosquitoes with altered immunity has been instrumental in deciphering major immune pathways and their role in antipathogen defense (7,(9)(10)(11). Transgenic mosquitoes with antipathogen refractory phenotypes have been developed by utilizing blood meal-activated, tissue-specific promoters, and effector molecules or RNAi-constructs (11)(12)(13)(14)(15).…”

“…Both these AMPs were expressed highly after immune challenge or ectopic expression of REL2, the mosquito orthologue of Drosophila Relish, in Ae. aegypti mosquitoes (11).…”