The Aedes aegypti toll pathway controls dengue virus infection

Abstract

Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference-based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway-associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway.

Figure 1. Functional classification of differentially expressed genes in the dengue-infected midgut and carcass at 10 days after blood meal.

The graph shows the functional class distributions in real numbers of genes that are regulated by virus infection (+ indicate induced and – indicate repressed). The virus infection responsive gene expression data are presented in Tables S1 and S2. Functional group abbreviations: IMM, immunity; R/S/M, redox, stress and mitochondrion; CSR, chemosensory reception; DIG, blood and sugar food digestive; PRT, proteolysis; C/S, cytoskeletal and structural; TRP, transport; R/T/T, replication, transcription, and translation; MET, metabolism; DIV, diverse functions; UNK, unknown functions.

Figure 2. Regulation of putative Toll signaling pathway genes by dengue virus infection.

Red color indicates infection responsive up-regulation and green color indicate infection responsive down-regulation. Non-colored gene boxes indicate lack of infection responsive regulation. The pathway was built with GenMapp software based on the immunogenomics prediction by Waterhouse et al 2007.

Figure 3. Comparative analysis of the dengue virus infection-responsive and Rel1 and Rel2 regulated transcriptomes.

A. Expression analysis of defensin (DEF), cecropin (CEC), Cactus (CAC), and Rel1 in Cactus, and Cactus and Rel1 depleted mosquitoes (upper panel) and in Caspar, and Caspar and Rel2 depleted mosquitoes. Bar represents standard error. B. Venn diagram showing uniquely and commonly regulated genes in dengue infected and Cactus and Caspar depleted mosquitoes. C. Cluster analysis of 131 genes that were regulated in at least two of four treatments: dengue-infected midgut and carcass, and whole mosquitoes upon Cactus (CAC(-)) or Caspar (CSP(-)) depletion. The expression data of immune genes, indicated by the number beside the panel are presented in Table 1, and all genes presented in the hierarchical cluster matrix are listed in Table S6. The primary data for the real-time qPCR assays are presented in Table S3.

Figure 4. Rel1 regulate anti-dengue activity.

Dengue virus loads in the midguts of Cactus, Caspar and MyD88 depleted mosquitoes, and GFP dsRNA treated control mosquitoes. A. Virus titers were measured by plaque assay in C6/36 cell. *, P<0.05, ***, P<0.001, in Student's t-test comparing to GFP control. B. Virus load is assayed through indirect immunofluorescence assay (IFA) in infected midguts. Error bar represents standard error.

Figure 5. Elimination of the mosquito's endogenous bacteria reduces basal levels of immune gene expression and increases the susceptibility to dengue virus infection.

A. LB agar plates at 20 hours after plating homogenized and diluted septic gut (SG) and whole mosquito (SW) from non-treated mosquitoes, and aseptic gut (AG) and whole mosquito (AW) from antibiotic treated mosquitoes B. fold change in the expression of selected immune genes in aseptic mosquitoes compared to septic mosquitoes; C. virus infection levels in aseptic and septic mosquitoes were measured and compared by plaque assay in C6/36 cells * P<0.05 in Student's t-test. D. Dengue virus distribution and loads in septic and aseptic mosquito midguts assayed through IFA. Error bar represents standard error. Primary data for the real-time qPCR assays are presented in Table S2.