Global cross-talk of genes of the mosquito Aedes aegypti in response to dengue virus infection

Abstract

Background: The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in humans, and DENV is the most important arbovirus across most of the subtropics and tropics worldwide. The early time periods after infection with DENV define critical cellular processes that determine ultimate success or failure of the virus to establish infection in the mosquito.

Methods and results: To identify genes involved in these processes, we performed genome-wide transcriptome profiling between susceptible and refractory A. aegypti strains at two critical early periods after challenging them with DENV. Genes that responded coordinately to DENV infection in the susceptible strain were largely clustered in one specific expression module, whereas in the refractory strain they were distributed in four distinct modules. The susceptible response module in the global transcriptional network showed significant biased representation with genes related to energy metabolism and DNA replication, whereas the refractory response modules showed biased representation across different metabolism pathway genes including cytochrome P450 and DDT [1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane] degradation genes, and genes associated with cell growth and death. A common core set of coordinately expressed genes was observed in both the susceptible and refractory mosquitoes and included genes related to the Wnt (Wnt: wingless [wg] and integration 1 [int1] pathway), MAPK (Mitogen-activated protein kinase), mTOR (mammalian target of rapamycin) and JAK-STAT (Janus Kinase - Signal Transducer and Activator of Transcription) pathways.

Conclusions: Our data revealed extensive transcriptional networks of mosquito genes that are expressed in modular manners in response to DENV infection, and indicated that successfully defending against viral infection requires more elaborate gene networks than hosting the virus. These likely play important roles in the global-cross talk among the mosquito host factors during the critical early DENV infection periods that trigger the appropriate host action in susceptible vs. refractory mosquitoes.

Figure 1. Modular expression patterns of Aedes aegypti genes in response to dengue virus infection.

The top panel shows a WGCNA transcriptional network of host responsive genes between the susceptible (MS) and the refractory (MR) strains at 3 hr and 18 hr post-infection times, followed by dynamic hybrid cutting by topology overlapping. The scale on the left shows the branch heights. The modules are shown as ‘A’ through ‘G’. The bar graphs in the bottom panel represent the overall gene expression pattern of genes in the respective modules among the test samples. The up-regulated and down-regulated expression of genes is shown by eigengene values (module eigengene is defined as the first principal component of the expression matrix of the corresponding module) (shown in the Y-axis) for each module.

Figure 2. Heat maps of gene expression in the modules.

Each row represents a gene in the module. The module IDs are indicated. The columns in the heat maps are labeled to indicate the mosquito strain and the time point to which the gene expression belongs to. The red color indicates up-regulation and green indicates down-regulation of gene expression.

Figure 3. A simplified illustration of inter-modular networking of Aedes aegypti genes responsive genes.

The expression modules are shown by colored circles (A through G). The number of genes belonging to each module is shown. The black lines connect the modules. The thickness of these line shows how closely similar they are to each other in the cluster tree (see Figure 1). The red colored circles and lines represent the refractory response modules (RRM), the green color circle represents the susceptible response module (SRM) and the blue colored circles and lines represent the core response modules (CRM).

Figure 4. Relative abundance of different signal transducing genes responsive to dengue infection in Aedes aegypti.

The percentage below each category represents the percentage of genes of that category with respect to all responsive genes related to signal transduction (based on KEGG pathways).

Figure 5. A pathway diagram of JAK-STAT cascades of dengue responsive genes.

The KEGG pathway template was used to generate this diagram by mapping the responsive genes (genes responsive in MS strain are in pink and genes responsive in MR strain are in green).

Figure 6. Validation of microarray expression of five genes by quantitative real-time PCR (qRT-PCR).

For each gene, comparisons are made between array data (MS and MR strains) and the qRT-PCR data (MS and MR strains). Also, two additional strains of A. aegypti, D2S3 and MD, were infected with dengue (see text) and then compared for expression of the same genes by qRT-PCR. The relative gene expression (in comparison to the control) is shown in the Y-axis along with standard errors. The X-axis shows the gene identity.