Wolbachia infections in Anopheles gambiae cells: transcriptomic characterization of a novel host-symbiont interaction

Abstract

The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria.

Figure 1. Anopheles gambiae gene regulation in response to Wolbachia infection.

A. Venn diagram of 690 Anopheles transcripts which display differential expression due to wAlbB or wRi infection. 104 transcripts were common to both strains, while 389 were down regulated and 320 up regulated due to Wolbachia infection. B. Scatter plot of regulated significant genes (>2 fold regulation; False discovery rate P value <0.05). Blue dots represent significant genes regulated by wRi only, red regulated by wAlbB only and purple, genes commonly regulated. C. Number of genes in each functional classes class up or down regulated in response to either wAlbB or wRi infection. Genes were classified into groups; transport (TRP), replication, transcription and translation (RTT), redox, stress and mitochondrial (RSM) proteolysis and digestion (PROT), metabolism (M) cytoskeletal and structural (CS) and immune (I) depicted in the first column, and diverse (D) and unknown (U), in the second column.

Figure 2. Validation of microarray data in cell culture and whole mosquitoes.

A. Log2 fold change for selected An. gambiae genes (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin6, Filamin, TEP3) comparing microarray and QPCR methods. B. Comparison of Anopheles gene expression in response to Wolbachia in cell culture and whole mosquitoes. Expression of 6 genes from wAlbB in Sua5B cells analyzed using microarrays (MA) compared to wAlbB somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). C. Microarray data from wRi infected Sua5B cells compared to wMelpop somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). qPCR gene expression is a ratio of Wolbachia infected (wAlbB or wRi) to Schneider's injected control. Error bars represent maximum and minimum range of expression.

Figure 3. Wolbachia strain-specific regulation of Anopheles gambiae immune pathways.

Anopheles immune networks regulated by wRi (A) and wAlbB (B). Pathways are models of the IMD and Toll pathways and components of the melanization regulatory module divided into the 4 broad categories of immune molecules. Blue color represents induction, while yellow color represents suppression. The intensity of coloring is proportional to the intensity of expression. Regulation is depicted to a maximum fold change of ±4. Some transcripts were greater than ±4 regulated. Abbreviations: LLR leucine rich repeats; FBNs fibrinogens; TEPs thioester containing proteins; GNBPs Gram-negative binding proteins; CTLs C type lectins; CLIPs clip-domain serine protease; PGRPs peptidoglycan recognition proteins; SRPNs serpins; CEC cecropins; Def defensins; PPO Prophenoloxidase; PO phenoloxidase; LYS lysozmyes.