Characterization of the Rel2-regulated transcriptome and proteome of Anopheles stephensi identifies new anti-Plasmodium factors

Abstract

Mosquitoes possess an innate immune system that is capable of limiting infection by a variety of pathogens, including the Plasmodium spp. parasites responsible for human malaria. The Anopheles immune deficiency (IMD) innate immune signaling pathway confers resistance to Plasmodium falciparum. While some previously identified Anopheles anti-Plasmodium effectors are regulated through signaling by Rel2, the transcription factor of the IMD pathway, many components of this defense system remain uncharacterized. To begin to better understand the regulation of immune effector proteins by the IMD pathway, we used oligonucleotide microarrays and iTRAQ to analyze differences in mRNA and protein expression, respectively, between transgenic Anopheles stephensi mosquitoes exhibiting blood meal-inducible overexpression of an active recombinant Rel2 and their wild-type conspecifics. Numerous genes were differentially regulated at both the mRNA and protein levels following induction of Rel2. While multiple immune genes were up-regulated, a majority of the differentially expressed genes have no known immune function in mosquitoes. Selected up-regulated genes from multiple functional categories were tested for both anti-Plasmodium and anti-bacterial action using RNA interference (RNAi). Based on our experimental findings, we conclude that increased expression of the IMD immune pathway-controlled transcription factor Rel2 affects the expression of numerous genes with diverse functions, suggesting a broader physiological impact of immune activation and possible functional versatility of Rel2. Our study has also identified multiple novel genes implicated in anti-Plasmodium defense.

Keywords: Innate immunity; Mosquitoes; Plasmodium; Proteome; Rel2; Transcriptome; Transgenesis.

Figure 1. Global changes in transcript levels in transgenic A. stephensi following Rel2 induction

A) The total number of genes significantly up- or down-regulated that are predicted to be in each GO category. Genes were considered significantly differentially regulated if the -fold change was>= 0.75 on a log₂ scale. B) Venn diagram comparing the total number of regulated transcripts between the midgut of CP line mosquitoes and fat body of VG line mosquitoes at 12 h PBM. Red arrows correspond to midgut samples, and green arrows correspond to fat body samples; the arrow direction indicates significant up- or down-regulation.

Figure 2. Global changes in protein levels in transgenic A. stephensi following Rel2 induction

A) The total number of proteins significantly up- or down-regulated that are predicted to be in each GO category. Genes were considered significantly differentially regulated if the ratio of transgenic to wild type was>= 0.75 on a log₂ scale. B) Venn diagram comparing the total number of regulated proteins between the midgut of CP line mosquitoes and fat body of VG line mosquitoes at 24 h PBM. Red arrows correspond to midgut samples, and green arrows correspond to fat body samples; the arrow direction indicates significant up- or down-regulation.

Figure 3. P. falciparum infection intensity following RNAi knockdown of immune genes

The number of oocysts per midgut of wild-type A. stephensi following RNAi-mediated depletion of GFP, SCRBQ1, bacterial response protein (AGBP1), Niemann-Pick type-C (NPC2), alpha-2-macroglobulin (A2MRAP), or leucine-rich transmembrane protein (LRTP). Depletion of both A2MRAP and LRTP led to a significant increase in the number of oocysts per mosquito midgut. Each circle represents a single midgut, and horizontal black bars represent the median of the sample. Significance was determined by a Kruskal-Wallis test followed by Dunn’s post-hoc test to compare immune-depleted mosquitoes to GFP controls. Significance was assessed at α=0.05. Supplementary data for this figure is given in table S2.

Figure 4. P. falciparum infection intensity following RNAi knockdown of protease genes

The number of oocysts per midgut of wild-type A. stephensi following RNAi-mediated depletion of: A) green fluorescent protein (GFP), serpin 10 (SRPN10), Rel2-responsive serine protease 1 (R2RSP1), Rel2-responsive serine protease 2 (R2RSP2), or serine protease precursor 1 (SEPRP1); and B) trypsin precursor (TRYPP), anigiotensin-converting enzyme precurser (ACEP), or serine protease precursor 2 (SEPRP2). Silencing of R2RSP2, ACEP, and SEPRP2 all led to significant increases in the number of oocysts per midgut. Each circle represents a single midgut, and horizontal black bars represent the median of the sample. Significance was determined by a Kruskal-Wallis test followed by Dunn’s post-hoc test to compare immune-depleted mosquitoes to GFP controls. Significance was assessed at α=0.05. Supplementary data for this figure is given in table S2.

Figure 5. Influence of novel anti-Plasmodium genes on midgut microbiota

The number of colony forming units of culturable bacteria in the midguts of females following RNAi-mediated knockdown of genes shown above to have anti-Plasmodium effects. A) Sugar-fed mosquitoes following depletion of GFP, A2MRAP, and LRTP. B) Sugar-fed mosquitoes following depletion of GFP, R2RSP2, ACEP, and SEPRP2. C) Blood-fed mosquitoes following depletion of GFP, A2MRAP, and LRTP showed that A2MRAP depletion leads to a significant increase in CFUs per midgut. D) Blood-fed mosquitoes following depletion of GFP, R2RSP2, ACEP, and SEPRP2. Significance was determined by a one-way ANOVA, followed by Dunnett’s multiple comparison test, with significance assessed at α=0.05. Bars represent the mean of three biological replicates, and error bars represent the standard error of the mean.