Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae

Abstract

We show that, in the malaria vector Anopheles gambiae, expression of Cecropin 1 is regulated by REL2, an NF-kappaB-like transcription factor orthologous to Drosophila Relish. Through alternative splicing, REL2 produces a full-length (REL2-F) and a shorter (REL2-S) protein isoform lacking the inhibitory ankyrin repeats and death domain. RNA interference experiments show that, in contrast to Drosophila Relish, which responds solely to Gram-negative bacteria, the Anopheles REL2-F and REL2-S isoforms are involved in defense against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria, respectively. REL2-F also regulates the intensity of mosquito infection with the malaria parasite, Plasmodium berghei. The adaptor IMD shares the same activities as REL2-F. Microarray analysis identified 10 additional genes regulated by REL2, including CEC3, GAM1, and LRIM1.

Fig. 1.

Genomic organization and alternative splicing of the Anopheles REL2 gene. Introns (lines) and exons (open boxes) are shown, based on Ensembl gene prediction (A) and actual data (B). The length (bp) of introns and exons is shown below and above the lines and boxes, respectively. ○, putative κB elements. (C) REL2 has two alternative 5′ exons. The 5′ end of REL2-S has not been confirmed and is indicated by dashed intron lines. Alternative splicing occurs at the 3′ end with a REL2-S-specific exon. Protein domains are depicted by shades of gray inside the exons. Q/H, glutamine and histidine-rich region; RHD, Relish homology domain; IPT/TIG, DNA-binding domain; NLS, nuclear localization signal; ANK, ANK-repeat domain; DD, death domain. Putative start- and stop-codons and target regions of dsRNA constructs (RHD dsRNA and ANK dsRNA) are indicated.

Fig. 2.

Expression profile of REL2 in mosquito cultured cells and developmental stages. (A) Relative abundance of REL2-F and REL2-S transcripts at different stages of mosquito development. (B) Transcriptional regulation of REL2 transcripts in immune-challenged cultured Sua1B cells. REL2-F was detected by using primer pairs targeting the ANK domain. REL2-S was detected with one primer specific to the REL2-S transcript (see Materials and Methods). PCR conditions: 30 cycles, 60°C annealing temperature, 1.5 min extension time, and 2 μg of total RNA used per reaction. *, a band which includes the intron sequence of REL2-S. It is unclear whether this transcript is part of an additional splice variant of REL2. The identity of REL2-S bands was confirmed by sequencing. Control RT-PCR with rpS7 showed no genomic DNA contamination. CTRL, control; B.b., B. bassiana; M.l., M. luteus; E.c., E. coli; ♂, males; ♀, females.

Fig. 3.

CEC1 promoter activity in cultured Anopheles cells. (A) REL2, but not REL1, is required for expression of CEC1 promoter in Sua1B cells, which is equally affected by dsRNAs encompassing the RHD and the ANK-repeats domain. (B) REL2, IMD, and CASPL1, but not IKK1 or IKK2, are required for CEC1 expression in Sua1B cells. (C) REL2, IMD, IKK1, and IKK2, but not CASPL1, are required for CEC1 expression in 4a3A cells. CTRL, control.

Fig. 4.

Efficacy of RNAi-mediated gene silencing. (A) KD of IKK1, IKK2, and CASPL1 genes in Sua1B cultured cells. RNAi effectiveness was determined by semiquantitative RT-PCR (Left). Quantification of PCR products (Right) was performed by using rpS7 as an internal reference and untreated cells as a calibrator. (B) REL2 gene silencing in adult mosquitoes after injections with either RHD or ANK dsRNA. Relative REL2-S and REL2-F transcript levels in KD compared with control GFP dsRNA-treated mosquitoes were determined in the carcass, midgut, and whole mosquitoes 4 days after injections by using quantitative RT-PCR.

Fig. 5.

Mosquito survival to bacterial infection. Survival of adult mosquitoes to Gram-positive S. aureus (A) and Gram-negative E. coli (B) infections after KD of IMD, REL2-RHD, and REL2-ANK. GFP dsRNA-treated mosquitoes were used as a control. The survival assay was repeated at least 3 and up to 10 times per gene. Error bars represent the standard error.

Fig. 6.

REL2- and IMD-mediated Plasmodium killing and melanization in the mosquito vector. (A) Averaged P. berghei oocyst load per midgut after KD of IMD, REL2-ANK, and REL2-RHD. Oocyst load after individual gene KD was normalized relative to oocyst load of parallel dsGFP treatment and presented as a percentage. Every gene KD was repeated three times or more. Black sections of columns represent melanized ookinete fraction. Error bars represent the standard error. (B) Representative sections of infected Anopheles gambiae midguts infected with GFP-tagged P. berghei. Arrows point to melanized ookinetes.