Anopheles gambiae immune responses to human and rodent Plasmodium parasite species

Abstract

Transmission of malaria is dependent on the successful completion of the Plasmodium lifecycle in the Anopheles vector. Major obstacles are encountered in the midgut tissue, where most parasites are killed by the mosquito's immune system. In the present study, DNA microarray analyses have been used to compare Anopheles gambiae responses to invasion of the midgut epithelium by the ookinete stage of the human pathogen Plasmodium falciparum and the rodent experimental model pathogen P. berghei. Invasion by P. berghei had a more profound impact on the mosquito transcriptome, including a variety of functional gene classes, while P. falciparum elicited a broader immune response at the gene transcript level. Ingestion of human malaria-infected blood lacking invasive ookinetes also induced a variety of immune genes, including several anti-Plasmodium factors. Twelve selected genes were assessed for effect on infection with both parasite species and bacteria using RNAi gene silencing assays, and seven of these genes were found to influence mosquito resistance to both parasite species. An MD2-like receptor, AgMDL1, and an immunolectin, FBN39, showed specificity in regulating only resistance to P. falciparum, while the antimicrobial peptide gambicin and a novel putative short secreted peptide, IRSP5, were more specific for defense against the rodent parasite P. berghei. While all the genes that affected Plasmodium development also influenced mosquito resistance to bacterial infection, four of the antimicrobial genes had no effect on Plasmodium development. Our study shows that the impact of P. falciparum and P. berghei infection on A. gambiae biology at the gene transcript level is quite diverse, and the defense against the two Plasmodium species is mediated by antimicrobial factors with both universal and Plasmodium-species specific activities. Furthermore, our data indicate that the mosquito is capable of sensing infected blood constituents in the absence of invading ookinetes, thereby inducing anti-Plasmodium immune responses.

Figure 1. Experimental Design and Global Gene Expression Patterns at the Different Conditions of Infection

(A) Model outlining the experimental design for assessing responses to the invading ookinetes (indicated as P.f. ookinete [P.f. o.] and P.b. ookinete [P.b. o.] in (B) and (C) by comparing transcription between mosquitoes fed on blood infected with a wt Plasmodium strain and those fed on blood infected with the invasion-incapable mutant Plasmodium strain CTRP⁻. Responses to infected blood (indicated as P.f. blood in [B] and [C]) in the absence of ookinete invasion were assessed by comparing gene expression between mosquitoes fed on blood infected with the P. falciparum invasion-incapable mutant and mosquitoes fed on noninfected (no Plasmodium) blood. (B) Gene regulation in midgut and carcass tissues triggered by P. falciparum ookinete invasion (P.f. ookinete), P. berghei ookinete invasion (P.b. ookinete), and P. falciparum strain CTRP⁻-infected blood lacking invasive ookinetes (P.f. blood). Colored arrows indicate genes that are up- or down-regulated in the various unique and overlapping sections. (C) Proportions and numbers of genes belonging to distinct functional classes which were up- or down-regulated by the various stimuli in the gut and carcass tissues. DIV: diverse; R/T/T: replication, transcription, translation; MET: metabolism; TRP: transport; CY/ST: cytoskeletal, structural; PR/DI: proteolysis, digestion; MIT: mitochondrial; RE/ST: oxidoreductive, stress-related; APO: apoptosis; P/A: putative immunity and apoptosis. Gene functions were predicted based on Gene Ontology data and manual sequence homology searches. (D) Same as in (C), but also including genes of diverse functions (DIV) and unknown functions (UKN).

Figure 2. Effects of Gene Silencing of 11 Selected Putative Immune Genes on P. falciparum and P. berghei Infection

The gene silencing efficiency values (KD %) are displayed in Table S6. The frequency distribution of oocysts pooled from three independent assays is displayed, with bars indicating the percentile of mosquitoes with the corresponding oocyst number in the range indicated on the x-axis. Equal numbers of midguts from all three experiments in each dataset were pooled. Bars with asterisks indicate the statistically significant differences at the 95% confidence level, based on the p value from two independent probability tests, the KS and Mann-Whitney tests (Tables S5 and S6). n: total midguts assayed; MI: mean intensity of infection (oocysts number); S.E.: standard error of mean intensity; p value: from Mann-Whitney test.

Figure 3. Comparison of Anti-Plasmodium and Antibacterial Activities for 16 Selected Immune Genes

(A) Effect of gene silencing on P. berghei development, as described in Table S6. For ease of comparison, only the mosquito portions with the highest P. berghei oocyst numbers (>200) are presented. The effect of gene silencing on bacterial infection is presented as the mosquito survival at d 6 after challenge with E. coli and S. aureus. After 6 d, the survival rates stabilized and did not change significantly until age-related mortality ensued. The baseline survival rate was set to that of the challenged GFP dsRNA-treated control mosquitoes (~70%). Standard error bars with asterisks indicate the results of two-way analysis of variance, with p < 0.05 considered statistically significant. The gene names are numbered for ease of comparison. (B) Mosquito survival rates for each silenced gene after challenge with E. coli and S. aureus. The numbers in parenthesis correspond to the numbers in (A). Open squares, dsGFP control-treated mosquitoes challenged with E. coli; solid squares, dsGFP control–treated mosquitoes challenged with E. coli; open triangles, gene-silenced mosquitoes challenged with E. coli; and solid triangles, gene-silenced mosquitoes challenged with S. aureus. Standard error bars from three replicate experiments are included for each time point.

Figure 4. AgMDL Gene Family

(A) A. gambiae MD2-like genes encode proteins ranging from 130 to 162 amino acids and include signal peptides and an ML lipid recognition domain. Alignment of AgMDL1 with the human homologues MD1, MD2, and Npc2, the mite allergen Der-P2, and the Bombyx mori promotor protein (BmPP). Two conserved cysteines, Cys95 and Cys105, that are essential for binding to TLR4 are indicated with asterisks. (B) Phylogenetic tree of MD2-like proteins from A. gambiae, D. melanogaster, B. mori, and humans. 1:1 orthologs and ortholog groups are highlighted with filled circles. Ag, Anopheles gambiae; Dm, D. melanogaster. The accession numbers for these genes are listed in Table S7.