Dual role of the Anopheles coluzzii Venus Kinase Receptor in both larval growth and immunity

Abstract

Vector-borne diseases and especially malaria are responsible for more than half million deaths annually. The increase of insecticide resistance in wild populations of Anopheles malaria vectors emphasises the need for novel vector control strategies as well as for identifying novel vector targets. Venus kinase receptors (VKRs) constitute a Receptor Tyrosine Kinase (RTK) family only found in invertebrates. In this study we functionally characterized Anopheles VKR in the Gambiae complex member, Anopheles coluzzii. Results showed that Anopheles VKR can be activated by L-amino acids, with L-arginine as the most potent agonist. VKR was not required for the fecundity of A. coluzzii, in contrast to reports from other insects, but VKR function is required in both Anopheles males and females for development of larval progeny. Anopheles VKR function is also required for protection against infection by Plasmodium parasites, thus identifying a novel linkage between reproduction and immunity in Anopheles. The insect specificity of VKRs as well as the essential function for reproduction and immunity suggest that Anopheles VKR could be a potentially druggable target for novel vector control strategies.

Figure 1

Genomic and protein structures of Anopheles VKR. (A) Exon-intron structure of VKR locus. Exons are represented as red boxes and introns as red lines. The arrow indicates the positions of the translational start (ATG) and stop (TAG) codons. The scale indicates the length in kb. (B) Protein sequence alignment of VFT from mosquitoes VKR (AgVKR ACF34410.1, CqVKR DAA06510.1, AaVKR DAA06509.1) and honey bee (AmVKR ACF34409.1) using the CLUSTAL W method. Lobe I and Lobe II (indicated by the upper black line) and the three linkers (black boxes) constitute the structure of VFT domains. (C) Sequence alignment of kinase domains of AgVKR, CqVKR, AaVKR and AmVKR. Numbers I to XI indicate the eleven subdomains conserved in protein kinase domains. Consensus sequences required for tyrosine kinase activity are indicated in red.

Figure 2

Functional characterization of Anopheles VKR in Xenopus oocytes. (A) Minimal L-AA concentrations to induce GVBD in Xenopus oocytes expressing Anopheles VKR (from 1 µM to 1 mM). Arginine is the most efficient L-AA to induce VKR activation. Mutation of Ser₅₀₅ present inside the ligand binding pocket strongly decreases the capacity of VKR to be activated by L-AA. (B) Effect of various tyrosine kinase inhibitors on VKR activity induced by arginine binding evaluated by the percentage of GVBD in oocytes expressing VKR. AG1024, AG538 and HNMPA (IR inhibitors) and AG1478 (IR and EGFR inhibitor) totally inhibit Anopheles VKR activation at 1 µM whereas SU11274 (Met receptor inhibitor) has no effect at this concentration. (C) Native Anopheles VKR^WT or mutant VKR^S505A expressed in oocytes were immunoprecipitated from membrane extracts of oocytes using anti-V5 antibodies and revealed by Western Blot using anti-V5 and anti-PY antibodies for detecting tyrosine phosphorylation. VKR^WT is found to be phosphorylated in the presence of the ligand arginine but not in the presence of the inhibitor AG1024. The autophosphorylating activity of the mutant VKR^S505A is strongly affected.

Figure 3

VKR does not affect A. coluzzii ovary structure and fecundity. (A) Median size and structure of A. coluzzii ovaries from dsGFP and dsVKR females at 48 h post-blood meal are not significantly different. (B) Median numbers of developed oocytes per ovary pair from dsGFP and dsVKR females at 48 h post-blood meal are not significantly different. The Differences in ovary sizes and in the number of developed oocytes between the two groups were tested using non-parametric Wilcoxon Mann-Whitney. (C) Eggs from pools of females: VKR has no effect on the number (Nb) of laid eggs reported to the number (Nb) of females (mothers) in four independent experiments (Chi-square p-value > 0.05). The number of female mothers for each group (dsGFP and dsVKR) is the same (40 females) for each biological replicate. (D) Eggs from individual female: VKR has no effect on the number of laid eggs per individual female (non-parametric Wilcoxon Mann-Whitney, p-value > 0.05).

Figure 4

VKR is required in larval growth. VKR silencing either in female (A) or in male (B) mosquitoes affects the larval development of their progenies, considering the proportion of emerging adults reported to the number of eggs put in water. Stars (*) and (***) represent Chi2 test p-value < 0.05 and p-value < 0.001 respectively.

Figure 5

VKR is required for protective function against Plasmodium parasites. VKR restricts permissiveness of mosquitoes to P. berghei infection, measured as oocyst infection prevalence (proportion of infected mosquitoes). For each independent experiment, Chi-square p-values related to the proportion of infected mosquitoes (infection prevalence) between the two groups (dsGFP and dsVKR) are mentioned. Using the meta-analytical approach of Fisher to combine individual p-value related to infection prevalence from the three independent replicates, we found a statistically significant combined p-value (p = 0.0008).

Figure 6

Proposed model for Anopheles VKR dual roles in both immunity and larval growth. As a tyrosine kinase receptor, activation of VKR in A. coluzzii would lead to activate kinase pathways, which are either involved in immunity (through potential activation of JNK and/or MAP pathways) or phosphorylate ecdysteroid (22P), which serve as a reservoir of ecdysteroids during the larval growth.