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Implication of mosquito peptidoglycan recognition proteins in immune regulation and anti-malarial defence.

Final Activity Report Summary - MOSQUITO IMMUNITY (Implication of mosquito peptidoglycan recognition proteins in immune regulation and anti-malarial defence)

Malaria is still a threat to human health and economic growth on a worldwide scale. Malaria infection of the mosquito results in the activation of an immune response that contributes to the killing of plasmodia. The recognition of 'non-self' leading to the activation of the known immune signalling pathways in the fruit fly have recently been shown to be mediated by members of a peptidoglycan recognition protein (PGRP) family that appeared to represent the major pattern recognition receptors of its immune surveillance system.

The role of the mosquito PGRPs in regulating its immune system and activating anti-malarial defence mechanisms was examined through the combination of reverse genetic and comparative transcriptomic approaches. Of the seven PGRP genes in a. gambiae PGRP-LC was the candidate of choice for the following reasons:
1. firstly, the homologous gene in drosophila melanogaster had a central role in the modulation of the immune mediated disease (IMD) immune response;
2. secondly, the PGRP-LC function in a. gambiae remained to be verified.

The level of transcription was assessed by means of quantitative polymerase chain reaction (qPCR). This technique ascertained the amount of each messenger ribonucleic acid (mRNA) transcript at different mosquito life stages, in different tissues, as well as in an infected versus a non-infected state. This was achieved by generating standard curves for the gene under investigation as well as the housekeeping gene used for normalisation. Similarly, a reverse genetic approach allowed for the determination of the endogenous role of this gene family. Constructs were developed to silence both all PGRP-LC isoforms, as well as each single isoform (PGRP-LC1, PGRP-LC2, and PGRP-LC3). The expressions for timing and location of the silencing machinery were of paramount importance. Therefore, the so far uncharacterised a. gambiae vitellogenin promoter was identified with a bioinformatical approach. The activity of this promoter was tested transiently by so far, but would be further characterised in constitutively integrated individuals and upon several generations.

Recent research developments on a. stephensi highlighted the role of the IMD pathway in the regulation of immune response to gram positive and negative bacteria as well as to p. berghei. The toll pathway constituted another major immunoregulatory pathway in insects. We generated cactus dominant negative constructs, which were introduced into the genome of a. stephensi. Moreover, homozygous lines of cactus dominant negative transgenic mosquitoes were isolated and infections with p. berghei parasites underlined any implication this immune regulating pathway might have in the immune response against malaria.