Immune regulation in the African malaria mosquito, Anopheles gambiae

Abstract

Anopheles gambiae is a primary vector of Plasmodium parasites that cause human malaria, an infectious disease that continues to pose a major public health threat in much of sub-Saharan Africa. Despite the availability of two effective, albeit non-sterilizing vaccines, mosquito population control remains the main prevention strategy for malaria transmission. Vector control relies primarily on the use of chemical insecticides. However, increasing insecticide resistance in mosquitoes demands alternative strategies that will rely on a deep molecular understanding of mosquito physiology. Knowledge of mosquito immunity is particularly important, given its critical role in mosquito fitness, vector competence, and resistance to entomopathogens. To fight pathogens that invade their body cavity, mosquitoes mount a vigorous humoral immune response. Humoral immunity largely depends on the activation of downstream signaling and effector responses such as phenoloxidase-based melanization and antimicrobial activity regulated by the Toll pathway. While melanization kills the pathogen through the deposition of melanin on its surface, antimicrobial activity promotes the rupturing of the pathogen’s membrane through the action of antimicrobial peptides (AMPs). Central to the regulation of these immune responses are extracellular protease cascades that involve clip-domain serine proteases and their non-catalytic homologs, that in mosquitoes are together known as CLIPs. Although more than a hundred CLIP genes are annotated in the An. gambiae genome, only a subset of them have been evaluated as regulators of melanization and organized into partially defined cascades. Furthermore, CLIPs involved in antimicrobial activity in mosquitoes remain largely uncharacterized, in part due to the lack of effective assays for detecting antimicrobial factors. As a result, the composition of the protease cascades that form the immunoregulatory network in An. gambiae is still not fully understood. In this dissertation, I aimed to (1) establish an assay that allows the detection of factors contributing to humoral antimicrobial activity in An. gambiae, and (2) evaluate the contribution of CLIPs to immunity against microbial pathogens. Towards my first aim, I developed an ex vivo zone-of-inhibition (ZOI) assay to determine the antimicrobial activity of hemolymph collected from adult An. gambiae after microbial challenges. By combining this assay with RNAi, I demonstrated that both Gram-positive and Gram-negative bacteria induce antimicrobial activity that is dependent on Toll pathway function. For my second aim, I designed an RNAi screening pipeline that integrates the ZOI assay with a previously established melanization assay, enabling me to identify 27 immunoregulatory CLIPs that form two distinct subnetworks regulating antimicrobial activity and melanization, respectively. By quantifying bacterial load and median survival post-infection, I established that the CLIPs regulating antimicrobial activity control infection resistance. Finally, by examining the transcription profiles of CLIPs upon infection, I established that while CLIPs are infection-responsive genes, their immunoregulatory roles are defined by their baseline co-expression rather than infection-induced upregulation. Taken together, the work in this dissertation contributes to our fundamental understanding of immune regulation in An. gambiae. It establishes an ex vivo medium throughput assay that can be used to determine intracellular and extracellular regulatory elements of antimicrobial activity in mosquito hemolymph. Importantly, it provides critical new insights into the complexity and general organization of the protease network that regulates humoral immunity in this mosquito species, offering a framework for future studies in other mosquito species. Lastly, this work uncovers a novel immunoregulatory mechanism that may be targeted for malaria control interventions.