Mechanisms of mosquito gut homeostasis and the role of NF-kappaB signalling

Similar to the human intestinal tract, the mosquito gut contains large and complex bacterial communities. The numbers of bacteria in the female mosquito gut increase drastically after ingestion of a bloodmeal. We have recently shown that strong immune reactions of the mosquito gut elicited by the bacterial increase are important in restoring the original gut conditions (homeostasis), and also attack and reduce the numbers of ingested animal and human blood-borne pathogens such as malaria parasites and viruses. These findings highlight the importance of the gut microbiota in the capacity of mosquitoes to transmit pathogens and led us postulate that interfering with the interactions between the mosquito gut and its microbiota could lead to new approaches for disease transmission control. This project aims to characterise the molecular mechanisms involved in the control of mosquito gut bacteria. The capacity to harbour gut bacteria differs from one mosquito to another due to genetic variation. We will take advantage of this variation to identify genes that are involved in the interactions with gut bacteria, and then inactivate each of these genes to examine their specific effects on the gut bacterial communities. Our previous research has shown that a protein recognising peptidoglycan, an important structural component of the bacterial cell wall, is important for defence reactions against gut bacteria. It also suggested that this receptor controls the levels of bacteria by quantitative sensing of peptidoglycan and tightly regulates activation of defence reactions. We will investigate this novel hypothesis using molecular and cell biological techniques.

The Anopheles gambiae gut is habitat to large and diverse bacterial communities. The total number of bacteria in the gut of a female mosquito increases several folds after ingestion of a bloodmeal. We have shown that the increase of gut bacteria triggers strong immune reactions of the REL2 NF-kappaB signalling pathway, which are important in reducing the bacterial numbers and restoring gut homeostasis, but also limit infections of blood-borne parasites and viruses. This project aims to investigate the molecular mechanisms involved in regulating the mosquito gut flora. To identify genes and mechanisms involved in responses to gut bacteria, we will carry out genome-wide association studies (GWAS) using an Affymetrix SNP chip that we have recently developed and three colonies of A. gambiae mosquitoes that will be orally infected with fluorescently tagged Serratia marcescens and Asaia. Candidate genes identified following sequencing of the GWAS highlighted regions will be silenced using RNAi, and their effect on the total size and composition of the gut flora as well as on infections with malaria parasites and the O'Nyong Nyong virus will be examined. We will also investigate whether these genes function through the REL2 pathway. We have previously shown that the peptidoglycan recognition receptor, PGRPLC, senses gut bacteria and triggers activation of the REL2 pathway. PGRPLC mediates both low-level constitutive activation of REL2 signalling prior to bloodmeal, which is important for homeostasis maintenance, and robust activation of the pathway after a bloodmeal, which is critical in restoring homeostasis. We will experimentally explore our hypothesis that the underlying molecular mechanism involves differential expression of PGRPLC isoforms, linked to infection-regulated alternative splicing of a pool of unspliced PGRPLC transcripts.

The proposed research aims to generate new knowledge about the regulatory mechanisms of the mosquito gut homeostasis and the interactions of the mosquito immune system with the gut microbiota. As identified elsewhere, our project could greatly benefit academic research in this field, in mosquitoes and other insects. In addition, as highlighted by earlier research on insect innate immunity, such discoveries could influence breakthrough discoveries in humans and other higher animals. A prime example is the identification of the immune function of the Drosophila Toll receptor that led to the discovery of the Toll-like receptors (TLRs) in mammals, which led to the Nobel Prize in Physiology and Medicine in 2011, with immense medical and pharmaceutical applications in animal and human health. As a large fraction of the genes found in the mosquito genome have orthologues or homologues in vertebrate animals, the genes (and mechanisms) identified through our genome-wide scans are likely to be present in these animals. Therefore, our proposed research, besides its potential importance in fundamental understanding of the function of the animal immune system and the host-microbe interactions, may aid in future development of therapies aiming to improve the human and animal health. Such translatable results could attract the commercial private sector, including the pharmaceutical industry.

Importantly, A. gambiae and other mosquitoes are vectors of parasites and viruses, which cause devastating diseases in humans and other animals. A central theme of our research is the tripartite, direct or indirect, interactions between the host immune system, the gut microbiota and blood-borne pathogens. We and others have shown that interfering with the gut microbiota, either directly or by manipulating the mosquito immune system can greatly affect the transmission of pathogens. Therefore, our proposed research could directly lead to the development of new approaches to improve the human and animal health, enhance the quality of life and improve the global economic performance, thus attracting both the commercial private and the public sector.