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

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description In the gut of malaria vector mosquitoes, bacteria and malaria parasites are tightly associated. Bacteria are critical modulators of the mosquito immune response against malaria parasites, while responses against bacterial infections can affect malaria parasites.

To simultaneously monitor the malaria parasite and bacterial dynamics in the mosquito gut we developed two new methods to discriminate between and quantify live and dead parasites and commensal bacteria. The first method is based on PCR and the second is based on flow cytometry. These methods can be valuable when studying the molecular interactions between the mosquito, the malaria parasite and midgut microbiota.

To characterize the processes that control mosquito gut bacteria thus possibly affecting the infection by malaria parasites, we combined mosquito gut infections with the bacterium Serratia marcescens with genome-wide discovery and phenotypic analysis of genes involved in antibacterial responses. We revealed complex genetic networks controlling the gut bacterial infection load and ecosystem homeostasis. These networks exhibit much higher specificity toward specific classes of bacteria than previously thought and include behavioural responses involved in antibacterial immunity.

We investigated what is the effect of gut bacteria on malaria infections by feeding mosquitoes on infected blood treated with antibiotics. We showed that elimination of gut bacteria enhances the mosquito infection with malaria parasites and confirmed this effect in semi-natural settings by feeding mosquitoes with antibiotic-treated blood sampled from children naturally infected with the deadly parasite Plasmodium falciparum. Antibiotic exposure additionally increases mosquito survival and fecundity. These findings suggest that malaria transmission may be exacerbated in areas of high antibiotic usage, and that regions targeted by mass antibiotic administration programs against communicable diseases may necessitate increased vector control.

To further study the role of the mosquito gut in controlling bacterial populations, we conducted a transcriptomic analysis of mosquito midguts with their native microbiota or after depletion of the microbiota by antibiotic feeding. We identified several genes that show different responses in the presence or absence of bacteria including genes involved in metabolic processes, immune responses and formation of the peritrophic matrix. The latter is an acellular structure composed of chitin and glycoproteins secreted by the gut epithelium after a blood meal, which physically separates the gut epithelium from its luminal contents. We showed that the synthesis and integrity of the peritrophic matrix is microbiota dependent, and revealed that the peritrophic matrix functions to (a) limit the growth and persistence of bacteria within the gut and (b) prevents seeding of a systemic infection. These findings led us to propose that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract.

Finally, we investigated how Peptidoglycan Recognition Proteins (PGRPs) control the levels of bacteria in the mosquito gut. We demonstrated that PGRPLC2 and PGRPLA are the main receptors of bacterial peptidoglycan on the gut epithelium and that activation of these receptors induced an epithelial antibacterial pathway that is equivalent to the mammalian TNF pathway. We also revealed that following a blood meal, which promotes growth of the gut microbiota, another PGRP, PGRPLB, negatively regulates the activation of this pathway, promoting tolerance for bacterial growth that is needed for the synthesis of essential metabolites such as vitamins.
Exploitation Route The two new techniques that we developed offer enormous potential and possibilities of integration with advanced molecular biochemical techniques for the study of the bacterial communities in disease vector mosquitoes and how these might interact with the malaria parasites and other pathogens.

The novel and exciting data on the role of peritrophic matrix in the mosquito antibacterial responses will be investigated further to understand the mechanisms by which the mosquito gut homeostasis is restored after a blood meal with the involvement of the peritrophic matrix. These data will be essential in the continuation of this project. In addition, this function of the peritrophic matric can be exploited towards vector and malaria transmission control.

Our data about the effect of antibiotic-treated blood meal on malaria transmission can have important implications for public health. Antibiotic usage including mass drug administration is shown to significantly reduce childhood mortality in malaria-endemic countries. Our data suggest that understanding the impact of these antibiotics on malaria transmission may add significant value to such treatments. For example, prescription of an antibiotic shown to enhance malaria transmission could be combined with a recommendation for increased bednet usage, further reducing the exposure to mosquito bites. Such antibiotics could be alternatively co-prescribed with drugs such as atovaquone, which not only combat malaria infection but also block its transmission. This is especially true for young children that are both highly vulnerable to malaria infection and very efficient at transmitting the parasite to mosquitoes. If possible, antibiotics that do not promote or even prevent malaria transmission could be prioritized.
Sectors Education,Environment,Healthcare

 
Description BBSRC IAA
Amount £39,290 (GBP)
Funding ID BB/S506667/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 12/2020
 
Description IBTEC, UNESP, Brazil 
Organisation Sao Paulo State University
Country Brazil 
Sector Academic/University 
PI Contribution This collaboration led to an award by the Brazilian National Council for Scientific and Technological Development of the Ministry of Science, Technology and Innovation (CNPq/MCTI) to dissect the interactions between the Amazonian malaria vector Anopheles darlingi and Plasmodium vivax, including dissecting the role of the midgut microbiota and homeostasis. This allowed the employment of a direct postdoctoral fellow in Brazil and co-supervision of two PhD students at UNESP. Through this collaboration, I visited Brazil several times as a Special Visiting Researcher and worked with my collaborator Sr Jayme Souza-Neto, the postdoc and students to design experiments and data.
Collaborator Contribution Dr Souza-Neto has sponsored the Special Visiting Researcher award and assisted with co-supervision of the postdoc and PhD students. He also visited Imperial several times to work together on data analysis. The postdoctoral fellow has visited Imperial in November 2016 for carrying out collaborative work.
Impact No publication yet. Research grant by the Brazilian National Council for Scientific and Technological Development of the Ministry of Science, Technology and Innovation (CNPq/MCTI) to dissect the interactions between the Amazonian malaria vector Anopheles darlingi and Plasmodium vivax (314577/2014-9)
Start Year 2015
 
Description University of Jimma 
Organisation University of Jimma
Country Ethiopia 
Sector Academic/University 
PI Contribution It enables data produced in the laboratory to be tested in field settings.
Collaborator Contribution It enables data produced in the laboratory to be tested in field settings.
Impact No publications or any other outputs yet from this collaboration.
Start Year 2013
 
Description VLIR Ethiopia 
Organisation University of Ghent
Department Department of Comparative Physiology and Biometrics
Country Belgium 
Sector Academic/University 
PI Contribution It facilitated access to infrastructures in developing countries so that data produced in the laboratory can be directly tested in the field.
Collaborator Contribution It facilitated access to infrastructures in developing countries so that data produced in the laboratory can be directly tested in the field.
Impact Academic outcomes, including 2 publications so far.
Start Year 2013