Aedes albopictus, Wolbachia, arbovirus tri-partite interaction : molecular and cellular mechanisms of microbial interference

L'émergence et la réémergence d'arboviroses au niveau mondial sont responsables de pathologies pouvant causer le décès des personnes infectées et elles sont transmises par des moustiques vecteurs. En l'absence de prophylaxies efficaces, les moyens de lutte actuels reposent essentiellement sur l'utilisation d'insecticides, mais ces derniers sont nocifs pour les écosystèmes et des résistances apparaissent chez la plupart des vecteurs. Il devient nécessaire de développer des moyens de lutte alternatifs. La découverte du potentiel antiviral de certaines bactéries symbiotiques des moustiques a ouvert de nouvelles voies pour le développement de la lutte biologique. Mon projet de thèse porte sur l'étude des mécanismes déclenchés dans l'interaction tripartite entre le moustique tigre Aedes albopictus, ses deux souches de Wolbachia, wAlbA et wAlbB, et deux arbovirus (Chikungunya et Dengue) dont il est un vecteur avéré. Originaire d'Asie du Sud Est, il est devenu une préoccupation majeure de santé publique, notamment pour son fort pouvoir invasif, ayant colonisé les quatre autres continents, ainsi que pour son statut de vecteur de nombreux arbovirus et certaines filaires. Les résultats de nos travaux ont montré un potentiel antiviral de la bactérie chez les moustiques infectés par Wolbachia en comparaison des moustiques aposymbiotiques. Les analyses du protéome et du transcriptome ont permis d'identifier certains mécanismes cellulaires et moléculaires du moustique spécifiquement modulés par la présence de Wolbachia ou des arbovirus, mais aussi deux partenaires microbiens simultanément. Ces approches ont permis de mettre en évidence le rôle important du métabolisme dans le maintien de l'intégrité cellulaire et du système immunitaire pour contrôler l'infection virale. En outre, nos résultats révèlent un patron de compétition entre bactérie et arbovirus contribuant à la compréhension du phénomène interférence observé chez les moustiquesThe emergence and reemergence of arboviruses in the world are responsible for pathologies that can cause the death of infected and they are transmitted by mosquitoes. In absence of effective prophylaxis, current control methods are essentially based on the use of insecticides, but they are harmful to ecosystems and resistances appear in most vectors. It becomes necessary to develop alternative means of control. Discovery of the antiviral potential of some symbiotic bacteria mosquito has opened new avenues for the development of biological control. My PhD project focuses on the study of mechanisms triggered in the tripartite interaction between the Asian tiger mosquito, Aedes albopictus, two strains of Wolbachia, and wAlbA wAlbB and two Arbovirus (Chikungunya and Dengue) of which he is a proven vector. Native to Southeast Asia, it has become a major public health concern, particularly for its highly invasive, have colonized the four other continents, as well as its status vector many arboviruses and some wire. The results of our work has shown antiviral potential of bacteria in mosquitoes infected with Wolbachia in comparison aposymbiotic mosquitoes. The analyzes of the proteome and transcriptome have identified certain cellular and molecular mechanisms of mosquito specifically modulated by the presence of Wolbachia or arboviruses, but also two microbial partners simultaneously. These approaches have helped to highlight the important role of metabolism in maintaining cell integrity and immune system to control the viral infection. In addition, our results reveal a pattern competition between bacteria and arbovirus contributing to understanding the phenomenon interference observed in mosquitoe

Native Wolbachia from Aedes albopictus Blocks Chikungunya Virus Infection In Cellulo

International audienceWolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". However, in some cases Wolbachia may have no effect or even enhance pathogen infection, depending on the host-Wolbachia-pathogen combination. The tiger mosquito Aedes albopictus is naturally infected by two strains of Wolbachia, wAlbA and wAlbB, and is a competent vector for different arboviruses such as dengue virus (DENV) and Chikungunya virus (CHIKV). Interestingly, it was shown in some cases that Ae. albopictus native Wolbachia strains are able to inhibit DENV transmission by limiting viral replication in salivary glands, but no such impact was measured on CHIKV replication in vivo. To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain. Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB. Using FISH, we observed CHIKV and wAlbB in the same mosquito cells, indicating that the virus is still able to enter the cell in the presence of the bacterium. Further work is needed to decipher molecular pathways involved in Wolbachia-CHIKV interaction at the cellular level, but this cellular model can be a useful tool to study the mechanism behind virus blocking phenotype induced by Wolbachia. More broadly, this underlines that despite Wolbachia antiviral potential other complex interactions occur in vivo to determine mosquito vector competence in Ae. albopictus

Comprehensive proteome profiling in Aedes albopictus to decipher Wolbachia-arbovirus interference phenomenon

International audienceBackground: Aedes albopictus is a vector of arboviruses that cause severe diseases in humans such as Chikungunya, Dengue and Zika fevers. The vector competence of Ae. albopictus varies depending on the mosquito population involved and the virus transmitted. Wolbachia infection status in believed to be among key elements that determine viral transmission efficiency. Little is known about the cellular functions mobilized in Ae. albopictus during co-infection by Wolbachia and a given arbovirus. To decipher this tripartite interaction at the molecular level, we performed a proteome analysis in Ae. albopictus C6/36 cells mono-infected by Wolbachia wAlbB strain or Chikungunya virus (CHIKV), and bi-infected.Results: We first confirmed significant inhibition of CHIKV by Wolbachia. Using two-dimensional gel electrophoresis followed by nano liquid chromatography coupled with tandem mass spectrometry, we identified 600 unique differentially expressed proteins mostly related to glycolysis, translation and protein metabolism. Wolbachia infection had greater impact on cellular functions than CHIKV infection, inducing either up or down-regulation of proteins associated with metabolic processes such as glycolysis and ATP metabolism, or structural glycoproteins and capsid proteins in the case of bi-infection with CHIKV. CHIKV infection inhibited expression of proteins linked with the processes of transcription, translation, lipid storage and miRNA pathways.Conclusions: The results of our proteome profiling have provided new insights into the molecular pathways involved in tripartite Ae. albopictus-Wolbachia-CHIKV interaction and may help defining targets for the better implementation of Wolbachia-based strategies for disease transmission control

Electron microscopy of <i>Wolbachia</i> in <i>Aedes albopictus</i> C6/36 cells.

<p>Low-magnification transmission electron micrograph of C6/36_TET cells with no bacterial signal in host cell cytoplasm (<b>A</b>) whereas <i>Wolbachia</i> (white arrowhead) are seen throughout the cytoplasm of C6/36_<i>w</i>AlbB cells (<b>B</b>). <i>Wolbachia</i> presumably is undergoing the process of cell division (<b>C</b>). High-magnification micrograph of <i>Wolbachia</i> in cytoplasm of the host cell showing a membranous structure surrounding the bacterium (black arrowhead) (<b>D</b>).</p

Identification of human skin bacteria attractive to the Asian Tiger mosquito

International audienceAedes albopictus is a vector of arboviruses and filarial nematodes. Originating from Asia, this mosquito has rapidly expanded its geographical distribution and colonized areas across both temperate and tropical regions. Due to the increase in insecticide resistance, the use of environmentally friendly vector control methods is encouraged worldwide. Using methods based on semiochemicals in baited traps are promising for management of mosquito populations. Interestingly, human skin microbiota was shown to generate volatile compounds that attract the mosquito species Anopheles gambiae and Aedes aegypti. Here, we investigated the composition of skin bacteria from different volunteers and the attractive potential of individual isolates to nulliparous Ae. albopictus females. We showed that three out of 16 tested isolates were more attractive and two were more repulsive. We identified dodecenol as being preferentially produced by attractive isolates and 2-methyl-1-butanol (and to a lesser extent 3-methyl-1-butanol) as being overproduced by these isolates compared with the other ones. Those bacterial volatile organic compounds represent promising candidates but further studies are needed to evaluate their potential application for baited traps improvement

Effect of <i>Wolbachia</i> on CHIKV replication and infectiosity.

<p>Kinetics at MOI 0.1 of CHIKV RNA titer measured by RT-qPCR on total cellular RNA (<b>A</b>) and CHIKV infectious titer in supernatant measured by FFA (<b>B</b>) in presence of <i>Wolbachia</i> (wAlbB) or in cells cured from the bacteria by tetracycline treatment (TET). Error bars represent the standard deviation of the mean of three independent samples.</p

Detection and localization of <i>w</i>AlbB and CHIKV <i>in cellulo</i> by FISH.

<p>Detection of <i>Wolbachia</i> 16S rRNA gene (red) and CHIKV <i>Env</i> RNA (green) using oligonucleotide probes labelled with Rhodamine and Alexa488, respectively. <i>Wolbachia</i> signal is detected in C6/36_<i>w</i>AlbB but not in tetracycline treated cells (C6/36_TET). CHIKV signal is detected only in CHIKV infected modality, in the absence or in the presence of <i>Wolbachia</i> where it co-localize with the bacteria in the cytoplasm of C6/36_<i>w</i>AlbB cells. Nuclei of host cells are shown in blue after DAPI labelling (bars = 10 μm).</p

Effect of tetracycline treatment on CHIKV growth in C6/36.

<p>Kinetics of CHIKV RNA titer at MOI 0.1 (<b>A</b>) and 3 (<b>B</b>) measured by RT-qPCR on total cellular RNA isolated from C6/36 cells (non infected by <i>Wolbachia</i>) treated with tetracycline (TET) or not (CTRL). Error bars represent the standard deviation of the mean of two independent samples.</p