EXPLORING BACTERIAL MOLECULAR FACTORS MODULATING THE SYMBIOTIC INTERACTIONS WITH INSECTS

Many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. Homeostatic interactions between insects and commensal microbes are widespread in nature. Commensal microbes have many roles in the biology and lifecycle of most insect species, affecting different aspects of their life. The recently recognized acetic acid bacteria (AAB) are the most abundant microorganisms in the insects and also metabolically linked to one another. These symbionts establish close interactions with their animal host, including insects and mosquitoes. In particular, gut microbiota establishes strict interactions with its host and, indeed, close interactions are established among the different microbial partners. Fine regulation of the immunity in the host gut is required for homeostasis of gut microbiota. A recent study highlighted the role of bacterium-secreted uracil as a signal molecule controlling immunity in the gut of flies. Moreover, diversity of microbial symbionts suggests that synergistic activity in their role is favored. This gut bacterial symbionts have a role in the fitness of insects that are vectors of the most severe diseases in plant, animal and human affecting the agricultural production and the environment. The mosquitoes Anopheles are vectors of Plasmodium parasites, the causative agents of malaria. Asaia is a useful model for the study of promising tools in the control of disease-transmitting mosquitoes like Anopheles and AAB symbionts. The aim of my Ph.D. research work was to explore the molecular factors of acetic acid bacteria (AAB) involved in the interaction with the host in order to unveil targets addressable for novel strategies of pathogen biocontrol. The purpose was challenged by a comparative proteomic approach considering Asaia SF2.1 as a model AAB-symbiont. Since this approach is based on the analysis of two-dimensional electrophoresis (2D-PAGE) protein profiles, it requires a consistent amount of bacterial-specific protein that cannot be achieved in the gut environment. To this end, hypoxia was applied as a model condition eliciting or suppressing pathways possibly involved in the symbiotic interaction. Hypoxia condition was applied to mimic the insect gut condition, where the oxygen concentration is usually low from 2% to 8%. Microbial population of model host insects (Drosophyla suzukii and Anopheles stephensi) was characterized by using 16S rRNA and ITS-PCR. The analysis showed that Asaia SF2.1 is among the most abundant acetic acid bacteria (AAB) species. Most abundant AAB species were selected from each genus (i.e. Gluconobacter, Acetobacter, Komagataeibacter and Asaia) and the biomass production was defined. Furthermore, for all strains, conditions for protein extraction methods were optimized followed by protein quality assessment and quantification. Asaia SF2.1 was selected for comparative proteomic approach based on the availability of genome sequence, protein quality and quantity. Different sample preparation methods were used for the setup of protein extraction. The best method for protein extraction was selected and was followed by optimization of the 2D-PAGE run conditions. Using bioreactor, hypoxia (6%) and normal (20%) condition (control) of Asaia SF2.1 were setup and growth data evaluated. 2D-PAGE protein profiles of more than 220 spots were obtained by applying the optimized protocol. Total 12 protein spots with altered expression differences were observed and the corresponding proteins were identified by mass spectrometry technique. MS analysis identified different proteins from the selected spots and functional categorization of proteins was carried out according to the annotated information. Protein spots were mostly identified as being involved in transcription, cellular respiratory function, cell wall biogenesis, protein biosynthesis, pentose-phosphate shunt. Gene ontology analysis suggested interesting putative candidate pathways involved in endosymbiosis, thus paving targets for biocontrol strategies.