IMPROVEMENT OF METAGENOMIC TECHNIQUES TO BETTER UNDERSTAND THE ROLE OF CANDIDATE PHYLA RADIATION IN FOOD ALLERGIES

Food allergies are local or systemic reactions that occur after the ingestion of normally innocuous food antigens (allergens) and can be triggered by various factors. The oral microbiota plays a critical role in the development of immune tolerance and is one of the factors involved. During my PhD I participated in a study in which the oral microbiota of food-allergic children was compared to healthy controls, in order to identify a microbial “signature” that characterizes this pathological state. The results of this study led us to continuing the investigation by focusing on a specific group of bacteria which we found to be enriched in the oral microbiota of allergic children: “Candidatus Saccharibacteria”. This group belongs to the Candidate Phyla Radiation (CPR), a category of peculiar bacteria that accounts for at least 25% of the whole bacterial domain. Several researches indicate that, within the human microbiota, CPR bacteria play a role in immune modulation and consequently they might too in food allergy development. However, the detection of CPR is challenging due to their unique characteristics: small size, almost unculturable, unusual aminoacidic code, peculiar 16S rRNA gene with introns and indels. Therefore, molecular methods currently used, such as 16S rRNA amplicon metagenomics, often underestimate CPR and prevent a fully understanding of their role in human physio-pathology. This led us to evaluate other existing methods to quantify CPR: we performed a comparative study between currently available qPCRs protocols targeting 16S rRNA and 23S rRNA genes for the quantification of “Candidatus Saccharibacteria”. Furthermore, we performed an in silico study to identify a novel target for amplicon metagenomics studies, capable of adequately quantifying CPR bacteria as well, which identified RecA as a novel gene target for pan-bacterial metagenomics studies. Finally, we compared different protocols for DNA extraction from human saliva to obtain an extract enriched in CPR DNA. This study led us to obtain an efficient laboratory protocol for enriching "Candidatus Saccharibacteria" in human saliva samples. This method will be necessary to perform genomic studies on these difficult-to-culture bacteria, in order to deepen our knowledge of their genomic features and better understand their involvement in human health and disease states.