L'évolution du phagosome

Phagocytosis is a cellular process by which large particulate material are internalized in a newly formed vesicule, the phagosome.  Once formed, the phagosome acquires its functional properties through a complex maturation process called phagolysosome biogenesis.  This pathway involves a series of rapid interactions with organelles of the endocytic apparatus, enabling the gradual transformation of newly formed phagosomes into phagolysosomes in which proteolytic degradation occurs.  The amoeba Dictyostelium discoideum uses phagocytosis as a predation mechanism for feeding, whereas multicellular organisms utilize this process as an immune mechanism where specialized cells named phagocytes internalize, kill and degrade phatogens found through the host, forming the basis of innate immunity.  In jawed verterbrates however, the phagosome links innate and adaptive immunity by processing and presenting antigenic peptides.  Despite its crucial role in immunity, little is known about the composition and the functional organization of the phagosome.  It is therefore essential to characterize in details the functional properties that are recruited to the phagosome.   High-throughput proteomics analysis of isolated phagosomes has been tremendously helpful for the molecular comprehension of this organelle.  Studies of our lab notably have revealed the first proteomics identification of mouse phagosomes without determining the roles of these proteins through the complex process of phagocytosis (Brunet et al, 2003; Garin et al, 2001).  In the first study of this thesis (Stuart et al, 2007), we characterized the functions of the entire drosophila phagosome proteome by combining high-throughput proteomics, interactive networks and RNAi.  By applying this strategy, we’ve identified 617 phagosomal proteins by mass spectrometry from which we’ve expanded this list by building the phagosome interactome.  The contribution of each protein to bacterial internalization was tested and validated by RNAi and led to the identification of a new regulator of phagocytosis, the exocyst complex.  In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.      In the second study of this thesis (Boulais et al, 2010), we characterized some of the key steps that contributed to the remodeling of phagosomes functional properties during evolution. To do so, we isolated this organelle from three distant organisms: the amoeba Dictyostelium discoideum, the fruit fly Drosophila melanogaster, and mouse (Mus musculus) that use phagocytosis for different purposes.  By performing and comparing proteomics and phosphoproteomics analyses of isolated phagosomes from the three species, we identified an ancient core of phagosomal proteins around which the immune function of this organelle have likely organized.  Our data indicate that a larger proportion of the phagosome proteome, has been acquired through gene duplication at periods coinciding with the emergence of innate and adaptive immunity.  Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.

Keyphrases: Proteomics, Evolution, Systems biology, Phagotrophy, Innate immunity, Adaptive immunity, Phosphoproteomics, Comparative genomics, Phagocytosis, Exocyst, Exocyst