First large-scale comparative proteomics/phosphoproteomics study characterizing some of the key steps that contributed to the remodeling of phagosomes that occurred during evolution. Comparison of profiling analyses of isolated phagosomes from three distant organisms (Dictyostelium, Drosophila, and mouse) revealed a protein core that defines a potential ‘ancient' phagosome and a set of 50 proteins that emerged while adaptive immunity was already well established.Gene duplication events of mouse phagosome paralogs occurred mostly in Bilateria and Euteleostomi, coinciding with the emergence of innate and adaptive immunity, and thus, provided the functional innovations needed for the establishment of these two crucial evolutionary steps of the immune system.Phosphoproteomics of isolated phagosomes from the same three distant species indicate that the phagosome phosphoproteome has been extensively modified during evolution. Still, some phosphosites have been maintained for >1.2 billion years, and thus, highlight their particular significance in the regulation of key phagosomal functions.
Phagocytosis is the process by which multiple cell types internalize large particulate material from the external milieu. The functional properties of phagosomes are acquired through a complex maturation process, referred to as 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 degradative environment encountered in the phagosome lumen has enabled the use of phagocytosis as a predation mechanism for feeding (phagotrophy) in amoeba, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in jawed vertebrates (fish), initiate a sustained immune response.
High-throughput proteomics profiling of isolated phagosomes has been tremendously helpful for the molecular comprehension of this organelle. This approach is achieved by feeding low buoyancy latex beads to phagocytic cells, enabling the subsequent isolation of latex bead-containing phagosomes, away from all the other cell organelles, by a single-isopicnic centrifugation in sucrose gradient. In order to characterize some of the key steps that contributed to the remodeling of phagosomes during evolution, 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, and performed detailed proteomics and phosphoproteomics analyses with unparallel protein coverage for this organelle (two- to four-fold enhancements in identified proteins).
In order to establish the origin of the mouse phagosome proteome, we performed comparative analyses among 39 taxa including plants/algea, unicellular organisms, fungi, and more complex animal multicellular organisms. These genomic comparisons indicated that a large proportion of the mouse phagosome proteome is of ancient origin (73.1% of the proteome is conserved in eukaryotic organisms) (Figure 2A). This stresses the fact that phagocytosis is a very ancient process, as shown by its possible involvement in the emergence of eukaryotic cells (eukaryogenesis). Indeed, we identified close to 300 phagosome mouse proteins also present on Drosophila and Dictyostelium phagosomes, defining a potential ‘ancient' core of proteins from which the immune functions of phagosomes likely evolved. Around 16.7% of the mouse phagosome proteins appeared in organisms that use phagocytosis for innate immunity (Bilateria to Chordata), whereas 10.2% appeared in Euteleostomi or Tetrapoda where phagosomes have an important function in linking the killing of microorganisms with the development of a specific sustained immune response following antigen recognition. The phagosome is made of molecules taken from a variety of sources within the cell, including the cytoplasm, the cytoskeleton and membrane organelles. Despite the evolution and diversification of these various cellular systems, the mammalian phagosome proteome is made preferentially of ancient proteins (Figure 2B). Comparison of functional annotation during evolution highlighted the emergence of specific phagosomal functions at various steps during evolution (Figure 2C). Some of these proteins and their point of origin during evolution are highlighted in Figure 2D. Strikingly, we identified in Tetrapods a set of 50 proteins that arose while adaptive immunity was already well established in teleosts (fish), indicating that the phagocytic system is still evolving.
Our study highlights the fact that the functional properties of phagosomes emerged by the remodeling of ancient molecules, the addition of novel components, and the duplication of existing proteins (paralogs) leading to the formation of molecular machines of mixed origin. Gene duplication is a process that contributed continuously to the complexification of the mouse proteome during evolution. In sharp contrast, paralog analysis indicated that the phagosome proteome was mainly reorganized through two periods of gene duplication, in Bilateria and Euteleostomi, coinciding with the emergence of adaptive immunity (in jawed fish), and innate immunity (at the split between Metazoa and Bilateria). These results strongly suggest that selective constraints may have favored the maintenance of phagosome paralogs to ensure the establishment of novel functions associated with this organelle at these two crucial evolutionary steps of the immune system.
The emergence of genes associated to the MHC locus in mammals that appeared originally in the genome of jawed fishes, contributed to the development of complex molecular mechanisms linking innate (our immune system that defends the host from infection in a non-specific manner) and adaptive immunity (the part of the immune system triggered specifically after antigen recognition). Several of the genes of this locus encode proteins known to have important functions in antigen presentation, such as subunits of the immunoproteasome (LMP2 and LMP7), MHC class I and class II molecules, as well as tapasin and the transporter associated with antigen processing (TAP1 and TAP2), involved in the transport and loading of peptides on MHC class I molecules (Figure 6). In addition to their ability to present peptides on MHC class II molecules, phagosomes of vertebrates have been shown to be competent for the presentation of exogenous peptides on MHC class I molecules, a process referred to as cross-presentation. From a functional point of view, the involvement of phagosomes in antigen cross-presentation is the outcome of the successful integration of a wide range of multimolecular components that emerged throughout evolution (Figure 6). The trimming of exogenous proteins into small peptides that can be loaded on MHC class I molecules is inherited from the phagotrophic properties of unicellular organisms, where internalized bacteria are degraded into basic molecules and used as a source of nutrients. Ancient processes have therefore been co-opted (the use of an existing biological structure or feature for a new function) for new functionalities. A summarizing model of the various steps that enabled phagosome antigen presentation is presented in Figure 6. This model highlights the fact that although antigen presentation is unique to evolutionary recent phagosomes (starting in jawed fishes about 450 million years ago), it uses and integrates molecular machines composed of proteins that emerged throughout evolution.
In summary, we present here the first large-scale comparative proteomics/phosphoproteomics study characterizing some of the key evolutionary steps that contributed to the remodeling of phagosomes during evolution. Functional properties of this organelle emerged by the remodeling of ancient molecules, the addition of novel components, the extensive adaption of protein phosphorylation sites and the duplication of existing proteins leading to the formation of molecular machines of mixed origin.
Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an ‘ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period 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.