Abstract
Macrophages are specialized cells of the immune system which mediate destruction and killing of invading micro-organisms. They do so by engulfing them by a process called phagocytosis. Microbes are then captured in an intracellular compartment, the phagosome, which gradually acquire molecules able to attack and degrade its cargo. Use of proteomics let us demonstrate the presence of flotillin-1 enriched microdomains (also called lipid rafts or membrane rafts) on the phagosomes. Our team
demonstrated the crucial importance of these rafts in the phagocytosis process. Indeed, survival of L. donovani correlates with its presence in a ‘raftless’ phagosome while a mutated L. donovani without LPG is rapidly killed in a phagosome containing lipid rafts.
To understand the membrane raft destabilisation mechanism mediated the LPG molecule, we induced phagocytosis of parasites devoid of LPG (LPG-) and compared it to the wild type parasite by microscopy. We first demonstrated that LPG alone is necessary to prevent normal maturation of the phagosome. Additionally, we discovered that the LPG molecule not only inhibits lipid rafts formation on the phagosome but also disorganise pre-existing lipid rafts. This effect of LPG is proportional to the number of repetitive sugar units (Gal( 1,4)-Man 1-PO4) which compose this molecule. Our work demonstrated for the first time an important role of
the membrane rafts in the phagosome maturation. Moreover, our conclusions will give new interesting leads for clinical studies on leishmaniosis.
The second goal of this work was to characterise them with proteomics and lipidomics tools. To do this, we undertook the systematic identification of proteins present on both subdomains of the phagosome (lipid rafts versus the rest of the phagosomal membrane). To achieve this, we purified phagosomes, from which we isolated lipid rafts by floating Triton X-100 insoluble membranes (DRMs for Detergent Insoluble Membranes). After that, we identified proteins by mass spectrometry. Because phagosome is not a static organelle and its protein composition is constantly changing, we did our analysis on three representative maturation time-points (early phagosomes, intermediate phagosomes and phagolysosomes). Thereby, we established a list of 921 phagosome-associated proteins including 352 associated to DRMs. These proteins have three different behaviours during phagosome maturation; some of them decrease in quantity, others increase, while the rest are transiently present. Each of these kinetic sub-groups covers about a third of the phagosome proteins, while the
group where proteins are increasing during maturation is particularly abundant in DRMs. This shows that early phagosomes contain either very few lipid rafts or rafts with low amounts of proteins, and these rafts are recruited during phagosome maturation. We also analysed phospholipids of the phagosome membrane and found that the proportions of the classes changes with time. Moreover, by looking specifically to different species of phospholipids, we noticed that the main species of the cell are not necessary the most important in the phagosome membrane.
Together, these results let us discover several potential functions of the phagosome membrane rafts (signalling, membrane fusion, microbicidal processes, transmembrane transport and actin dynamics). Furthermore, kinetics of the proteins acquisition on the membrane rafts reveals that these are playing their roles mainly on matures phagosomes. This increasing number of proteins in microdomains is accompanied by a
modulation of the phospholipids, which let us think that membrane rafts assemble themselves gradually on the phagosome and this is not only proteins that are imported into them.
Keywords : phagosome, lipid rafts, membrane rafts, proteomics, Leishmania, immunity