Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum

Abstract

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. Molecular

Proteome and Structural Organization of the Knob Complex on the Surface of the Plasmodium Infected Red Blood Cell

“…While the two samples were separately analysed by MS, nowhere in the entire study it is indicated if PfEMP1 was detected from the soluble fraction or the membrane fraction. Another study by Yam et al in 2013 described proteomics analysis of total detergent resistant membrane microdomains in the trophozoite stage and this study also does not report detection of PfEMP1 by MS . The above data from published literature suggests that the detection of PfEMP1 by mass spectrometric approaches has been possible only from soluble fractions of the parasite during its transport and not from detergent resistant membranes where it is ultimately located.…”

“…PfEMP1, the chief cytoadherent protein known to be present on the detergent‐resistant membranes (DRMs) of infected erythrocytes, was not detectable in our proteomics analysis. A detailed analysis of existing literature on proteomics studies in the parasite reveals that till date PfEMP1 has been determined through MS approaches only in the soluble fractions of the parasite and never in DRMs . This is in spite of overwhelming evidences from literature that have established beyond doubt that PfEMP1 proteins are integral components of knobs which are present in DRMs.…”

Interaction of pathogens with host cholesterol metabolism

“…to deplete it of rafts, rather than to satisfy a need in cholesterol for the parasite. Trophozoites recruit cholesterol from lipid rafts of red blood cells disrupting signalling and possibly affecting immune response toward infected cells [37]. …”

An Integrated Approach to Explore Composition and Dynamics of Cholesterol-rich Membrane Microdomains in Sexual Stages of Malaria Parasite

“…Sample Preparation for Proteomic Analysis-Proteins extracted from DRM fractions 2-8 were loaded on SDS-PAGE (home-made 5% stacking-12% resolving bis-tris-PAGE) and run just to allow the pro-tein marker (BenchMark™ Pre-Stained Protein Ladder, Invitrogen, Carlsbad, CA) to enter the resolving gel. After Coomassie Staining (Novex, Colloidal Blue Staining gel, Invitrogen) unresolved bands were excised and in-gel tryptic digestion was performed as already described (16) with slight modifications. Briefly, gel slices were destained by washings in acetonitrile (ACN)/50 mM NH4CO3 (1:1), treated with 10 mM DTT (40 min at 56°C) and 55 mM iodoacetamide (30 min in the dark at RT) to reduce and alkylate cysteines, shrunken with ACN and rehydrated for 40 min on ice with a solution of 12,5 ng/l trypsin (Promega, Madison, WI) in 50 mM NH 4 CO 3 and protein digestion was carried out overnight at 37°C.…”

“…Previous studies on Plasmodium asexual blood stages indicated that CH-rich membrane microdomains participate to various biological processes, such as protein sorting/trafficking, host cell invasion, organelle biogenesis and membrane remodeling with a key function in parasite virulence and pathogenesis (13)(14)(15)(16). A specific role of these assemblies in sexual stages has not been investigated so far.…”