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

Federica Fratini,Carla Raggi,Gabriella Sferra,Cecilia Birago,Anna Sansone,Felicia Grasso,Chiara Currà,Anna Olivieri,Tomasino Pace,Stefania Mochi,Leonardo Picci,Carla Ferreri,Antonella Di Biase,Elisabetta Pizzi,Marta Ponzi

doi:10.1074/mcp.m117.067041

Abstract

Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.

Highlights

From the ‡Istituto Superiore di Sanita, Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate; §Istituto Superiore di Sanita, Dipartimento di Biologia Cellulare e Neuroscienze; ¶Consiglio Nazionale delle Ricerche, I.S.O.F. – Bio Free Radicals; ʈIstituto Superiore di Sanita, Dipartimento di Sanita Pubblica Veterinaria e Alimentare
A Pipeline for the Generation of a “detergent resistant membranes (DRMs) Barcode”—In this study we propose and validate a method based on label-free quantitative proteomics and lipid analysis to define DRM components, and their flotation properties, following enrichment by sucrose gradient centrifugation (Fig. 1)
To gain insights into conserved and stage-specific functions associated with these membrane assemblies, we compared DRM proteome and lipid components of sexual stages with those obtained from the asexual trophic stage

Summary

Introduction

From the ‡Istituto Superiore di Sanita , Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate; §Istituto Superiore di Sanita , Dipartimento di Biologia Cellulare e Neuroscienze; ¶Consiglio Nazionale delle Ricerche, I.S.O.F. – Bio Free Radicals; ʈIstituto Superiore di Sanita , Dipartimento di Sanita Pubblica Veterinaria e Alimentare. Biological reproducibility of PAPs was assessed by probing DRM gradient fractions of two independent preparations of mature gametocytes (MG-B and C) and trophozoites (T-B and C) with antibodies against the proteins under study (supplemental Fig. S3).

Results

Conclusion