Malaria parasites release vesicle subpopulations with signatures of different destinations

Abstract

Malaria is the most serious mosquito-borne parasitic disease, caused mainly by the intracellular parasite Plasmodium falciparum. The parasite invades human red blood cells and releases extracellular vesicles (EVs) to alter its host responses. It becomes clear that EVs are generally composed of sub-populations. Seeking to identify EV subpopulations, we subject malaria-derived EVs to sizeseparation analysis, using asymmetric flow field-flow fractionation. Multi-technique analysis reveals surprising characteristics: we identify two distinct EV subpopulations differing in size and protein content. Small EVs are enriched in complement-system proteins and large EVs in proteasome subunits. We then measure the membrane fusion abilities of each subpopulation with three types of host cellular membranes: plasma, late and early endosome. Remarkably, small EVs fuse to early endosome liposomes at significantly greater levels than large EVs. Atomic force microscope imaging combined with machine-learning methods further emphasizes the difference in biophysical properties between the two subpopulations. These results shed light on the sophisticated mechanism by which malaria parasites utilize EV subpopulations as a communication tool to target different cellular destinations or host systems.

Extracellular vesicles from malaria‐infected red blood cells: not all are secreted equal

“… Using different biophysical techniques, Abou Karam et al ( 2022 ) characterize two distinct extracellular vesicle (EV) subpopulations with different sizes, protein content, membrane biophysical properties, and membrane fusion capabilities. The smaller F3‐EVs (30–70 nm) had more densely packed lipid membranes and demonstrated better fusion capability to early endosomal conditions as compared to the larger F4‐EVs (70–300 nm), suggesting that each EV subpopulation may traffic to different recipient cells or subcellular locations, where they could mediate divergent functions in the host–pathogen interaction and disease.…”

“…In this issue of EMBO Reports, Abou Karam and colleagues take advantage of recent advancement in EV isolation methods to separate and characterize two EV subpopulations generated from red blood cells infected with the protozoan parasite Plasmodium falciparum (Pf) (Abou Karam et al , 2022 ). These parasites are the causative agent of malaria, which represents a huge global public health burden.…”

“…In this study, Abou Karam et al ( 2022 ) employed a sophisticated combination of techniques to separate and characterize EVs generated by Pf ‐infected red blood cells. They used asymmetrical flow field‐flow fractionation (AF4), a method that separates extracellular nanoparticles based on their hydrodynamic size, (Phillips et al , 2021 ), to identify two EV subpopulations (Fig 1 ) with distinct size ranges of 30–70 nm (F3‐EVs) and 70–300 nm (F4‐EVs).…”

“…The sizes of the Pf‐ iRBC EVs were further confirmed using atomic force microscopy (AFM) and cryo‐transmission electron microscopy. Liquid chromatography with tandem mass spectrometry (LC–MS‐MS) of these subpopulations identified 132 proteins in total in both fractions, 23 of which were P. falciparum ‐derived proteins, and 109 of which were human, including proteins that may mediate vesicle fusion (Abou Karam et al , 2022 ). Of these, 66 proteins had differential abundance in the two EV fractions: 6 P. falciparum proteins and 60 human proteins, demonstrating that F3‐EVs and F4‐EVs contain different protein cargos that include both parasite‐ and human‐derived factors.…”

“…The two EV subpopulations also had distinct membrane characteristics, as demonstrated by Laurdan staining, which is sensitive to the polarity and fluidity of membranes, and atomic force microscopy puncture analysis, which measures the force required to punch small holes in the EV membrane and serves as a readout for the mechanical properties of the membrane (Abou Karam et al , 2022 ). Both assays indicated that the membranes of smaller F3‐EVs had denser lipid packing compared to F4‐EVs, which could impact EV tolerance to different subcellular environments and could impact their fusion properties.…”