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

Extracellular vesicles (EVs) mediate the transfer of molecules between cells and play diverse roles in host–pathogen interactions. Malaria is an important disease caused by intracellular Plasmodium species that invade red blood cells and these red blood cells release EVs. The EVs from infected cells have diverse functions in the disease and an obstacle in understanding how they exert their functions is that multiple EV types exist. In this issue of EMBO reports, Abou Karam and colleagues use sophisticated biophysical techniques to isolate and characterize two EV subpopulations produced by red blood cells infected with Plasmodium falciparum (Abou Karam et al, 2022). The authors show that these EV subpopulations have distinct sizes, protein content, membrane packing, and fusion capabilities, suggesting that EV subpopulations from infected cells could target different cell types and subcellular locations. This work underscores the concept that understanding EV heterogeneity will go hand in hand with understanding EV functions.


Frances Blow & Amy H Buck*
Extracellular vesicles (EVs) mediate the transfer of molecules between cells and play diverse roles in host-pathogen interactions. Malaria is an important disease caused by intracellular Plasmodium species that invade red blood cells and these red blood cells release EVs. The EVs from infected cells have diverse functions in the disease and an obstacle in understanding how they exert their functions is that multiple EV types exist. In this issue of EMBO reports, Abou Karam and colleagues use sophisticated biophysical techniques to isolate and characterize two EV subpopulations produced by red blood cells infected with Plasmodium falciparum (Abou Karam et al, 2022). The authors show that these EV subpopulations have distinct sizes, protein content, membrane packing, and fusion capabilities, suggesting that EV subpopulations from infected cells could target different cell types and subcellular locations. This work underscores the concept that understanding EV heterogeneity will go hand in hand with understanding EV functions. EMBO Reports (2022) 23: e55499 See also: P Abou Karam et al (2022) E xtracellular vesicles (EVs) are small lipid bilayered structures released by cells into the environment, where they can be taken up by other cells as a form of cell-to-cell communication that impacts normal and disease physiology. There has been an explosion of interest in studying the functions of EVs in a range of disease contexts, with excitement that harnessing or inhibiting EVs could help diagnose and treat disease. This also extends to infectious diseases, since many pathogens exploit EVs as a communication mechanism to move their molecules into host cells, to signal to other pathogens, or to alter the host environment to favor pathogen survival. In the case of malaria, EVs derived from red blood cells infected with the Plasmodium species have been shown to activate immune cells, communicate information between parasites, and alter a range of host cell types to enable infection (reviewed in Opadokun & Rohrbach, 2021). Work in the last 5 years from various biological systems has shown that multiple EV types can be simultaneously released from cells, which have different functional properties. An obstacle in the field is separating the different EV types; studying a heterogeneous pool of EVs has been equated to trying to do immunology before there was the capacity to separate cell types using technologies such as Fluorescence-Activated Cell Sorting (FACS).
There are at least three classes of EVs, which are generated by distinct biogenesis pathways: those derived from the endosomal system (exosomes), those that bud from the plasma membrane (microvesicles), and those that derive from cells undergoing apoptosis (apoptotic bodies) (Phillips et al, 2021). Extensive work in the last decade has shown that EVs derived from different biogenesis pathways cannot always be distinguished by size, or even specific protein markers (Kowal et al, 2016). Furthermore, EVs generated through one biogenesis pathway can also include multiple subpopulations with distinct properties (Kowal et al, 2016;Willms et al, 2016). While this sophistication in EV biology is exciting, it also represents a massive technical challenge that has led to the invention and/or adaption of more diverse methods to characterize EVs (Phillips et al, 2021).
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. The EVs released from infected cells (termed "Pf -iRBC-EVs") have previously been shown to contribute to malaria-associated clinical symptoms, including severe disease cerebral malaria (reviewed in Sampaio et al, 2017). This could be linked to the EV role in manipulating immune cells; however, Pf-iRBC-EVs have also been shown to play a role in parasite cell-cell communication, promoting differentiation to sexual forms or gametocytes which has implications in Plasmodium transmission from the human host to the mosquito vector (reviewed in Babatunde et al, 2020). Given the so-far unexplained diversity in functional effects induced by malaria-derived EVs, it is plausible that different EV subpopulations underpin different functions. Indeed, recent findings indicate that EVs produced by red blood cells infected with different life stages of P. falciparum contain different protein cargos (Opadokun et al, 2022).
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 cryotransmission 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. Complement-associated proteins were enriched in F3-EVs, and proteolysis-related proteins including proteasome subunits were enriched in F4-EVs, potentially describing a mechanism by which EV subpopulations may induce different phenotypic effects in recipient cells (Fig 1).
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. A Förster resonance energy transfer (FRET)-based membrane mixing assay was also used to investigate membrane fusion properties and showed that maximal fusion probability for both EV subpopulations occurs under conditions (pH and membrane makeup) that mimic the plasma membrane. Both EV subpopulations demonstrate preferential fusion to the plasma membrane, with F3-

Plasmodium falciparum-infected donor red blood cell
Pf  EVs demonstrating better fusion capability to early endosomal conditions as compared to F4-EVs (Abou Karam et al, 2022). These differences in EV characteristics suggest that each EV subpopulation could have different targeting properties, in terms of recipient cells as well as subcellular compartments. This work provides a potential explanation for how different EV subpopulations could show different specificities in uptake, trafficking, and stability in recipient cells, which could also underpin different phenotypic responses. While the study by Abou Karam demonstrated that Pf-iRBCs generate EV subpopulations with distinct protein cargos and distinct lipid properties, it remains to be elucidated how this is achieved, and how this affects trafficking to recipient cells or subcellular compartments. The need for further characterization of EV subpopulations interfaces with reports in the last year that propose additional diverse mechanisms by which EVs from Pf-iRBC could impact malaria infections: from priming na€ ıve RBCs to enable parasite invasion (Dekel et al, 2021) to regulating cytokine levels in immune cells (Ofir-Birin et al, 2021). It will be timely to investigate the EV biogenesis mechanisms that differentiate EV subpopulations and the role of the parasite factors in driving this, since P.falciparum release EVs while growing inside their organelle-free host cells. Due to the diversity of phenotypic effects induced by EVs during Plasmodium infections (Babatunde et al, 2020), this model provides the potential to link EV heterogeneity with measurable phenotypic outcomes, represent-ing a valuable tool for further investigation of the role of EV heterogeneity in disease outcomes.