Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.

A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, deformability of red blood cells (RBCs) is key to their function and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single-cell methods provide very valuable information, they are often technically challenging and lack the high data throughput needed to distinguish differences in heterogeneous populations, while fluid-flow high-throughput methods miss the accuracy to detect subtle differences. Here we present a new method for multiplexed single-cell mechanical probing using acoustic force spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

Studying cellular mechanics allows important insights into its cytoskeletal composition, developmental stage, and health. While many force spectroscopy assays exist that allow probing of mechanics of bioparticles, most of them require immobilization of and direct contact with the particle and can only measure a single particle at a time. Here, we introduce quantitative acoustophoresis (QAP) as a simple alternative that uses an acoustic standing wave field to directly determine cellular compressibility and density of many cells simultaneously in a contact-free manner. First, using polymeric spheres of different sizes and materials, we verify that our assay data follow the standard acoustic theory with great accuracy. We furthermore verify that our technique not only is able to measure compressibilities of living cells but can also sense an artificial cytoskeleton inside a biomimetic vesicle. We finally provide a thorough discussion about the expected accuracy our approach provides. To conclude, we show that compared to existing methods, our QAP assay provides a simple yet powerful alternative to study the mechanics of biological and biomimetic particles.

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