Reversible host cell remodeling underpins deformability changes in malaria parasite sexual blood stages

Abstract

The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin crossmembers and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer's clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.gametocyte | deformability | spectrin/actin skeleton | AFM | molecular dynamics simulation T he most virulent of the human malaria parasites, Plasmodium falciparum causes ∼440,000 deaths annually (1). Pathology is associated with asexual multiplication within red blood cells (RBCs). The trophozoite (growing) and schizont (dividing) stages (∼24-48 h after invasion) sequester in deep tissue using adhesive proteins presented on platform-like structures called "knobs" at the infected RBC surface. Cytoadhesion enables the parasite to avoid passage through the splenic sinuses and thus mechanical clearance from the circulation. Unfortunately, complications associated with sequestration of infected RBCs in the brain are responsible for much of the malaria-related mortality and morbidity.After a period of asexual cycling, a proportion of blood-stage parasites commit to sexual development (gametocytogenesis). The intraerythrocytic gametocyte develops through five distinct stages (I-V) over a period of 10-12 d, eventually adopting the characteristic crescent (falciform) shape that gives P. falciparum its name. Elongation is driven by assembly of a sheath of microtubules, attached to an inner membrane complex, underneath the parasite plasma membrane. From stage II to IV, gametocytes disappear from the circulation (2, 3); however, the mechanism of sequestration is not well understood. Upon maturation, the microtubule cytoskeleton is disassembled, and stage V gametocytes re-enter the circulation (2, 3). Ingestion of mature gametocytes by an Anopheles mosquito triggers release from the RBCs, followed by sexual recombination in the insect gut, and eventual transmission.

Quantitative mass spectrometry of human reticulocytes reveal proteome‐wide modifications during maturation

“…(B) Atomic force microscopy ( AFM ) imaging of cytosolic side‐ exposed cells revealed significant nano‐structural differences between immature reticulocytes and normocytes, (C) shrinkage of spectrin filaments during reticulocyte maturation was estimated from skeletonized images obtained through AFM (Scale bar: 100 nm). While the average length of spectrin filament was 48 nm in CD 71 + reticulocytes, it was reduced to 41 nm in CD 71 − cells [value of CD 71 − cells is in agreement with prior reports (Dearnley et al , )]. [Colour figure can be viewed at wileyonlinelibrary.com]…”

“…(B) Atomic force microscopy (AFM) imaging of cytosolic side-exposed cells revealed significant nano-structural differences between immature reticulocytes and normocytes, (C) shrinkage of spectrin filaments during reticulocyte maturation was estimated from skeletonized images obtained through AFM (Scale bar: 100 nm). While the average length of spectrin filament was 48 nm in CD71 + reticulocytes, it was reduced to 41 nm in CD71 À cells [value of CD71 À cells is in agreement with prior reports (Dearnley et al, 2016)]. [Colour figure can be viewed at wileyonlinelibrary.com] differences in proteome composition to bio-physical property changes as well as identifying host components relevent to blood infections, such as preferential invasion of reticulocytes by human malaria parasites (Mons, 1990;Clark et al, 2014), require further targeted investigations, our data form an avenue to explore and formulate a starting hypothesis towards this.…”

The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum

“…We then applied controlled shear forces to eliminate most of the cell structure and leave behind the cell membrane, cytoskeleton, and other structures firmly attached to it (Dearnley et al, 2016;Shi et al, 2013). To test whether there are physical interactions of GEXP02 with the cytoskeleton, 3D7/GEXP02-HA parasites were induced for gametocytogenesis, treated with GlucNac to remove asexual parasites and were bound to pretreated glass slides on Day 6 of sexual development.…”

“…Sheared GIEs as described by Dearnley et al (2016) were blocked with 3% BSA in PBS and subsequently incubated with antibodies. The shearing of infected erythrocytes was done as previously described (Dearnley et al, 2016;Shi et al, 2013). Immediately after shearing, the slides were blocked in 3%BSA/PBS and then probed with antibodies.…”

“…Although, exported Plasmodium proteins are expected to drive many of the involved modifications and interactions with the GIE cytoskeleton, there is a clear lack of knowledge about the role of exported proteins in host cell remodelling during gametocyte development (Dearnley et al, 2016;Naissant et al, 2016).…”