RBC Membrane Biomechanics & Plasmodium falciparum Invasion: Probing Beyond Ligand-Receptor Interactions

Abstract

A critical step in malaria blood-stage infections is the invasion of red blood cells (RBCs) by merozoite forms of the Plasmodium parasite. Much progress has been made in defining the parasite ligands and host receptors that mediate this critical step. However, less well understood are the RBC biophysical determinants that influence parasite invasion. In this review we explore how Plasmodium falciparum merozoites interact with the RBC membrane during invasion to modulate RBC deformability and facilitate invasion. We further highlight RBC biomechanics-related polymorphisms that might have been selected for in human populations due to their ability to reduce parasite invasion. Such an understanding will reveal the translational potential of targeting host pathways affecting RBC biomechanical properties for the treatment of malaria.

RBC Membrane Biomechanics and Merozoite Invasion

Malaria is a deadly mosquito-transmitted disease caused by obligate intracellular Plasmodium species parasites, where P. falciparum, the most common species, causes the highest disease burden. Plasmodium parasites transverse a variety of host cell types over the course of their complex life cycles, causing the most severe disease pathology in humans during blood stage malaria. This intraerythrocytic stage begins with red blood cell (RBC) penetration by invasive merozoites (see Glossary) that develop into trophozoites and then replicate into schizonts that undergo segmentation to form 16-20 merozoite progeny that burst free to begin additional rounds of invasion [1]. RBCs are equipped with a specialized and highly deformable spectrin-actin cytoskeleton to allow for squeezing through tight capillaries & vessels. These minimal cells are incapable of endocytosis, the mode of entry for many pathogens [2]. To circumvent this obstacle, Plasmodium merozoites possess an actomyosin motor that allows them to mechanically infiltrate RBCs, contorting the RBC membrane around themselves to form a nascent parasitophorous vacuole (Figure 1). From within, P. falciparum progressively decreases the deformability and stability of the RBC membrane in preparation for egress, heavily influencing the pathogenesis of malaria through vascular adhesion, splenic retention, and anemia—reviewed in [3] [4–10].

Figure 1. P. falciparum invasion into its red blood cell (RBC) host during blood stage malaria infection.

(A) P. falciparum entry into host RBCs begins during pre-invasion when parasite ligand / host receptor interactions mediate attachment and reorientation of invasive merozoites to their apical end, a process accompanied by RBC deformation. Qualitative deformation scores are utilized to describe the strength of this deformation (inset adapted from live microscopy images from Reference [16]). (B) During the subsequent recoil phase, a final strong deformation event mediated by the merozoite actomyosin motor (white arrow) initiates the commitment step of merozoite invasion, which involves merozoite recoil, pore formation between the merozoite and RBC host, the release of merozoite rhoptry contents into the RBC, and insertion of the RON complex into the RBC membrane (shown in purple). This phase coincides with a calcium influx observed in most successful invasion events. (C) Merozoite internalization, driven by the merozoite motor, subsequently occurs when merozoites pass through the tight junction (mediated by AMA1 binding to the RON complex), wrapping the host RBC membrane around themselves to form a nascent parasitophorous vacuole membrane (PVM) along the way. RBC membrane cholesterol becomes enriched at the site of merozoite contact preceding & during internalization. (D) Sealing of the PVM, mediated by SUB2, is accompanied by merozoite motor-driven merozoite twisting. All four steps take less than 60 seconds. RBC echinocytosis proceeds for several minutes following calcium influx before the RBC returns to its original biconcave shape.

While the merozoite motor overcomes energy barriers posed by the RBC membrane during invasion (Figure 1, arrows), many recent studies show that P. falciparum parasites augment this activity by exploiting endogenous mechanisms RBCs utilize to modulate RBC membrane stability and deformability [11,12]. Highlighting the significance of the relationship between RBC membrane biomechanics and merozoite invasion, there is an RBC tension threshold above which merozoite invasion does not occur. Additionally, mutations affecting RBC membrane biomechanics can protect against P. falciparum invasion and malaria, as exemplified by the recently characterized Dantu polymorphism [13]. This review aims to provide an updated view of merozoite invasion; describe how P. falciparum targets the RBC cytoskeleton and its associated lipid bilayer to modulate RBC deformability during invasion; summarize the known molecular and biomechanical consequences of host receptor ligation; and highlight RBC mutants affecting RBC deformability that protect against Plasmodium invasion and malaria.

Merozoite Invasion: A Balance of Force and Deformation

For almost two decades, live video microscopy in the presence and absence of blocking antibodies, peptides, and genetic perturbations has revealed details regarding the sequence and kinetics of merozoite invasion and shed light on the ligand-receptor interactions involved at each step [12,14–20]. Much progress has been made in defining the parasite ligands and host receptors that mediate attachment and reorientation to the apical end during merozoite invasion. Distinct phases of invasion have been identified through live video microscopy: Pre-invasion (RBC attachment & merozoite reorientation); the recoil phase (final deformation & recoil), internalization, and sealing of the parasitophorous vacuole membrane (PVM) (Figure 1) [16,20,21]. On average, completion of these phases takes less than 60 seconds—although there is variability in the timing of each step. This variability could stem from heterogeneities in the parasites or the RBCs, depend on the orientation the merozoite originally contacts the RBC, or variability in the abundance of RBC receptors at the site of merozoite contact [16,20].

Several well-characterized merozoite ligands are sequentially secreted by merozoite organelles (rhoptries and micronemes) to tightly attach and reorient the merozoite to its apical end on the RBC surface in preparation for entry (Figure 1A). At the molecular level, these pre-invasion events begin when merozoite surface proteins (MSPs) mediate initial, low affinity attachment, triggering the merozoite surface expression of members of the erythrocyte binding-like (EBL), reticulocyte binding-like (RBL) families, and apical membrane antigen (AMA-1) [15,16,18]. The majority of RBL and EBL adhesins mediating attachment during this strong attachment stage have redundant function in reorienting the parasite but are collectively essential. According to a study modeling these interactions, apical reorientation is mediated by RBC binding to a concentration gradient of adhesins—including EBA175, Rh4, and EBA140—such that highest concentration lies on the apical end. These binding interactions alone are thought to be sufficient for reorientation of the merozoite via RBC membrane wrapping [11].

Beyond mediating attachment to the RBC, ligand-receptor interactions initiate signaling to the RBC membrane: A hallmark of merozoite invasion videos are major contortions of the RBC membrane at the site of adhesion preceding internalization of the merozoite, followed by a strong deformation event that is highly correlated with successful invasion [16,20]. These RBC deformations can be phenotypically categorized by deformation scores from 0-3 (Figure 1) [16]. While successful reorientation to the apical end is essential for the subsequent commitment step of merozoite invasion, a range of deformation strengths and durations triggered by pre-invasion lead to successful invasion [20]. After apical reorientation, there is a final, intense RBC deformation and merozoite recoil event (recently coined the recoil phase) that occurs in all successful invasion events prior to internalization (Figure 1B) [20]. This coincides with a plethora of molecular events, including the binding of the Rh5/CyRPA/Ripr complex to its receptor basigin (BSG) on the host RBC. Rh5/BSG binding is the only confirmed essential binding event, is the irreversible commitment step for merozoite entry into the RBC, and serves as the trigger for all subsequent invasion events [17,22–24]. Evidence suggests that the Rh5-binding event opens a fusion pore between merozoite rhoptries and host RBC, triggering a calcium flux into the RBC and the release of rhoptry contents [16,17,20,25]. Previously, this calcium influx was shown to occur in only 45% of invasion events [16,19], and more recently in up to 89% of invasion events utilizing more a sophisticated live video microscopy approach, reviewed in [20,26]. However, it is unclear whether calcium influx into the RBC during this commitment step is significant for downstream signaling events in the RBC during merozoite entry. Nevertheless, it serves as a useful temporal reference during video microscopy for the release of rhoptry contents into the RBC and commitment step of merozoite invasion.

Following calcium influx into the RBC, the cell begins to adopt a shrunken, spiky morphology in a process called echinocytosis (Figure 1). This process was previously thought to initiate post-internalization but was recently shown to begin during internalization in some instances, with varying degrees of delay [20]. Hypothesized triggers for echinocytosis include perturbed osmotic balance following calcium influx; parasite-induced membrane remodeling following insertion of new parasite proteins and rhoptry bulb lipids into the RBC membrane; and biophysical forces involved in internalization [12,20]. Like calcium influx, it is not clear whether echinocytosis is an essential driver of invasion or simply a downstream un-related marker of invasion progression.

Once the irreversible step of invasion has occurred, merozoite rhoptries inject RON2 and associated proteins into the host RBC membrane to serve as sites of adhesion for merozoite-surface bound AMA1 (Figure 1B). This interaction, known as the tight junction, serves as an anchor point for merozoite internalization [27,28]. The merozoite moves through the tight junction, powered by merozoite actomyosin motor activity and this AMA1/RON2 interaction, leading ultimately to the formation of the parasitophorous vacuole (PV) (Figure 1C). The PV membrane was recently confirmed to originate primarily from the RBC membrane [20]. Internalized merozoites exhibit a twisting motion within the newly formed PVM [12,20]. This twisting is thought to seal the PVM and complete PVM scission from the RBC membrane, although this has not been experimentally proven. Other factors such as SUB2 have also been recently linked to PVM sealing [29] (Figure 1D). RBC echinocytosis continues beyond internalization, after which the RBC returns to its biconcave shape.

Merozoites possess an actomyosin motor that is required to deform the RBC membrane during pre-invasion, and overcome additional energetic barriers posed by this membrane during merozoite internalization and sealing of the nascent PVM (Figure 1, white arrows) [12,16]. However, attachment in the absence of motor activity also leads to an RBC deformation event [16,20], supporting the notion that RBC ligation alone can initiate signaling to the RBC membrane. Indeed, several merozoite adhesins have been shown to initiate signal transduction within the RBC to alter RBC membrane deformability during pre-invasion, pointing to a balance of forces required for deformation. Several lines of evidence corroborate the merozoite’s ability to exploit physiological mechanisms for altering RBC membrane deformability to facilitate its entry into the RBC.

Primed for Entry: P. falciparum-Induced Modification of the RBC Membrane

The RBC membrane consists of the RBC lipid bilayer and its associated pseudohexagonal spectrin-actin cytoskeleton [30]. RBC deformability is heavily influenced by the stability of this membrane, which depends on how strongly this cytoskeleton is tethered to the lipid bilayer (for vertical stability); and how well connected the spectrin meshwork is to its actin nodes (for horizontal stability) [31] (Box 1). Two complexes in the RBC cytoskeleton contribute to RBC membrane stability: The ankyrin-containing complex and the actin junctional complex (Figure 2B). Vertical membrane stability is conferred through a number of transmembrane proteins that connect the RBC cytoskeleton to the lipid bilayer in both complexes whereas horizontal stability is largely conferred by proteins that connect spectrin to the actin junctional complex [32]. RBC membrane stability can be measured semi-quantitatively by visualizing changes in mesh size and spectrin length via atomic force microscopy or quantitatively through osmotic stability assays [33,34]. Disruption of this stability—particularly through post-translational modification of many of the aforementioned cytoskeletal factors—alters the tension and viscoelasticity of the RBC membrane, ultimately leading to altered RBC deformability [32]. Both tension and viscoelasticity are biomechanical metrics of deformability that can also be measured experimentally (Box 1).

Box 1. Biomechanical Determinants of RBC Deformability.

RBC Deformability describes an RBC’s ability to change its shape. Many biological processes signal through RBC membrane to influence RBC membrane deformability, namely by altering the vertical and/or horizontal stability of the RBC cytoskeleton or changing the composition of the lipid bilayer. These biological processes result in altered RBC membrane biomechanics, which can be quantified experimentally though changes in tension & viscoelasticity (Figure I). These metrics have been measured in the presence and absence of specific parasite ligands to determine their influence on RBC deformability.

RBC Membrane Tension:

RBC Tension, or surface stress, is a metric that characterizes how tightly the red blood cell membrane is pulled across its surface. The native RBC cytoskeleton is slightly stretched and under tension, as maintained by non-muscular myosin IIA (NMMIIA), a force-generating, actin junctional complex-associated motor protein that dynamically regulates the curvature & deformability of RBC [81,82]. Increased RBC tension leads to less deformable RBCs. Conversely, cytoskeletal instability can decrease RBC tension via the relaxation of spectrin [31,83] Flickering spectroscopy is the most commonly used method for measuring RBC tension, and was recently used to demonstrate that there is a tension threshold above which merozoite invasion does not occur, irrespective of genetic background [13].

RBC Membrane Viscoelasticity:

Viscoelasticity is a metric of RBC membrane deformability that can be broken down into membrane elasticity and viscosity. Purely elastic materials return to their original shape almost instantaneously upon deformation. However, the RBC membrane is considered to be viscoelastic because while its spring-like cytoskeleton confers membrane elasticity, its lipid bilayer component has viscous characteristics that delay RBC membrane recovery following deformation [84]. Membrane viscoelasticity strongly influences merozoite invasion. The RBC membrane’s resistance to specific types of deformation can be quantified by elastic moduli such as Young’s modulus, shear modulus, and bending modulus. These moduli are conceptually interchangeable with “membrane stiffness” and have been used most often to measure/describe how Plasmodium parasites modulate RBC deformability. Atomic force microscopy, magnetic twisting cytometry, and ektacytometry can be used to determine the multi-point, single cell and bulk Young’s Modulus of an RBC population. While Young’s modulus is primarily determined by cytoskeletal stability, bending modulus is more sensitive to changes in lipid bilayer composition, and can be experimentally determined by flickering spectrometry [85].

For more comprehensive reviews on methods to study RBC deformability during Plasmodium invasion, I refer readers to References [86] & [87].

Figure I.

Biophysical Determinants of RBC Membrane Deformability

Figure 2. Primed for Entry: P. falciparum induced modification of the RBC membrane during merozoite invasion.

(A) P. falciparum-induced stimuli postulated to alter RBC membrane biomechanics to facilitate merozoite invasion include ligand-receptor initiated phosphorylation of the RBC cytoskeleton (highlighted in 2B & 2C), the internalization of schizont-derived extracellular vesicles (EVs) containing cytoskeleton-targeted proteins, merozoite actomyosin motor-mediated mechanical disruption of the RBC membrane & the opening of mechanosensitive ion channels, and the secretion of parasite factors capable of altering RBC lipid composition. Intracellular consequences of these stimuli include post-translational modification of the RBC cytoskeleton and the induction of a calcium influx. (B) Much progress has been made in defining the parasite ligands and host receptors that mediate merozoite invasion. Several of these RBC host receptors reside in the RBC cytoskeleton, a pseudohexagonal mesh of spectrin anchored to the lipid bilayer through the actin junctional and ankyrin complexes. Many cytoskeletal proteins become phosphorylated in the context of merozoite invasion (colored to match 2C), altering RBC membrane stability. (C) Phosphoproteomics studies conducted following RBC ligation by merozoites [37], or their recombinant ligands (EBA175 [38] or Rh5 [25]) have identified many overlapping cytoskeletal substrates (color coded to match 2B). Starred substrates were also identified by a phosphoproteomics study following RBC incubation with schizont-derived EVs.

During the process of invasion, merozoites have local access to the RBC membrane at sites of contact, through which they can hijack endogenous RBC pathways for modulating RBC deformability to facilitate entry. Parasite stimuli postulated to initiate these pathways include ligand-receptor initiated phosphorylation of the RBC cytoskeleton, internalization of schizont-derived extracellular vesicles (EVs) containing cytoskeleton-targeted proteins, mechanical stimulation, and the secretion of parasite factors capable of altering RBC lipid composition (Figure 2A). All of these biological processes are postulated to disrupt RBC membrane biomechanics (Box 1; Table 1).

Table 1.

The Biomechanical Consequences of RBC Membrane Receptor Ligation

		Preinvasion				Recoil Phase
			Reorientation
	Parasite Ligand	MSPs	EBA140	EBA175	Rh4	Rh5
	Host Receptor	?	GPC	GPA	CR1	BSG
Stability	Cytoskeletal Phosphorylation	?	?	Yes [38]	Yes [58]1	Yes [25]
	Calcium Influx Induction	Unlikely [19]	Unlikely [19]	Yes [38] No [45] Unlikely [19]	Yes [58]2 Unlikely [19]	Yes [17,25]
	Cytoskeletal Morphology	?	?	?	?	↑ Mesh Size [25]
	Osmotic Fragility	?	?	?	?	?
Viscoelasticity	Young’s Modulus	↓, [38]	↓, [38]	↓↓, [38]	↓, [38]	↓, [38] ↓, [62]3
	Bending Modulus	?	?	↓, [45]	?	?
Tension	Tension	?	?	↑, [51,52]4 ↑, high [EBA175] ↓, low [EBA175] [45]	?	?
RBC Deformability (cellular)		?	?	↑ Elongation Index [38] ↓, Microfiltration [50]5	↑, Microfiltration [58]6↑, splenic filtration [57]7	?

¹Ligand: Anti-CR1 antibody

²Ligand: Anti-CR1 antibody

³Ligand: Anti-CD147/BSG antibody; murine RBCs

⁴Ligand: Anti-GPA Antibody

⁵Ligand: Complement (C4d)

⁶Ligand: Anti-CR1 antibody

⁷Ligands: Complement-tagged microbes, apoptotic/necrotic debris, immune complexes

RBC ATP is required for merozoite invasion, implicating cytoskeletal phosphorylation as an essential component of invasion [35–37]. Supporting this notion, extensive increases in RBC cytoskeletal phosphorylation following merozoite attachment, invasion, or ligation with recombinant merozoite ligands have been detected by phosphoproteomics studies, suggesting that the merozoite signals to the RBC cytoskeleton to modulate RBC deformability during the pre-invasion and the commitment recoil phases [25,37,38]. Targets include various cytoskeletal proteins responsible for maintaining vertical and horizontal stability of the RBC membrane and mechanosensitive ion channel, PIEZO1 (Figure 2C) [25,37,38]. This cytoskeletal phosphorylation occurs even in the absence of merozoite-applied mechanical force, and RBCs treated with static beads incapable of ligating RBCs via the same receptors are unable to initiate phosphorylation of the RBC cytoskeleton [25,37]. Collectively, these data demonstrate that cytoskeletal phosphorylation events are not driven by mechanical stimulation exclusively but are instead dependent on signal transduction initiated by specific ligand-receptor interactions. Because merozoite attachment alone induces these changes, host kinases are postulated to mediate these phosphorylation events [37,38].

Interestingly, a recent paper has suggested a role for extracellular vesicles (EVs) in delivering host and parasite kinases and 20S proteasomes to uninfected RBCs, priming them for parasite entry. While not essential for invasion, these EVs alter the phosphorylation of many of the same RBC substrates as seen during RBC incubation with merozoite ligands (Figure 2A; Figure 2C, starred). Provocatively, most of these phosphorylated host cytoskeletal proteins are 20S proteasome substrates, including mediators of vertical and horizontal stability that are lost from the RBC cytoskeleton following Rh5 ligation [25,39].

A striking feature of merozoite invasion is the associated calcium influx into the RBC [16,17,20] (Figure 1; Figure 2A). Ligand-receptor interactions alone are sufficient for inducing this calcium influx, which could originate from the merozoite or the surrounding media [16,25,38]. Physiologically, mechanosensitive ion channels initiate a calcium influx in response to sheer stress in the vasculature. This influx activates calcium-sensitive effectors (such as protein kinase C, calpain proteases, and calmodulin) that destabilize the cytoskeleton via phosphorylation, degradation, and the dissociation of RBC cytoskeletal proteins, globally increasing RBC deformability—reviewed in [40]. This has led to the hypothesis that the calcium influx into the RBC observed during the commitment recoil phase increases RBC deformability by hijacking these mechanisms [20]. Indeed, mechanosensitive calcium channels TRPM7 and PIEZO1 have been linked to P. falciparum invasion—although their mechanistic roles are largely uncharacterized [38,41] However, additional studies are needed to corroborate a functional role for the calcium influx observed during invasion, as calcium chelation still leads to successful invasion and calcium influx leads to secondary effects known to affect merozoite invasion, including RBC dehydration [16,42,43].

Merozoite invasion can also be affected by artificially altering the lipid composition and bending modulus of the RBC membrane (Box 1). Incorporating 7-ketocholesterol into the RBC membrane, for example, reduces lipid order/packing in the RBC bilayer, reducing the bending modulus of the RBC membrane and enhancing invasion [44,45]. Conversely, artificial depletion of membrane cholesterol reduces the RBC bending modulus and inhibits P. falciparum invasion [46]. Indeed, RBC membrane cholesterol becomes enriched at the site of merozoite contact and in the nascent PVM during invasion. This enrichment is correlated with higher local membrane curvature, and is hypothesized to facilitate RBC membrane wrapping during internalization of the merozoite [20]. It is unclear which parasite stimuli are responsible for modulating host RBC membrane cholesterol, or whether merozoites are capable of synthesizing cholesterol de novo, although calcium levels can alter the lipid composition of the lipid bilayer [47].

Molecular & Biomechanical Consequences of Host Receptor Ligation

Historically, there has been a relatively exclusive focus on ligand-receptor interactions as sites of adhesion for Plasmodium parasites with little attention toward downstream pathways in the RBC initiated by ligand-receptor binding. However, in the past five years the field has begun exploring the influence of these ligand-receptor interactions on signaling pathways within the RBC that modulate RBC deformability. This section will synthesize known ligand-receptor induced molecular mechanisms that signal to the RBC cytoskeleton & lipid bilayer during merozoite invasion, and their biomechanical consequences (summarized in Table 1).

EBA175 / Glycophorin A Binding Alters RBC Deformability

EBA175 is released from micronemes into the merozoite membrane during pre-invasion to mediate apical reorientation of the merozoite [15] (Figure 1). Glycophorin A (GPA), a transmembrane RBC protein with a cytosolic tail for signaling, is the host receptor for EBA175 [48]. This protein contributes to the vertical stability of the RBC membrane through association to Band 3 (SLC4A1), both within and outside of the RBC membrane complexes (Figure 2B). When GPA is endogenously ligated by autologous antibodies and excess complement in the blood stream, RBC deformability decreases (Table 1). This occurs in part through formation of a membrane skeleton-linked protein complex that increases the vertical stability of the RBC membrane [49–51]. GPA binding by antibody moieties in vitro also leads to a concentration-dependent increase in RBC tension and rigidity [52,53]. This decrease in RBC deformability is thought to occur through the GPA cytoplasmic tail, which associates with the RBC cytoskeleton upon GPA ligation [52]. Reactive oxygen species (ROS) production is also triggered by the GPA ligation, which has a detrimental effect on RBC membrane deformability and lipid mobility [54].

Consistently, GPA ligation by recombinant EBA175 (rEBA175) at high, likely saturating, concentrations increases RBC tension [45]. However, RBC ligation by lower concentrations of rEBA175 increases RBC deformability: RBC tension is reduced, RBC elongation under shear stress increases, and both bending and Young’s moduli decrease (Table 1) [38,45]. These data support the paradigm that Plasmodium parasites locally signal to the cytoskeleton during invasion to increase RBC deformability and facilitate invasion. Indeed, RBC ligation by rEBA175 increases cytoskeletal phosphorylation (Figure 2C). Both phosphorylation and altered deformability phenotypes are reversed upon inhibition of host calcium and magnesium channel/kinase, TRPM7. Interestingly, EBA175 is not the only recombinant parasite ligand capable of signaling through TRPM7 to increase RBC deformability. EBA140, Rh4, and—to a lesser extent—Rh5 also alter RBC biomechanics through TRPM7. This suggests that ligand-receptor interactions signal to the cytoskeleton through shared effectors [38].

Rh4 / Complement Receptor 1 Binding Increases RBC Deformability

Complement Receptor 1 (CR1) ligation by parasite ligand Rh4 also mediates specific attachment and apical reorientation of the merozoite during pre-invasion [16,55]. CR1 biochemically partitions in the cytoskeleton fraction but has not been observed in actin junctional or ankyrin complexes [56]. In vivo, complement-tagged microbes, apoptotic/necrotic debris, and immune complexes ligate CR1, increasing RBC deformability [57]. Consistently, CR1 ligation by anti-CR1 antibodies triggers a calcium influx into the RBC through TRPC6, globally increasing RBC deformability (Table 1). This calcium influx is hypothesized to mediate increased RBC deformability through phosphorylation of beta-spectrin by casein kinase II (CKII), which also increases upon CR1 ligation. Interestingly, the extent of both global RBC deformation and calcium influx following CR1 ligation are directly correlated to the amount of CR1 expressed on the RBC surface [58].

RBC ligation by recombinant Rh4 binding increases RBC deformability through TRPM7, presumably via CR1 binding [38]. It is not known whether this biomechanical phenotype is stimulated by calcium influx through TRPC6 and beta spectrin phosphorylation by CKII. The extent of RBC deformation in response to CR1 ligation positively correlates to CR1 surface availability [58]. Intriguingly, CR1 surface levels also influence the invasion efficiencies of both P. falciparum and P. vivax, suggesting that CR1 ligation is a shared mechanism for modulating the biomechanics of the RBC membrane to facilitate Plasmodium invasion [59,60].

Rh5 / Basigin Binding Increases RBC Deformability

While various EBA and Rh ligations initiate early RBC membrane deformations during pre-invasion, basigin (BSG/CD147) ligation by Rh5 during the commitment recoil phase is the only essential ligand-receptor interaction for merozoite invasion [22]. This binding event correlates with a strong, final deformation event observed in all successful invasion events [20]. BSG, one of the few genetically validated P. falciparum host receptors, is an integral membrane glycoprotein and extracellular matrix metalloprotease inducer that plays roles in adhesion [61]. BSG ligation with anti-CD147 Ab in mice leads to mechanical trapping of RBCs in the spleen, presumably due to compromised viscoelasticity [62]. While it is unknown whether BSG associates with the RBC cytoskeleton, BSG is associated with both calcium signaling and altered cytoskeletal architecture in other cell types [63–65]. BSG also functionally interacts with known cytoskeleton interactor and genetically validated host determinant, CD44, in other mammalian cell lines, suggesting that a BSG complex is important for modulating deformability [30,66,67].

Rh5, found in a complex on the merozoite surface with CyRPA and Ripr proteins, binds to BSG following apical reorientation of the merozoite [17]. Rh5/BSG binding is thought to open a pore between the merozoite and the RBC, leading to the release of rhoptry contents into the RBC [16,17,20,25]. This event coincides with a calcium influx into the RBC that can be blocked by antibody disruption of the Rh5/BSG interaction; and can be initiated by recombinant Rh5 binding [25]. Rh5 binding decreases the Youngs Modulus—or membrane stiffness—of RBCs, albeit to a lesser extent than binding of MSPs, Rh4, EBA140, or EBA175 [38]. Additionally, as revealed by increased cytoskeletal mesh size upon binding, Rh5/BSG binding decreases membrane stability (Table 2) [25]. Analysis of the RBC phosphoproteome in the presence of recombinant Rh5 reveals several cytoskeletal substrates (Figure 2C). While some studies have hypothesized a mechanistic link between the calcium influx and the cytoskeletal alterations observed during Rh5/BSG ligation, it remains unclear how BSG signals to the cytoskeleton during invasion. Surprisingly, complementation of a BSG knockout RBC line with a truncated mutant indicates that the cytosolic tail of basigin is dispensable for P. falciparum invasion [61].

Table 2.

Some natural and engineered RBC membrane biomechanical mutants that protect against Plasmodium infection

RBC Membrane Protein	Parasite Ligand(s)	RBC Disorder	Mutation / Perturbation	Biomechanical RBC Phenotype	Restricts P. falciparum invasion?	Protective against malaria?	References
CR1	Rh4	Knops blood group	Expression polymorphism (decreased expression)	↓deformability	Yes	Yes: P. falciparum & P. vivax	[58,60,88]
GPA	EBA175	En(a-) (MNS blood group)	GYPA null	Unknown	Yes	Unclear	[89]
GPB	EBL-1	S-s-U- (MNS blood group)	GYPB null	Unknown	Yes	Unclear	[90,91]
GPA/GPB	EBA175	Dantu Blood Group (DUP4)	GPA (extracellular)/GPB tail fusion	↑tension	Yes	Yes	[13,69]
GPC	EBA140	Gerbich-negative modification	Exon 3 deletion	↑deformabilit y; elliptocytosis	Yes	Yes	[92]
BSG	Rh5	Ok blood group;	Binding interface polymorphism	Unknown	Unknown	Unknown	[22]
	Rh5	BSG KO	CRISPR knockout	Unknown	Yes	Unknown	[61,66]
Band3/ SLC4A1 / AE1	Unknown	Southeast Asian Ovalocytosis (SAO)	Decreased Expression	↑ tension; ↓deformability	Yes	Yes: placental & cerebral malaria	[72–76]
ATP2B4	Unknown	N/A	Multiple SNPs including enhancer SNPs	High intracellular calcium; [altered] RBC hydration	Unknown	Yes: Severe malaria (GWAS & P. berghei)	[70,93–95]
PIEZO1	Unknown	Hereditary Xerocytosis	Gain of function mutations	RBC dehydration	Yes	Yes	[43,96]
Ankyrin (Ank1)	N/A	N/A	Non-functional mutants	↓ RBC deformability; ↑ osmotic fragility; ↑ clearance	Unknown	Yes: P. chabaudi (restricts invasion)	[78]
Spectrin (Sptb)	N/A	N/A	CRISPR mutagenesis of ankyrin binding site	↓deformability; ↑clearance	Unknown	Yes: P. chabaudi (invasion-independent)	[97]
Ferroportin/SLC40A1/FPN	Unknown	Hemochromatosis	Q248H mutationn	↑ osmotic fragility	Unknown	Yes: P. yoelli, P. chabaudi	[98]
	Unknown	Hemochromatosis	FPN knockout	Unknown	Unknown	No	[99]

RBC Membrane Mutations Protective Against Plasmodium Infection

As discussed, several recent studies have corroborated the ability of the merozoite to alter the biomechanical properties of the RBC membrane and facilitate merozoite entry (Figure 2; Table 1). Conversely, naturally occurring mutations in proteins that impact RBC membrane biomechanics have been shown to protect against malaria in several instances (Table 2). This underscores the potential ongoing evolutionary dynamics between select RBC host receptors and parasite invasion ligands. Such studies have the potential to elucidate the mechanistic roles of host proteins during invasion and may provide targets for intervention.

A prominent example is the recently described Dantu hybrid, a genomic rearrangement and duplication in the GYPA/GYPB locus that results in the expression of a GPA extracellular domain / GPB tail fusion protein that does not interact with the RBC cytoskeleton [68–70]. Mechanistically, the Dantu mutation leads to a global increase in RBC membrane tension that protects against P. falciparum invasion, supporting a role for the GPA cytoplasmic domain in signaling to the cytoskeleton. This study also led to the insight that there is a critical RBC membrane tension threshold beyond which parasite entry is restricted, even in wildtype RBCs. [13]. The Dantu mutation is prevalent in specific East African human populations and is associated with reductions in the risk for severe malaria, comparable to that afforded by the sickle cell trait.

Another striking example—Southeast Asian Ovalocytosis (SAO)—involves a 27bp deletion in Band3, an essential cytoskeletal protein in the actin junctional and ankyrin complexes (Figure 2B). This mutant results in ovalocytic RBCs that have reduced deformability and restrict the invasion of most P. falciparum strains [71]. SAO is associated with clinical protection against severe cerebral malaria and placental malaria [72–76].

Indeed, a plethora of mutations in the RBC membrane have been identified to protect against malaria (Table 2) In many cases the mechanism of protection and effect on RBC deformability is unclear. However, some mutations lead to RBC hydration changes (e.g., PIEZO1, ATP2B4) or modified osmotic stability (e.g., ferroportin) that can have global effects on RBC deformability. Understanding the mechanistic links between these protective mutations and RBC deformability changes is a key next challenge.

Beyond naturally occurring polymorphisms, the roles of RBC membrane and cytoskeletal proteins has been investigated via genetic perturbations in mouse models (reviewed in [77] (Table 2). For example, non-functional mutations in ankyrin in mouse RBCs leads to decreased RBC deformability and stability and reduced invasion of the mouse malaria parasite P. chabaudi [78]. The recent establishment of genetically tractable human RBCs supporting the invasion of multiple Plasmodium species has enabled functional genetic validation for a handful of RBC membrane proteins involved in merozoite invasion, and provides a powerful system for future studies investigating the influence of specific host factors on RBC membrane biomechanics [61,66,67,79,80].

Concluding Remarks

The relationship between P. falciparum invasion and RBC deformability is challenging to investigate experimentally given the short duration of merozoite invasion, the technical difficulty of obtaining cells in physiologically relevant states of PTM or deformation, and the phenotypic heterogeneity of RBCs. Nevertheless, several recent technical advances have corroborated the parasite’s ability to exploit endogenous cellular pathways that signal to the RBC membrane to alter its biomechanical properties and facilitate merozoite entry (Figure 2C). Future studies are needed to answer outstanding questions surrounding the comprehensive parasite stimuli, host factors & PTMs involved in facilitating merozoite invasion (see Outstanding Questions), and to characterize the biomechanical consequences of receptor ligation more completely (Table 1)

Outstanding Questions.

• Which parasite/host kinases, proteases, and enzymes are involved in remodeling the RBC membrane during invasion?

• How does receptor density at the site of merozoite adhesion influence early deformation events and the success of invasion? Do merozoites recruit host receptors to local sites of adhesion?

• Which parasite stimuli are responsible for recruiting host RBC membrane cholesterol to sites of adhesion during merozoite invasion?

• Do ligand/receptor interactions work cooperatively to induce deformation changes and facilitate entry?

• How do members of the RBL and EBL ligand families outside of EBA175, Rh4, and Rh5 affect RBC deformability?

• How are signals transduced to the RBC membrane during merozoite invasion?

• Do ligand-receptor interactions initiate cytoskeletal PTMs other than phosphorylation during merozoite invasion?

• Does calcium influx into the RBC play a functional role in facilitating merozoite invasion?

• What role does PIEZO1 play in invasion?

• Are RBC membrane biomechanical requirements conserved between different Plasmodium species? How do these requirements differ between differently aged RBCs?

• What RBC membrane polymorphisms are associated with protection against clinical malaria?

Furthermore, many known natural polymorphisms affecting RBC biomechanics are protective against multiple Plasmodium species, suggesting that targeting cellular pathways signaling to the RBC membrane is a viable therapeutic strategy for restricting P. falciparum invasion that could lead to broad spectrum antimalarials. Moreover, RBC biomechanical properties and membrane protein composition can vary between differently aged RBCs, which are differentially infected by divergent Plasmodium species. In particular, P. vivax demonstrates a strict tropism for reticulocytes, the youngest of RBCs, which have distinct biomechanical properties from the better studied normocytes [34]. We anticipate that a thorough understanding of RBC heterogeneity and critical pathways signaling to the RBC membrane will more richly describe the contribution of the RBC to parasite invasion, and that identification of essential and druggable molecules could inform the development of novel host-directed targets for blood stage malaria.

Highlights.

• Merozoite ligands signal to the red blood cell (RBC) membrane through multiple pathways to prime host RBCs for invasion

• Merozoite attachment alters the biomechanics of the RBC membrane

• Evolution has selected for mutations affecting RBC biomechanics to combat malaria

Glossary

Actomyosin motor

A force generating, molecular complex underlying the merozoite membrane that powers invasion by a myosin motor, including PfMyoA, and actin

Adhesins / Ligands

Attachment factors that mediate merozoite anchoring and signaling through host receptors in the RBC membrane during invasion. These factors include merozoite surface proteins (MSPs), erythrocyte binding-like (EBL) proteins, reticulocyte binding (RBL) proteins, and tight junction adhesin, apical membrane antigen 1 (AMA-1)

Merozoite

The invasive form of the Plasmodium parasite, which enters host red blood cells during the asexual blood-stage of malaria

Micronemes

Small, rhoptry-associated vesicles at the apical end of the merozoite that release adhesins during invasion, including EBA175 and AMA1

Parasitophorous Vacuole (PV)

A parasite-housing vacuole where replication occurs to produce daughter merozoites; formed by RBC membrane invagination during merozoite internalization

Phases of Invasion

Pre-invasion (attachment & reorientation), the commitment recoil step (final deformation, recoil, pore formation, and rhoptry release), internalization, PV membrane sealing, and echinocytosis

RBC Cytoskeleton

A flexible pseudohexagonal mesh of spectrin filaments and actin nodes that underlies the structural integrity of the RBC membrane. Associates with the RBC lipid bilayer via vertical & horizontal interactions through the actin junctional and ankyrin complexes

RBC Deformability

The biomechanical term to describe a red blood cell’s ability to change its shape under an applied force. The baseline global deformability of an RBC is influenced by its age and genetic background (among other factors), which in turn impacts the magnitude of transient, localized, deformation events

RBC Membrane

The RBC lipid bilayer and its associated spectrin-actin cytoskeleton

RBC Membrane Biomechanics

Quantitative biophysical descriptions that characterize how the RBC membrane responds to deformation & other applied forces

Rhoptries

Club shaped, membrane-bound organelles at the apical end of the merozoite that fuse with the RBC membrane to release parasite factors into the RBC host during invasion, including RBL adhesins and RON complex members

Tight junction

A moving interface between the invading merozoite and RBC that provides traction for active merozoite internalization via interactions between merozoite surface-bound AMA1 and RON2 inserted in the RBC membrane