Gliding motility of Plasmodium merozoites

Abstract

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways.

Keywords: gliding; invasion; malaria; merozoite; plasmodium.

Fig. 1.

Gliding motility of P. falciparum merozoites. (A) Time-lapse imaging for P. falciparum merozoite gliding motility and erythrocyte invasion. Still images from Movie S1. Arrowhead indicates a merozoite gliding on the coverslip (5 and 10 s), followed by erythrocyte deformation (15 and 20 s) and merozoite internalization (30 s to 50 s). (Scale bar, 5 μm.) (B) Each merozoite was traced in different colors, and gliding speed was evaluated from Movie S2. (Scale bar, 5 μm.)

Fig. 2.

Gliding motility of P. knowlesi merozoites. (A) The percentage of merozoites within a P. knowlesi schizont which exhibit motility, for both DMSO-treated parasites (mean = 62.5%) and 0.1 μM cytochalasin D (CyD; IC₅₀ = 0.023 ± 6.7 nM)-treated parasites (no gliding observed). A “motile” merozoite was defined as having demonstrated directional forward motion along the surface of the coverslip for at least five continuous seconds. Each dot is representative of one schizont (n = 20). Error bars denote ±1 SD. (B) The total time each motile P. knowlesi merozoite (n = 109; median = 15 s) spent gliding during the 10-min imaging window post egress. Error bars indicate interquartile range. (C) Number of rotations that merozoites completed plotted against the distance traveled for each glide (n = 10). As the number of rotations increased, so did the distance traveled forward, indicating rotation drives forward motion (Pearson correlation coefficient, R = 0.88). (D) Time-lapse imaging demonstrating a P. knowlesi merozoite rotating as it glides. Red arrows indicate a dark spot located to one side of the wider end of the merozoite, which shifts to the opposite side (shown in subsequent frames), as it turns, and then back to the original position to complete a full rotation (Movie S6). (Scale bar, 5 μm.) (E) Time lapse imaging depicting a P. knowlesi merozoite with mNeonGreen-tagged AMA1 invading an erythrocyte. Left and Left Center demonstrate reorientation of the wider end of the merozoite to align with the erythrocyte membrane. This is followed by (Right Center) the formation of the moving junction, depicted as two green dots at the merozoite-erythrocyte interface, and, finally, (Right) entry into the host cell. (Scale bar, 5 μm.) (F) Schematic illustrating gliding and erythrocyte invasion. Gliding proceeds with the wider, apical end of the merozoite leading. During gliding, merozoites stretch, and a pointed protrusion can be seen at the wider end of the zoite (left-hand bright-field image), which engages with the erythrocyte membrane upon reorientation and internalization. Reorientation of the zoite to make a contact of wider end (green tick), and not the thinner end of the zoite as previously hypothesized (red cross), with the erythrocyte membrane occurs prior to entry. During internalization, constriction of the apical end of the zoite causes the basal end to expand. Finally, after entry is complete, the parasite resides in a parasitophorous vacuole where its development continues.

Fig. 3.

The effects of chemical compounds and parasite genetic modifications on P. falciparum merozoite gliding motility. Purified P. falciparum schizonts were seeded on the coverslip, and merozoites were left to egress for 1 h. (A) The distance of the merozoite (Mz) nucleus (DAPI) from hemozoin (Hz) (black pigment) was measured (green line, Mz–Hz distance). (B and C) Where indicated in the y axes, the relative Mz–Hz distances compared to DMSO control obtained from each schizont with their median and interquartile range are shown. The number of analyzed schizonts from three independent experiments is indicated in parentheses. (Scale bar, 5 μm.) (B) Effects of 0.1% DMSO, 0.1, 1, or 10 μM cytochalasin D (CyD, IC₅₀ = 0.085 ± 0.029 μM), or jasplakinolide (JAS, IC₅₀ = 0.110 ± 0.019 μM) were evaluated for merozoite gliding motility; ** and *** indicate P < 0.001, and < 0.0001, respectively. (C) Inhibition of gliding motility in rapamycin (RAP)-treated ACT1- or GAP45-deleted P. falciparum parasites. The indirect immunofluorescence assay with specific antibodies indicated ACT1 or GAP45 were not detected in RAP-treated transgenic parasites; *** indicates P < 0.0001 by the Mann–Whitney test. (Scale bar, 5 μm.) (D) Purified P. falciparum schizonts were treated with BAPTA-AM (IC₅₀ = 0.992 ± 0.187 μM), A23187 (IC₅₀ = 0.588 ± 0.029 μM), U73122 (IC₅₀ = 0.271 ± 0.085 μM), U73343 (IC₅₀ = 5.444 ± 0.199 μM), R59022 (IC₅₀ = 4.678 ± 0.392 μM), or propranolol (IC₅₀ = 0.551 ± 0.135 μM), and merozoite gliding assays were performed. The relative Mz–Hz distances compared to DMSO control obtained from each schizont with their median and interquartile range are shown. The number of analyzed schizonts from three independent experiments is indicated in parentheses; *, **, and *** indicate P < 0.05, < 0.001, and < 0.0001, respectively. (E) Overview of molecular mechanisms for gliding motility of P. falciparum merozoite. After merozoite egress from the erythrocyte, merozoite adhesin(s) are secreted from micronemes (green) via a signaling pathway involving PI-PLC and DAG kinase (DGK) and bind to environmental substrates including the erythrocyte membrane. A pathway involving PI-PLC and Ca²⁺ activates calcium-dependent protein kinases (CDPKs) and phosphorylates the components of the glideosome machinery (–35, 64). Gray, nucleus; blue, rhoptries. Gliding motility is powered by an actomyosin motor of the glideosome machinery, and the merozoite movement is transferred to the erythrocyte membrane, causing erythrocyte deformation upon merozoite attachment. ACT1, actin-1; PKG, cyclic GMP-dependent protein kinase; GAP45, glideosome-associated protein 45; MyoA, myosin-A; and GAC, glideosome-associated connector.

Fig. 4.

A role for gliding motility in facilitating merozoite–erythrocyte interactions. (A and B) Erythrocyte deformation and merozoite internalization events were seen for DMSO-treated ACT1- or GAP45-floxed P. falciparum parasites, but not detected after RAP treatment (***P < 0.001 by two-tailed Fisher′s exact test). Arrowhead indicates a merozoite. (Scale bar, 5 μm.) (C) Still images taken from Movie S7, depicting a P. knowlesi merozoite beginning to deform a human erythrocyte as gliding motility is initiated along the surface of the host cell. (Scale bar, 5 μm.) (D) A median 80% of merozoites within a given schizont demonstrate gliding motility along human erythrocytes (n = 18 schizonts). In contrast, no gliding is observed at all on either TNF stimulated or nonstimulated HUVECs (n = 16 and 10 schizonts, respectively) and dermal endothelial cells (n = 16 and 10 schizonts, respectively). Comparisons were made by one-way ANOVA, using a Kruskal–Wallis test; ***P < 0.0001. (E) The total number of unique erythrocyte contacts each invading merozoite made was significantly lower for macaque (n = 41 invasions) vs. human (n = 38 invasions) erythrocyte invasions. Comparisons were made using Poisson regression (Poisson coefficient 0.076, 95% CI: 0.37 to 0.52; P < 0.001).