Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission

Abstract

Malaria-causing parasites rely on an actin-myosin-based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.

Keywords: malaria; mosquito; myosin; ookinete; sporozoite.

Figure 1. Myosin motor is required at multiple stages throughout the Plasmodium life cycle

1. When an Anopheles mosquito feeds from an infected mammal, it takes up parasites with the bloodmeal. The parasites transform into motile ookinetes in the midgut. Myosin is essential for traversal of the midgut epithelium. Ookinetes transform into oocysts and develop into hundreds of sporozoites. Sporozoites egress from oocysts to float in the hemolymph. Active motility allows sporozoites to invade the salivary glands from where they are transmitted into the skin with the next bite. They traverse the skin and enter into a blood vessel. With the bloodstream, they are transported until they reach the liver and actively invade a hepatocyte to develop into thousands of merozoites. These merozoites are released back into the bloodstream where they depend on myosin to actively invade red blood cells (RBCs) and multiply. Red and blue arrows indicate active and passive movements of the parasite, respectively.

2. The motor protein MyoA localizes to the periphery of motile Plasmodium stages. Images show parasites expressing GFP‐tagged MyoA. Nuclei were stained with Hoechst. Scale bars, 5 µm.

Figure 2. Effect of 3’UTR replacement of myoA by the 3’UTR of dhfs on life cycle progression and myoA expression

1. Scheme showing simplified myoA loci in transgenic parasite lines.

2. Blood stage growth rate of mutated parasite lines. A single parasite was injected i.v. into a mouse, and the growth rate was calculated from parasitemia at day 6–9 for each mouse (black dots). Bars represent the mean of 4–7 mice.

3. Infected midgut showing oocysts stained with mercurochrome; scale bar, 200 μm. The graph shows oocyst numbers per infected mosquito. Numbers of mosquitoes that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers).

4. Ratio of salivary gland to midgut sporozoites of the indicated parasite lines. Black dots: individual experiments with 10–20 mosquitoes. Bars: mean.

5. Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging. Arrow indicates direction of a motile sporozoite. The graph shows the fraction of motile hemolymph sporozoites. Black dots: individual experiments with 10–20 mosquitoes. Bars: mean.

6. qRT–PCR analysis of schizonts and oocysts of the PbMyoA 3’dhfs parasite line. Individual data points correspond to the mean of three technical replicates.

Data information: Significance for (B), (D), and (E) determined by one‐way analysis of variance with Tukey’s multiple comparison test. Significance for (C) determined by Kruskal–Wallis test with Bonferroni’s multiple comparison test. Source data are available online for this figure.

Figure 3. Phosphorylation of MyoA at serine 19 is important for ookinete motility

1. Zoom of the PfMyoA structure showing important amino acid interactions for stabilization of the rigor‐like state that are thought to influence the kinetic properties of MyoA. The N‐terminal extension is depicted in purple. It is located in proximity to Switch I (green) and the connectors Switch II (orange) and Relay (yellow). Phosphorylated serine 19 (SEP19) in the N‐terminal extension interacts with lysine 764 (K764) in the converter and glutamic acid 6 (E6) in the N‐terminal extension interacts with arginine 241 (R241) and phenylalanine 476 (F476) from switch I and II. Image taken from Robert‐Paganin et al (2019). The image was published under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

2. Scheme of mutated MyoA versions that are expressed in clonal parasite lines at the endogenous myoA locus. Either the complete N‐terminal extension is deleted (19 amino acids) or point mutations within amino acids that are located in the N‐terminal extension are introduced.

3. Blood stage growth rate of clonal parasite lines. A single parasite or 100 iRBCs were injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6–9. Black dots: individual mice; bars: mean.

4. Proportion of motile ookinetes. Black dots: Individual movie; bars: mean. Data pooled from three independent biological replicates with 1–3 movies taken per experiment. See also Movie EV1.

5. Speed of ookinetes of the different lines. Numbers of analyzed parasites are shown above the graph. Data pooled from three independent experiments (biological replicates). Violin plots show median (line) and quartiles (dashed lines).

6. Number of oocysts per infected mosquito. Numbers of analyzed mosquitoes are shown above the graph. Data pooled from at least three independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers).

Data information: Statistical analysis: (C) One‐way analysis of variance with Tukey’s multiple comparison test. (D), (F) Kruskal–Wallis test with (D) Dunn’s multiple comparisons test or (F) Bonferroni’s multiple comparison test. Source data are available online for this figure.

Figure 4. Phosphorylation of MyoA at serine 19 is important for salivary gland invasion of sporozoites

1. Ratio of hemolymph to midgut sporozoites. Black dots: individual experiments with 10–20 mosquitoes. Bars: mean.

2. Ratio of salivary gland to midgut sporozoites. Black dots: individual experiments with 10–20 mosquitoes. Bars: mean.

3. Blood stage infection of 3–8 mice after transmission by mosquito bite from one to two biological replicates. Shown is the mean ± standard deviation.

4. Blood stage infection of 4‐8 mice after i.v. injection of 1,000 sporozoites from one to two biological replicates. Shown is the mean ± standard deviation.

Data information: Statistical analysis: (A) and (B) One‐way analysis of variance with Tukey’s multiple comparison test. Source data are available online for this figure.

Figure 5. Reduced speed during in vitro sporozoite gliding motility of parasite lines expressing mutated MyoA

1. A, B

   Fraction (left) and speed (right) of motile hemolymph (A) or salivary gland‐derived (B) sporozoites on glass. Dots correspond to individual experiments and bars indicate the mean. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Numbers indicate analyzed sporozoites. Significance for (A left) and (B left) determined by one‐way analysis of variance with Tukey’s multiple comparison test. Significance for (A right) and (B right) determined by Kruskal–Wallis test with Bonferroni’s multiple comparison test.

2. C

   Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min. Scale bar, 10 μm.

3. D

   Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min. Scale bar, 10 μm.

Source data are available online for this figure.

Figure 6. Lower force generation capacity and 3D motility of parasite lines expressing mutated MyoA

1. Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap. The bead is placed at the apical end of the sporozoite. The graph shows the fraction of sporozoites that pulled the bead out of the optical trap. Black dots: individual experiments. Bars: mean. 73–131 sporozoites were probed for each line. Significance determined by one‐way analysis of variance with Tukey’s multiple comparison test.

2. Scheme showing a sporozoite moving through a polymeric network in 3D. The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance determined by Kruskal–Wallis test with Bonferroni’s multiple comparison test.

3. Selected trajectories of 20 moving sporozoites observed over a period of 3 min. Scale bar, 10 μm.

Source data are available online for this figure.

Figure 7. Cartoon portraying importance of MyoA phosphorylation during transmission of Plasmodium

Wild‐type myosin is phosphorylated in early sporozoites allowing the generation of optimal force transduction and gliding motility. Dephosphorylation is also important for optimal force generation as S19D shows lower force and speed. Non‐phosphorylated myosin still allows sporozoite gliding but at reduced speed with too little force generated for efficient salivary gland invasion. Disruption of the interaction of the N terminus with the Switch I and II (E6R mutation) has similar impact as constitutive phosphorylation as mimicked by S19D.