Johanna Ripp

Plasmodium sporozoites are the highly polarized and motile forms of the malaria parasite that are transmitted by mosquitoes to the vertebrate hosts. Sporozoites use myosin molecular motors to generate retrograde flow of actin filaments. These are linked to plasma-membrane spanning adhesins, which in turn bind to the extracellular environment, resulting in forward directed gliding motility. The gliding motility machine of sporozoites leads to high speeds in the range of micrometer per second, which are essential for efficient migration in the skin. Yet, it is not clear how the individual parts of the machinery work together to generate force during migration. Sporozoites are elongated and curved cells and move on circular tracks in vitro . Sporozoites lacking the adhesin thrombospondin related anonymous protein (TRAP) like protein, TLP, can still migrate in the skin, but at a lower level. TLP lacking sporozoites generate a lower force on the dorsal (non-substrate facing) surface as measured by laser tweezers. Here we use traction force microscopy to investigate motile sporozoites and the forces they produce during migration on their ventral surface. Both wild type and tlp(-) sporozoites show distinct foci of force generation, but tlp(-) sporozoites generating overall lower forces. Our findings demonstrate that TLP is an important element of the force-generating machinery during sporozoite gliding motility.

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.

During transmission of malaria‐causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane‐associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin‐GFP localizes at the cytoplasmic periphery and concavin(−) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(−) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(−) sporozoites are transmitted. Strikingly, motile concavin(−) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here we report on a newly identified protein, concavin, that is important for maintenance of the sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localized at the cytoplasmic periphery of sporozoites and concavin(−) sporozoites progressively rounded up upon entry of salivary glands. These rounded concavin(−) sporozoites failed to pass through the narrow salivary ducts and were hence rarely ejected by mosquitoes. However, normally shaped concavin(−) sporozoites could be transmitted and migrated in the skin or skin like environments. Strikingly, motile concavin(−) sporozoites could disintegrate while migrating through narrow strictures in the skin leading to parasite arrest or death and decreased transmission efficiency. We suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. SIGNIFICANCE: Malaria parasites are transmitted by Anopheles mosquitoes and rely on rapid migration for establishing an infection. We identified and characterized a protein, named concavin, essential for maintaining the shape of the sporozoite. Concavin is a membrane associated protein facing the cytoplasm suggesting that it contributes to riveting the plasma membrane to the subtending inner membrane complex. Sporozoites lacking concavin can round up in the salivary glands, are less well transmitted to mice and disintegrate while migrating in the skin. Hence, concavin is essential for parasite transmission and infectivity. Highlights: - A membrane associated protein is essential for Plasmodium shape maintenance -Migrating parasites disintegrate in the absence of concavin -First protein essential for cellular integrity of Plasmodium sporozoites -Thickened and deformed Plasmodium sporozoites fail to be transmitted by mosquitoes

Malaria-causing parasites rely on an actin-myosin based motor for the invasion of different host cells as well as 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 serine 19 is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry and migration of salivary gland sporozoites in both 2D and 3D environments. Our data suggests that entry into salivary glands represents the strongest barrier in parasite transmission and hence is the key determinant for evolution of the motility and invasion machinery of these parasites. Highlights: The unusual N-terminal extension of Plasmodium myosin A is important for efficient gliding motility Altering the kinetics of the myosin A power stroke impacts Plasmodium life cycle progression and sporozoite motility Myosin A phosphorylation at serine 19 is important for malaria transmission by mosquitoes Salivary gland invasion emerges as key selection step for evolution of the parasite motor

Transmission of malaria‐causing parasites to and by the mosquito relies on active parasite migration and constitutes bottlenecks in the Plasmodium life cycle. Parasite adaption to the biochemically and physically different environments must hence be a key evolutionary driver for transmission efficiency. To probe how subtle but physiologically relevant changes in environmental elasticity impact parasite migration, we introduce 2D and 3D polyacrylamide gels to study ookinetes, the parasite forms emigrating from the mosquito blood meal and sporozoites, the forms transmitted to the vertebrate host. We show that ookinetes adapt their migratory path but not their speed to environmental elasticity and are motile for over 24 h on soft substrates. In contrast, sporozoites evolved more short‐lived rapid gliding motility for rapidly crossing the skin. Strikingly, sporozoites are highly sensitive to substrate elasticity possibly to avoid adhesion to soft endothelial cells on their long way to the liver. Hence, the two migratory stages of Plasmodium evolved different strategies to overcome the physical challenges posed by the respective environments and barriers they encounter.

Transmission of malaria-causing parasites to and by the mosquito rely on active parasite migration and constitute bottlenecks in the Plasmodium life cycle. Parasite adaption to the biochemically and physically different environments must hence be a key evolutionary driver for transmission efficiency. To probe how subtle but physiologically relevant changes in environmental elasticity impact parasite migration, we introduce 2D and 3D polyacrylamide gels to study ookinetes, the parasite forms emigrating from the mosquito blood meal and sporozoites, the forms transmitted to the vertebrate host. We show that ookinetes adapt their migratory path but not their speed to environmental elasticity and are motile for over 24 hours on soft substrates. In contrast, sporozoites evolved more short-lived rapid gliding motility for rapidly crossing the skin. Strikingly, sporozoites are highly sensitive to substrate elasticity possibly to avoid adhesion on soft endothelial cells on their long way to the liver. Hence the two migratory stages of Plasmodium evolved different strategies to overcome the physical challenges posed by the respective environments and barriers they encounter. Highlights: Plasmodium ookinetes can move for over 24 hours on very soft substrates mimicking the blood meal Plasmodium ookinetes change their migration path according to substrate stiffness Plasmodium sporozoites are highly sensitive to subtle changes in substrate elasticity Sporozoite may have evolved to not attach to the soft endothelium to help reach the liver