RESUMEN
Malaria is caused by unicellular parasites of the genus Plasmodium, which reside in erythrocytes during the clinically relevant stage of infection. To separate parasite from host cell material, haemolytic agents such as saponin are widely used. Previous electron microscopy studies on saponin-treated parasites reported both, parasites enclosed by the erythrocyte membrane and liberated from the host cell. These ambiguous reports prompted us to investigate haemolysis by live-cell time-lapse microscopy. Using either saponin or streptolysin O to lyse Plasmodium falciparum-infected erythrocytes, we found that ring-stage parasites efficiently exit the erythrocyte upon haemolysis. For late-stage parasites, we found that only approximately half were freed, supporting the previous electron microscopy studies. Immunofluorescence imaging indicated that freed parasites were surrounded by the parasitophorous vacuolar membrane. These results may be of interest for future work using haemolytic agents to enrich for parasite material.
Asunto(s)
Eritrocitos/parasitología , Hemólisis/efectos de los fármacos , Plasmodium falciparum/fisiología , Saponinas/farmacología , Estreptolisinas/farmacología , Proteínas Bacterianas/farmacología , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/parasitología , Eritrocitos/efectos de los fármacos , Vesículas Extracelulares/parasitología , Humanos , Estadios del Ciclo de Vida/fisiología , Microscopía , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma membrane-spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here, we show that different mutations in profilin, that do not affect actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.
Asunto(s)
Malaria , Parásitos , Actinas/genética , Animales , Plasmodium berghei , Profilinas/genética , Proteínas Protozoarias/genéticaRESUMEN
Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like ß-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.
Asunto(s)
Actinas/metabolismo , Malaria/parasitología , Plasmodium berghei/citología , Plasmodium berghei/metabolismo , Profilinas/metabolismo , Proteínas Protozoarias/metabolismo , Actinas/genética , Animales , Movimiento Celular , Femenino , Humanos , Ratones Endogámicos C57BL , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Profilinas/genética , Unión Proteica , Proteínas Protozoarias/genética , Esporozoítos/citología , Esporozoítos/crecimiento & desarrollo , Esporozoítos/metabolismoRESUMEN
Migration of malaria parasites is powered by a myosin motor that moves actin filaments, which in turn link to adhesive proteins spanning the plasma membrane. The retrograde flow of these adhesins appears to be coupled to forward locomotion. However, the contact dynamics between the parasite and the substrate as well as the generation of forces are complex and their relation to retrograde flow is unclear. Using optical tweezers we found retrograde flow rates up to 15 µm/s contrasting with parasite average speeds of 1-2 µm/s. We found that a surface protein, TLP, functions in reducing retrograde flow for the buildup of adhesive force and that actin dynamics appear optimized for the generation of force but not for maximizing the speed of retrograde flow. These data uncover that TLP acts by modulating actin dynamics or actin filament organization and couples retrograde flow to force production in malaria parasites.
Asunto(s)
Movimiento Celular/fisiología , Malaria/parasitología , Plasmodium berghei/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Esporozoítos/fisiología , Actinas/química , Actinas/metabolismo , Animales , Fenómenos Biomecánicos , Ratones , Plasmodium berghei/química , Esporozoítos/químicaRESUMEN
BACKGROUND: The virulence of Plasmodium falciparum malaria is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density. METHODOLOGY/PRINCIPAL FINDINGS: We used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from â¼20 h to â¼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions. CONCLUSIONS/SIGNIFICANCE: The density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host.