Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments.

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.

Toxoplasma gondii motility and host cell invasiveness are drastically impaired by jasplakinolide, a cyclic peptide stabilizing F-actin.

Actin polymerization and actin-myosin coupling activity most likely provide the driving force that the protozoan parasite Toxoplasma gondii has to exert to propulse itself during gliding and host cell entry. Nevertheless, little information is available on T. gondii tachyzoite actin dynamics, and in particular, the presence of actin filaments remains largely uncharacterized. Here, we report that the marine sponge peptide jasplakinolide, known to bind to filamentous actin, does indeed stabilize a pool of a parasite detergent-insoluble actin. This pool is likely to be formed by a dynamic assembled actin complex: first, it is competent for assembly/disassembly and secondly, it is sensitive to nucleotide phosphate concentration. In addition, T. gondii tachyzoites contain molecules which inhibit actin assembly and destabilize actin filaments. Thus, these activities could account for the remarkably low amount of the myosin-containing F-actin pool we describe here. Furthermore, when parasites are treated with cell-permeant jasplakinolide, they display a significant loss of both motility and host cell invasiveness. These data suggest that in vivo, the detergent-insoluble pool of actin is dynamic.

The use of nocodazole in cell cycle analysis and parasite purification from Theileria parva-infected B cells.

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.

[Cytoskeletal actin and its associated proteins. Some examples in Protista]. / L'actine cytosquelettique et ses protéines associées. Quelques exemples chez les protistes.

Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin IB at the leading edge of E. histolytica. ABP-120 organizes F-actin in a network and myosin IB participates in the pseudopod formation. Similar approaches using T. vaginalis resulted in the discovery of an actin-binding protein that participate in the F-actin reorganization during adhesion of parasites to target cells. This protein is homologous to alpha-actinin from other eukaryotic cells. Finally, by using cell biology approaches, F-actin was observed in the cytoplasm as well as in the nucleus of Dinoflagellates. The recent developments in the molecular genetics of protozoa will provide new insights to understand the roles of actin-binding proteins during cytoskeleton activities.

Actin-binding proteins of invasive malaria parasites and the regulation of actin polymerization by a complex of 32/34-kDa proteins associated with heat shock protein 70kDa.

Movement of the malaria parasite into a host erythrocyte during invasion is thought to involve polymerization of parasite actin. We have used F-actin affinity chromatography to isolate actin-binding proteins from Plasmodium knowlesi merozoites, in an attempt to identify proteins responsible for regulating parasite actin polymerization during invasion. Five major proteins, of molecular masses 75, 70, 48, 40 and 34 kDa, were reproducibly eluted from the F-actin columns. The 70 kDa actin-binding protein was identified by tryptic peptide microsequencing as heat shock protein-70 kDa (HSC70); this identification was confirmed by Western blotting with anti-HSC70 antibody, and binding of the protein to ATP-agarose. A doublet of 32/34-kDa proteins coeluted with parasite HSC70 from the F-actin and ATP-agarose columns; a complex of these three proteins was also observed by gel filtration chromatography Highly enriched fractions containing the Plasmodium HSC70/32/34 complex inhibited the polymerization of rabbit skeletal muscle actin, in vitro. This capping activity was calcium-independent, and abrogated by phosphatidylinositol 4,5-bisphosphate. The average length of the actin filaments polymerized in presence of the HSC70/32/34-kDa complex was significantly shorter than in the absence of the complex, consistent with a capping activity. The capping or uncapping of actin filament ends by the HSC70/32/34-kDa complex during invasion could provide a mechanism for localized actin filament growth and movement of the parasite into the host cell.

A Plasmodium falciparum novel gene encoding a coronin-like protein which associates with actin filaments.

Plasmodium falciparum, the major causative agent of human malaria, is an Apicomplexa protozoan parasite which invades in its intermediate host hepatocytes and erythrocytes. The driving force underlying internalization into the host cell is thought to involve both polymerization of parasite actin, as entry is inhibited by the cytochalasins, and an actin motor-associated protein. In the related Apicomplexa parasite, Toxoplasma gondii, the involvement of parasite actin during both processes of motility and host cell entry has been genetically established. In a search for molecules that can regulate actin dynamics within Apicomplexa parasites, we have identified a P. falciparum homologue of the actin associated protein called coronin originally described in the amoeba Dictyostelium discoideum. The single copy gene displays a strong homology with the amoeba sequence and with the bovine and human coronin homologues recently cloned. This homology lies not only within the N-terminus containing the five WD repeats that characterize coronin but also extends in the C-terminal part. Furthermore, using an affinity-purified mouse monoclonal antibody against D. discoideum coronin, we have detected in extracts of P. falciparum young and mature schizonts a 42-kDa polypeptide which binds this antibody and is present in a Triton insoluble fraction that also contains parasite actin filaments. In addition, the recombinant protein encoded by the homologue nucleotidic sequence of P. falciparum coronin is indeed recognized by the antibody against D. discoideum coronin. Finally, the cross-reactive polypeptide displays the ability to cosediment with exogenous F-actin, a property which fits with its involvement in actin dynamics.

Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients.

Trypanosoma cruzi enters cells by a unique mechanism, distinct from phagocytosis. Invasion is facilitated by disruption of host cell actin microfilaments, and involves recruitment and fusion of host lysosomes at the site of parasite entry. These findings implied the existence of transmembrane signaling mechanisms triggered by the parasites in the host cells before invasion. Here we show that infective trypomastigotes or their isolated membranes, but not the noninfective epimastigotes, induce repetitive cytosolic-free Ca2+ transients in individual normal rat kidney fibroblasts, in a pertussis toxin-sensitive manner. Parasite entry is inhibited by buffering or depleting host cell cytosolic-free Ca2+, or by pretreatment with Ca2+ channel blockers or pertussis toxin. In contrast, invasion is enhanced by brief exposure of the host cells to cytochalasin D. These results indicate that a trypomastigote membrane factor triggers cytosolic-free Ca2+ transients in host cells through a G-protein-coupled pathway. This signaling event may promote invasion through modulation of the host cell actin cytoskeleton.

Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells.

Trypanosoma cruzi invades most nucleated cells by a mechanism distinct from classical phagocytosis. Although parasites enter at the lysosome-poor peripheral cell margins, lysosomal markers are immediately incorporated into the parasitophorous vacuole. No accumulation of polymerized actin was detected around recently internalized parasites, and disruption of microfilaments significantly facilitated invasion. Lysosomes were observed to aggregate at the sites of trypanosome attachment and to fuse with the vacuole at early stages of its formation. Experimentally induced, microtubule-dependent movement of lysosomes from the perinuclear area to the cell periphery enhanced entry. Conditions that deplete cells of peripheral lysosomes or interfere with lysosomal fusion capacity inhibited invasion. These observations reveal a novel mechanism for cell invasion:recruitment of lysosomes for fusion at the site of parasite internalization.

Oral susceptibility of Aedes albopictus to dengue type 2 virus: a study of infection kinetics, using the polymerase chain reaction for viral detection.

Female Aedes albopictus mosquitoes, aged 1 week, were infected with DEN-2 dengue virus. The kinetics of infection in mosquito brain and mesenteron were monitored using DNA probes with polymerase chain reaction (PCR) amplification of target DNA sequences coding for DEN-2 virus envelope protein, compared with the standard immunofluorescence assay technique (IFA). Rates of virus detection in the mesenteron of orally infected mosquitoes rose to 38% by day 4 post-inoculation, then declined until day 8, followed by irregular peaks around days 11-14 and subsequently. In mosquito head squashes, virus was detected from day 4 onwards, reaching 38% positive by day 18. Salivary glands of all the same females were found to be positive for virus by day 8 onwards. Parenterally infected Ae.albopictus females were all positive for DEN-2 in the brain and salivary glands 8 days post-inoculation. In every case, results obtained with the PCR matched those from the IFA. Our DNA probe with PCR procedure can therefore be utilized as a sensitive and reliable method for studies of DEN-2 vectors.

Analysis of inheritance of oral susceptibility of Aedes aegypti (Diptera: Culicidae) to dengue-2 virus using isofemale lines.

Isofemale lines were compared to determine possible genetic variation in oral susceptibility within the Fare strain of Aedes aegypti (L.) to dengue-2 (DEN-2) virus. Three groups of 12 isofemale lines each were tested statistically using the SAS CATMOD procedure of analysis of variance. The "isofemale line" effect was highly significant, demonstrating genetic variability in oral susceptibility among females. The length of time eggs were stored before hatching influenced the oral susceptibility of the adult mosquitoes for DEN-2 review.

Variation among strains of Aedes aegypti in susceptibility to oral infection with dengue virus type 2.

We compared 18 Aedes aegypti strains for oral susceptibility to dengue virus type 2 (DEN-2) using a feeding protocol in which all parameters remained constant, including the titer of the infectious bloodmeal. For most strains, no significant variation between replicates was observed. Comparisons between pairs of strains showed variation of different degrees, and allowed us to characterize the strains with respect to their oral susceptibility to DEN-2.