RESUMO
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.
Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Proteínas de Capeamento de Actina , Animais , Sequência de Bases , Citosol/metabolismo , DNA de Protozoário , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/genéticaRESUMO
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.
Assuntos
Actinas/química , Depsipeptídeos , Peptídeos Cíclicos/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/fisiologia , Actinas/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Genes de Protozoários , Proteínas dos Microfilamentos/genética , Plasmodium falciparum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Humanos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Aedes/microbiologia , Vírus da Dengue/isolamento & purificação , Reação em Cadeia da Polimerase , Animais , Sequência de Bases , Encéfalo/microbiologia , Vírus da Dengue/genética , Sistema Digestório/microbiologia , Feminino , Dados de Sequência Molecular , Glândulas Salivares/microbiologiaRESUMO
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.
Assuntos
Aedes/microbiologia , Vírus da Dengue/fisiologia , Insetos Vetores/microbiologia , Análise de Variância , Animais , FemininoRESUMO
The identification of Borrelia strains isolated from ticks or relapsing fever patients is not easy. Seizing the opportunity of recent isolation of such strains from Western Africa, we tried to evaluate the interest and the limits of a method classically proposed for that aim; cross protection test in mouse. This technique is proving rather difficult to perform because of different technical reasons; the results are critical to read. In some cases, these results seem undeniable, but the observed differences in protection levels are often weak, inconstant, insufficiently reliable to constitute a diagnostic tool. So it appears important to develop other identification methods, based on molecular analysis of Borrelia DNA, which will be more subtle and more specific.
Assuntos
Infecções por Borrelia/microbiologia , Borrelia/isolamento & purificação , Carrapatos/microbiologia , África Ocidental , Animais , CamundongosRESUMO
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.
Assuntos
Aedes/microbiologia , Vírus da Dengue/fisiologia , Insetos Vetores/microbiologia , Animais , Vírus da Dengue/crescimento & desenvolvimento , Feminino , Ensaio de Placa Viral , Replicação ViralRESUMO
Permanent cell lines of Quail embryo fibroblasts appear in cultures of cells infected with a wild type strain of Rous sarcoma virus (SR-RSV) or with its temperature sensitive transformation mutants (ts-T) (NYts68 and PA101) following a three step process. In step one, infected cells grow twice as fast as the control. The second step consists of a crisis during which the cell population is stationary for four to five weeks. Towards the fourth week several foci of cell growth are observed in the flasks. Respreading of the content of these flasks yields permanent lines. This constitutes the third step of the population evolution. In step one the growth rate of the infected cells is the same irrespective of the incubation temperature (36 degrees C or 41 degrees C) whereas the level of the pp60v-src activity is considerably depressed at 41 degrees C for NYts68 and PA101. Foci do not appear at restrictive temperature in the ts infected population and permanent lines are not recovered under that condition. These lines grow ony at 36 degrees C. It can be shown that the virus which they produce is not modified with respect to the temperature sensitivity of the src gene expression since newly infected fibroblasts grow equally well in step one at both 36 degrees C and 41 degrees C, and stop after the same number of generations. This finding suggests that the events which, during the crisis period, lead to the establishment of permanent lines, take place at the cellular level but depend on the activity of the pp60v-src protein for their occurrence or their expression.
Assuntos
Vírus do Sarcoma Aviário/genética , Linhagem Celular , Oncogenes , Animais , Transformação Celular Viral , Coturnix/embriologia , Fibroblastos , Mutação , TemperaturaRESUMO
Quail embryo skin fibroblasts infected with wild type Rous sarcoma virus SR-RSV grow, at 36 or 41 degrees C, twice as fast (30 hours. mean division time) as the uninfected controls or the cells infected with RAV-1 virus. At 36 degrees C the SR-RSV infected cells stop growing after the same delay as the controls, thus having undergone twice as many doublings, (38 to 40 instead 18 to 20). However, at 41 degrees C, SR-RSV infected cells stop 5 to 6 divisions earlier. This cannot be reversed by shifting the cultures to 36 degrees C. Rising to 41 degrees C those cultures which grew previously at 36 degrees C reduce the number of residual divisions. No such effect is observed with NYts68 or PA101 viruses which display a reduced level of pp60v-src activity at 41 degrees C.