Purification and Characterization of the Alkaline Phosphatase from Echinococcus granulosus Cyst Membranes

International audienceThe purification to homogeneity and the characterization of Echinococcus granulosus alkaline phosphatase (AP; EC 3.1.3.1) from hydatid cyst membranes are described. After n-butanol extraction, the parasite enzyme was sequentially purified by affinity chromatography on concanavalin A-sepharose followed by gel filtration. The purified protein (210 kDa) had a tetrameric structure composed of 4 56-kDa subunits. Its isoelectric point (4.8) and its kinetic parameters were determined (Km = 0.24 ± 0.05 mmol/L; Vm = 173 ± 21 nmol/min/mg protein for p-nitrophenylphosphate). The parasite enzyme differed from the host liver enzyme in its thermal stability, optimum reaction temperature, optimum pH, and catalytic parameters, but not in its apparent molecular weight. Furthermore, sera from patients infected with E. granulosus recognized the parasite AP on immunoblots, whereas uninfected controls were negative. These results as well as the role of this enzyme in the host-parasite relationship emphasize its potential importance as a diagnostic and prognostic antigen in the monitoring of hydatid infection

Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design

International audienceIts natural resistance to antiprotozoal chemotherapy characterizes the intestinal protozoan parasite Cryptosporidium parvum and the P-glycoprotein-related multidrug resistance proteins such as CpABC3 could be involved. In order to design and study specific inhibitors of the CpABC3 nucleotide-binding domain, a hexahistidine-tagged recombinant protein encompassing the N-terminal cytosolic NBD1 domain was over-expressed in E. coli and purified. The 45 kDa H6-NBD1 displayed intrinsic fluorescent properties consistent with the presence of two Trp residues in a hydrophobic environment. The binding of ATP and the fluorescent analogue TNP-ATP produced a dose-dependent quenching as well as progesterone and the flavone quercetin. The extrinsic fluorescence of TNP-ATP was enhanced upon binding to H6-NBD1, which was only partially displaced by the natural substrate ATP. The recombinant protein hydrolyzed ATP (K m = 145.4 ± 18.2 M), but ADP (K m = 4.3 ± 0.6 mM) and AMP (K m = 5.4 ± 1.5 M) were also substrates. TNP-ATP is a competitive inhibitor of the catalytic activity (K i = 36.6 ± 4.5 M), but quercetin and progesterone were not inhibitors, evidencing different binding sites. The recombinant C. parvum H6-NBD1 should be a valuable tool for rational drug design and will allow the discrimination between specific inhibitors of the catalytic site and molecules binding to other sites

Use of Percoll for the infection of cells in vitro with Cryptosporidium parvum oocysts

International audienceA method for the infection of non-adherent THP-1 cells and adherent MDBK cells with Cryptosporidium parÕum oocysts using isotonic Percoll solutions was developed. Excystation was maximal after 2 h, but toxicity increased with the oocystrcell ratio and the incubation time. The infection rates did not increase with the oocystrcell ratio and both cell types were equally parasitized.

Fertilization in the cestode Echinococcus multilocularis (Cyclophyllidea, Taeniidae)

Fertilization in the taeniid cestode Echinococcus multilocularis with uniflagellate spermatozoa was examined by means of transmission electron microscopy (TEM). Fertilization in this species occurs in the oviduct lumen or in the fertilization canal proximal to the ootype, where the formation of the embryonic capsule precludes sperm contact with the oocytes. Cortical granules are not present in the cytoplasm of the oocytes of this species, however, several large bodies containing granular material where frequently observed. Spermatozoa coil spirally around the oocytes and syngamy occurs by lateral fusion of oocyte and sperm plasma membranes. In the ootype, one vitellocyte associates with fertilized oocyte, forming a membranous capsule which encloses both cell types. In this stage, the spirally coiled sperm body adheres partly to the external oocyte surface, and partially enters into the perinuclear cytoplasm. The electron-dense sperm nucleus becomes progressively electron-lucent within the oocyte cytoplasm after penetration. Simultaneously with chromatin decondensation, the elongated sperm pronucleus changes shape, forming a spherical male pronucleus, which attains the size of the female pronucleus. Cleavage begins immediately after pronuclear fusion

Echinococcus multilocularis Leuckart, 1863 (Taeniidae): new data on sperm ultrastructure

The present study establishes the ultrastructural organisation of the mature spermatozoon of Echinococcus multilocularis, which is essential for future research on the location of specific proteins involved in the sperm development in this species and also in Echinococcus granulosus. Thus, the ultrastructural characteristics of the sperm cell are described by means of transmission electron microscopy. The spermatozoon of E. multilocularis is a filiform cell, which is tapered at both extremities and lacks mitochondria. It exhibits all the characteristics of type VII spermatozoon of tapeworms, namely a single axoneme, crested bodies, spiralled cortical microtubules and nucleus, a periaxonemal sheath and intracytoplasmic walls. Other characteristics observed in the male gamete are the presence of a >900-nm long apical cone in its anterior extremity and only the axoneme in its posterior extremity. The ultrastructural characters of the spermatozoon of E. multilocularis are compared with those of other cestodes studied to date, with particular emphasis on representatives of the genus Taenia. The most interesting finding concerns the presence of two helical crested bodies in E. multilocularis while in the studied species of Taenia, there is only one crested body. Future ultrastructural studies of other species of the genus Echinococcus would be of particular interest in order to confirm whether or not the presence of two crested bodies is a characteristic of this genus

Toxoplasma gondii: Identification and immune response against a group of proteins involved in cellular invasion

International audienceToxoplasma gondii is an ubiquitous intracellular parasite, causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. A group of proteins secreted by tachyzoites during host-cell invasion was isolated from the interaction medium. It induced the permeability of the cells as assessed by alpha-sarcin and consequently facilitated the entry of the parasite into the cells. SDS-PAGE of the purified proteins showed a pattern of four proteins of 67, 42, 32 and 27 kDa. MRC-5 cells incubated with the total protein and the different electroeluted bands endured a high cellular death in presence of alpha-sarcin. BALb/C mice immunized with the group of proteins had a mixed Th1/Th2 response and were protected upon challenge with the parasites

Echinococcus multilocularis (Cestoda, Cyclophyllidea, Taeniidae): origin, differentiation and functional ultrastructure of the oncospheral tegument and hook region membrane

Both the oncospheral tegument and the hook region membrane (HRM) of Echinococcus multilocularis hexacanths originate from a syncytial binucleate complex that appears in the early stage of morphogenesis and organogenesis of the hexacanth larva. The primordium of this binucleate complex forms a binucleate syncytial cap or "calotte" situated beneath the inner envelope at one pole of the developing embryo. During oncospheral differentiation, the binucleate perikaryon of the syncytial cap is sunk progressively deeper into the central part of the embryo, but remains always connected with the distal cytoplasm by a tendrillar cytoplasmic connection or bridge. Following migration or sinking of the binucleate perikaryon, numerous cytoplasmic vesicles appear in the distal cytoplasm. These vesicles fuse progressively together and form a single large cavity or lacuna. The walls of this cavity are becoming at this point the walls of two delaminated layers: (1) the distal anucleated cytoplasmic layer is transformed into the oncospheral tegument and (2) the proximal thin cytoplasmic layer is transformed into the "hook region membrane". This delamination of the initially compact layer of distal cytoplasm into two layers seems to be closely associated with differentiation of oncospheral hooks, the elongating blades of which protrude progressively into a newly formed cavity. The pressure of hook blades on the hook region membrane appears to facilitate its further separation from the basal layer of distal cytoplasm which is transformed into the peripheral layer of oncospheral tegument. In the mature oncosphere, the surface of this peripheral layer forms a regular brush border of cytoplasmic processes or microvilli and represents the true body covering of the hexacanth. The very thin cytoplasmic connection between the peripheral layer of tegument and binucleate perikaryon appears only very seldom in the ultrathin sections as a narrow cytoplasmic strand and has a plasma membrane that is reinforced by a single row of cortical microtubules. The HRM covers only one pole of the oncosphere and is attached to the oncosphere surface. The HRM is clearly visible in the mature oncosphere and is draped over the hook blades, the sharp points of which are protected by moderately electron-dense caps. Comparison of the above morphology with that of TEM study of the tegument of adult cestodes shows a great similarity as well as homology in the body covering of both larval and adult cestodes

Echinococcus multilocularis (Cestoda, Cyclophyllidea, Taeniidae): oncospheral hook morphogenesis

Ultrastructural characteristics of the oncospheral hook morphogenesis in the taeniid cestode Echinococcus multilocularis Leuckart, 1863, a parasite of medical and veterinary importance, are described. Oncospheral hook primordia appear at the preoncospheral phase of the embryonic development. Within six specialised cells of the so-called oncoblasts, high concentration of mitochondria, numerous ribosomes and extended Golgi regions are involved in hook development. During hook growth, the blade and base gradually protrude outside the oncoblast plasma membrane. The nucleated oncoblast persists around the handles of fully formed hooks. Simultaneously with the hook primordium elongation and transformation into a blade, handle and base, the hook material differentiates into an electron-dense cortex and a less dense inner core. The exit of the blade of eachmature hook, protruding from the oncosphere, is surrounded by a circular, septate desmo some and two rigid, dense rings on either side. The pattern of oncospheral hook morphogenesis in E. multilocularis is compared with that of other previously examined cyclophyllidean cestodes. Though oncoblasts have never been observed around the mature hooks, their remnants are often still visible in the fully developed infective oncospheres in particular in some taeniid species so far examined in this respect. The origin and formation of oncospheral hooks in E. multilocularis, evidently differs from that of the rostellar hooks. Thus, although the hooks may have slight similarity at the gross level, they are neither analogous nor homologous structures