Rapid transport of the acidic phosphoproteins ofPlasmodium berghei andP. chabaudi from the intraerythrocytic parasite to the host membrane using a miniaturized fractionation procedure

Abstract

A miniaturized procedure for the separation of the host erythrocyte membrane from malarial parasites based on saponin lysis and density-gradient centrifugation with Percoll is described. The procedure requires only 20-35 microliters packed infected erythrocytes, is simple to perform, needs no sophisticated equipment, and can be completed in less than 2 h. Analysis of the isolated erythrocyte membranes and parasites using marker enzymes and electron microscopy revealed that both the purity and the yield of these fractions were relatively high. Erythrocyte membrane proteins, including spectrin, ankyrin, and band 4.1, were not found on the parasitophorous vacuolar membrane, which remained associated with some but not all of the isolated parasites. Application of this method to pulse-chase experiments indicated that the acidic phosphoproteins of Plasmodium berghei and P. chabaudi were rapidly transported from the parasite to the erythrocyte membrane immediately after their synthesis. The rapid export of these acidic phosphoproteins from the parasite distinguishes them from other proteins exported by the malarial parasite.

Recognition of the parasite infected cell surface determinants by homologous antiserum raised against infected cell membranes

“…Parasite derived antigens have been reported in P. berghei-infected eryrthrocyte membranes (Wiser et al 1988;Schetters et al 1988). Many among those were localized towards the cytoplasmic face of the membranes (Wiser et al 1988), while others were suggested to be present on the infected cell surface (Schetters et al 1988).…”

“…Many among those were localized towards the cytoplasmic face of the membranes (Wiser et al 1988), while others were suggested to be present on the infected cell surface (Schetters et al 1988). Our recent studies on immunoliposomes mediated delivery of chloroquine in vivo to the infected erythrocytes (Singh et al 1993;Owais et al 1995) supported the presence of neo-antigenic determinant(s) on the surface of infected erythrocytes.…”

“…The infected cell surface determinant(s) have been mostly identi®ed by using, either non-immune/immune sera (Newbold et al 1982;Schetters et al 1988;van Schravendijk et al 1991) or monoclonal antibodies generated by heterologous immunizations (Hall et al 1983;Crandall and Sherman 1991;Wiser et al 1988). However, these methodologies have limited sensitivities for detection of multiple antigenic determinants.…”

Identification and Characterization of Putative Secreted Antigens fromBabesia microti

“…Free parasites were isolated from infected hamster RBCs as previously described (38). A lysate was made from isolated B. microti organisms by washing the pellet three times in phosphate-buffered saline, with a final resuspension in 2 ml of phosphate-buffered saline, for a starting blood volume of 4 ml (75% parasitemia).…”

Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

“…The cells were washed twice with HBS and erythrocytes were lysed with saponine 0.15% in HBS. Erythrocyte membranes were eliminated after centrifugation over a Percoll cushion (Wiser & Lanners 1992). Free parasites were washed three times with HBS; the weight of the pellet was evaluated and the parasites were resuspended 1:10 w/v in extraction buffer (Hepes 5 mM pH 7.5, EDTA 0.5 mM, EGTA 0.5 mM, Nonidet P40 0.05%, supplemented with the following mixture of protease inhibitors: PMSF 1 mM, Benzamidine 0.01 mM, Phenantroline 1 µg/ml, Aprotinin 10 µg/ml, Leupeptin 10 mg/ml and Pepstatin A 10 mg/ml) and frozen at -70ºC.…”