Seymour Schulman

Plasmodium berghei and Plasmodium knowlesi: Serum binding to sporozoites

Abstract Plasmodium berghei salivary gland and oocyst sporozoites were examined with fluorescein isothiocyanate (FITC)-lectins to determine if sporozoites had carbohydrate-containing molecules on their surfaces. None of the eight fluorescein isothiocyanate-lectins bound to the sporozoites. However, incubation of sporozoites in mouse serum permitted subsequent binding of concanavalin A and Ricinus communis agglutinin I. In general, serum binding occurred when sporozoites were incubated in serum from hosts susceptible to sporozoite infection. Sporozoites of the rodent parasite, P. berghei, tended to bind rodent but not primate serum, while sporozoites of the monkey parasite, Plasmodium knowlesi, tended to bind primate but not rodent serum. The serum component(s) that bound to sporozoites were concentrated considerably by ammonium sulfate precipitation followed by concanavalin A—Sepharose affinity chromatography.

EFFECTS OF NEOGLYCOPROTEINS ON PENETRATION OF PLASMODIUM FALCIPARUM MEROZOITES INTO ERYTHROCYTES IN VITRO

Sugars conjugated to bovine serum albumin (neoglycoproteins) were tested for their ability to inhibit Plasmodium falciparum merozoite invasion of red blood cells in an in vitro inhibition assay. The inhibitory effects of the neoglycoproteins on merozoite invasion of erythrocytes were determined by assessing 3H-hypoxanthine incorporation into the cultures. The only neoglycoprotein that caused significant inhibition N-acetyl-glucosamine-bovine serum albumin. A comparison of the inhibitory effects of N-acetyl-glucosamine and N-acetyl-glucosamine-bovine serum albumin showed that N-acetyl-glucosamine-bovine serum albumin was 84 fold more effective than the free sugar in causing 50% inhibition of 3H-hypoxanthine incorporation.

ROLE OF A SERUM FACTOR IN ENHANCEMENT OF IN VITRO INTERACTIONS BETWEEN PLASMODIUM BERGHEI SPOROZOITES AND HAMSTER PERITONEAL MACROPHAGES

Interactions between Plasmodium berghei sporozoites and hamster peritoneal macrophages were studied. Hamster serum was shown to enhance the percentage of sporozoites tha attached to macrophages, thus confirming previous studies by other workers using mouse macrophages and mouse serum. The enhancement factor within hamster serum was concentrated by a fractionation procedure consisting of ammonium sulfate precipitation followed by Concanavalin A-Sepharose affinity chromatography. This serum fraction has been shown previously to contain component(s) that bind to P. berghei sporozoites.

In vitro Invasion of Host Cells by Plasmodium berghei Sporozoites in Serum-Free Medium

The development of techniques for the in vitro invasion of Plasmodium berghei sporozoites into continuous cell lines of WI38 human embryonic lung fibroblasts (Hollingdale et al., 1981, Science 213: 1021-1022) and HepG2 human hepatoma cells (Hollingdale et al., 1983, American Journal of Tropical Medicine and Hygiene 32: 685-690) has made it possible to investigate many aspects of sporozoite-host cell interactions during the process of sporozoite invasion. We recently used this in vitro procedure to demonstrate a direct correlation between the motility of sporozoites and their ability to invade both of these cell lines (Stewart et al., 1986, Infection and Immunity 51: 859-864). Agents and procedures that inhibited sporozoite motility invariably prevented sporozoites from invading. We had previously demonstrated that sporozoites suspended in culture medium containing serum or albumin undergo active motility when these sporozoites are pipetted onto a microscope slide and examined by phase-contrast microscopy (Vanderberg, 1974, Journal of Protozoology 21: 527-537); sporozoites thoroughly rinsed and suspended in protein-free medium are immotile. Because active sporozoite motility appears to be necessary for sporozoite invasion into target host cells in vitro (Stewart et al., 1986, loc. cit.), we were interested to see whether the absence of serum in the medium would affect sporozoite invasiveness into HepG2 and WI38 cells. Sporozoites were obtained from the salivary glands of P. berghei-infected mosquitoes that had fed on infective hamster blood 18 days previously (Vanderberg, 1977, Experimental Parasitology 42: 169-181). The salivary glands were pooled in ice-chilled Medium M199 (GIBCO Laboratories, Grand Island, New York) and triturated in a glass homogenizer to release the sporozoites. After a low-speed centrifugation step to pellet the mosquito debris, the sporozoites in the supernatant were thoroughly washed in Medium M199 and kept on ice until used. We found it necessary to wash the sporozoites because the concentrate from triturated mosquitoes appeared to contain an undefined soluble component that induced a degree ofsporozoite motility. For our invasion studies, we inoculated 3,000 sporozoites previously rinsed with medium (with or without 10% fetal bovine serum [FBS]) onto 12-mm-diameter target cell monolayers incubated in media (Eagles Minimal Essential Medium [MEM], GIBCO, Grand Island, New York) supplemented with or without FBS (Stewart et al., 1986, loc. cit.). These sporozoites effectively invaded both HepG2 and WI38 cells when the cultured cells were incubated in medium containing serum (data not shown). However, sporozoites can invade HepG2 cells effectively even in the absence of serum, although the absence of serum significantly inhibits the entry of sporozoites into WI38 cells (Table I). That P. berghei sporozoites invade HepG2 cells more effectively than WI38 cells confirms previous observations (Hollingdale et al., 1983, loc. cit.; Stewart et al., 1986, loc. cit.). These results appeared to be directly contradictory to our hypothesis that sporozoite motility is necessary for invasiveness, because serum albumin is normally required for the induction of sporozoite motility. To determine whether the sporozoites that invaded cultured cells in medium lacking serum are actively motile when in contact with the surface of cultured cells, we inoculated M199-rinsed sporozoites onto con-

Dextran Density Gradient Purification of Plasmodium Sporozoites

Several procedures have been described for isolating relatively pure preparations of Plasmodium sporozoites from homogenates of infected mosquitoes. These have included gradient centrifugation (on continuous or discontinuous diatrizoate gradients), column purification (with ion-exchange or lectin affinity columns), and filtration through pores of defined size (review and comparative assessment of these techniques in Vanderberg, 1980, In The in vitro cultivation of the pathogens of tropical diseases, W.H.O. Tropical Diseases Research Series, 3, Schwabe & Co., Basel, pp. 77-89). We now report a new gradient procedure, which yields better and more consistent results than any previously described. Plasmodium berghei sporozoites were obtained from Anopheles stephensi mosquitoes infected 18-22 days previously (procedures as in Vanderberg, 1977, Experimental Parasitology 42: 169-181). Generally, 3 separate batches of 100200 mosquitoes were gently homogenized for 2 min with 1.5 ml Medium M199 (GIBCO Labs., Grand Island, New York) in an ice-chilled mortar. The pooled homogenates were transferred to a 15-ml centrifuge tube, and additional medium added to make a total of 10 ml. The preparation was centrifuged at 20 g for 6 min, after which the supernatant suspension was transferred to a 12-ml glass Sorvall centrifuge tube. The pellet, containing residual sporozoites, was returned to a mortar and gently ground with 2 ml M199 for 2 min; the resulting homogenate was transferred to a 15-ml centrifuge tube, additional M199 to