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

Abstract

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 (Eagle's 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-