Evidence for infectious merozoites of Plasmodium falciparum from natural isolates of cultured hepatoma cells infected with sporozoites

Abstract

Previous cell culture systems using various human hepatoma cell lines established that the intra-hepatic stages of Plasmodium falciparum could be studied ex vivo. However, only one of these culture systems yielded infective merozoites that subsequently completed the parasite's life cycle outside a human host. We hypothesized that a major limitation is the use of laboratory-adapted P. falciparum blood stages for sporozoites generation. Plasmodium falciparum sporozoites were generated by membrane-feeding of gametocyte-infected blood samples from hospital patients to Anopheles arabiensis. Subsequently, cultured HepG2 cells were infected with the sporozoites. From 6 days post-sporozoite inoculation, liver merozoites could be harvested from the cell supernatants. When co-cultured with O + erythrocytes, these merozoites established a blood infection and yielded erythrocytic stage parasites that re-infected erythrocytes. To confirm that the erythrocytic parasites generated were P. falciparum, RNA expressed by the erythrocytic parasites was isolated and used as control in microarray analysis against RNA expressed by irradiated erythrocytic parasites; subsequently, P. falciparum genes were identified. The cultured HepG2 cells permitted the full intra-hepatic maturation of P. falciparum parasites from natural isolates. Infective merozoites were yielded which gave rise to the erythrocytic stage P. falciparum post-infection into O + erythrocytes. The full intra-hepatic maturation of the naturally isolated P. falciparum parasites in a HepG2 cell culture system is possible. This finding has important implications for malaria research and vaccine development.

Fig 1. A) HepG2 cells’ growth kinetics in DMEM complete media.

Cell population was highest on day 5 (120hrs) reaching a peak of 11.825 ×  105 cells/25 cm² flask – an 18.2-fold population increase from 0-hr. B) Saturation curve for the HepG2 G-6-Pase specific activity with increasing concentrations of HepG2 protein. The peak G6Pase specific activity before enzyme’s active sites were completely saturated was 9.1ηmol/min-mgprot. C) SDS-PAGE result for the HepG2 cell line’s secreted proteins. Lane M (Control) shows the serum proteins, albumins, and globulins. Lanes 1-6, showing the proteins synthesized and stored in the cells’ cytosol, give a similar pattern for the cells from the three flasks. γ-globulins were detected while albumin, α−and β−globulins secretions were not seen.

Fig 2. A) Plasmodium falciparum gametocytes from blood samples obtained from hospital patients in Khartoum, Sudan. B) P. falciparum sporozoites isolated from the infected Anopheles arabiensis mosquitoes 15-18 days post-gametocyte blood feeding.

Fig 3. A) Extracellular parasites, merozoites that emerged from the infected HepG2 cells 6 days post-sporozoite inoculation. The Giemsa-stained films detected the entire sequence of the parasite’s infection into the erythrocytes. (a) Shows the merozoites locating the erythrocytes. (b) Shows the initial recognition events, the subsequent attachment to the erythrocyte membrane and junction formation on the red cells. (c-d) Shows the erythrocyte’s invagination and engulfing of the merozoite; multiple infection of the erythrocytes (unique to P. falciparum) can also be seen. (e-f) Shows the merozoite now fully internalized into the erythrocyte. B) Giemsa-stained micrographs of ring-stage Plasmodium falciparum detected 48, 96 and 144hrs post-erythrocyte addition. Parasites have a defined chromatin dot with a bluish cytoplasm. Note the multiply infected erythrocytes in (a) and (b). C) Giemsa-stained micrographs of Plasmodium falciparum trophozoites and schizonts detected 48, 96 and 144h post-erythrocyte addition. (a and b) Trophozoites’ cytoplasms are denser and their ring shapes have gradually given way to a more amoeboid structure. (c) A ruptured schizont and a developing one. (d) Another ruptured schizont and a developing one.