Gene expression in Plasmodium berghei ookinetes and early oocysts in a co-culture system with mosquito cells
Introduction
Malaria is caused by parasites of the genus Plasmodium, whose complex life cycle takes place in two different hosts, the vertebrate and the mosquito. Considering the need for novel, alternative strategies for the control of this devastating disease, the period spent by Plasmodium parasites in the anopheline mosquito’s midgut has been recognized as one possible target for intervention. After the insect vector ingests malaria parasites with an infected blood meal a series of developmental processes are initiated. The motile ookinete resulting from fertilization traverses the peritrophic membrane, penetrates the midgut epithelial cells and comes to rest underneath the basal lamina, where it transforms into the immotile oocyst. During the following days nuclear divisions take place and thousands of sporozoites are finally formed. The main processes taking place during these developmental stages have been studied in some detail during the last few years [1], however, only little is known about the molecular events occurring in either of the two organisms. An obstacle in the analysis of the midgut stages of the malaria parasite is the difficulty to study, in detail, the biology of the molecular interactions due to technical difficulties. One of these, precise timing, can now be alleviated using the recently developed in vitro co-culture system of purified ookinetes and insect cells [2]; this method mimics the developmental events taking place in the midgut of an infected mosquito including penetration of insect cells by the mature ookinete, its transformation into an oocyst and early development of the latter. Using this system in conjunction with microarray technology, we investigated the gene expression profiling of Plasmodium berghei during the stages of insect cells invasion and early sporogony. Some 350 different parasite genes showed a differential pattern of expression; these findings provide an insight into the molecular processes taking place in this important transitional phase in the life cycle of the parasite.
Section snippets
Ookinete cultures and immunofluorescence staining of co-cultures
Plasmodium berghei parasite (wild type clone 2.34) infected blood was drawn from TO mice (obtained from Harlan, UK). The infection of the mice and the culture of the ookinetes [3] as well as the details of the conditions of the co-culture have been described earlier [2]. The mature ookinetes were purified using magnetic beads (Dynabeads) coated with the 13.1 monoclonal antibody to the P. berghei P28 protein. The Aedes aegypti cell line used was Mos20 [4] (ATCC # CCL-125). Cells were grown in
Hybridization and data analysis
We prepared parasite RNA from samples of a co-culture [2] of mature ookinetes and A. aegypti Mos20 cells after different periods of incubation. The four different time-points chosen for the analysis corresponded to milestones in the development of the ookinete. At the first time-point (3 h after the initiation of the co-culture) extracellular ookinetes were in a loose association with the insect cells, while at 20 h, the second time point, the parasites were either intimately associated to the
Acknowledgements
We thank N. Hall for allowing us access to the unpublished sequence data for P. berghei and D. Raine and R.E. Sinden for generously sharing their unpublished proteome analysis data. We are grateful to P. Topalis for help with the bioinformatic analysis and to G. Vrentzos and L. Spanos for expert technical assistance. This work was supported by a grant from the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR) to C.L., an EU “return
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