Purification and in Vitro Selection of Rosette-Positive (R+) and Rosette-Negative (R-) Phenotypes of Knob-Positive Plasmodium falciparum Parasites

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  • Laboratory of Infectious Diseases, Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Institute of Pathology, Case Western Reserve University, Palo, Alto, California

We have developed methods for in vitro selection of Plasmodium falciparum parasites that bear knob protrusions (K+) and are either of the rosette-positive (K+ R+) or rosette-negative (K+ R-) phenotypes. Cryopreserved parasites from spleen-intact Aotus monkeys that were K+, C32 cell adherence-positive (C+), CD36 adherence-positive, and R- with Aotus erythrocytes were adapted to continuous growth in human erythrocytes, and selected initially for adherence to C32 melanoma cells. In the absence of independent selection for rosettes, K+ R - C+ parasites were produced that adhered to both C32 cells and CD36. Without selection for the C+ phenotype, K+ R- C- parasites eventually predominated in such cultures. The R+ parasites were selected using differences in sedimentation behavior of rosette-infected cells versus non-rosette-infected cells. Methods were devised for selection of the R+ or R- phenotypes and for the purification of R+ or R- infected cells of high parasitemia that were suitable for molecular studies. With the repeated selection for K+ R+ parasites, we were able to maintain the K+ R+ phenotype for several months in vitro. These methods will allow systematic study of the molecular basis of the K+ R+ and K+ R- phenotypes.

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