CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants of Plasmodium falciparum invasion.
Jones, Patrick L
Duraisingh, Manoj T
Proceedings of the National Academy of Sciences of the United States of America
National Academy of Sciences
MetadataShow full item record
Kanjee, U., Grüring, C., Chaand, M., Lin, K., Egan, E., Manzo, J., Jones, P. L., et al. (2017). CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants of Plasmodium falciparum invasion.. Proceedings of the National Academy of Sciences of the United States of America, 114 (44), E9356-E9365. https://doi.org/10.1073/pnas.1711310114
During malaria blood stage infections, Plasmodium parasites interact with the red blood cell (RBC) surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34+ hematopoietic stem cells (cRBCs). Here, we report the development of a robust in vitro culture system to produce RBCs that allow for generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line. JK-1 cells spontaneously differentiate, generating cells at different stages of erythropoiesis, including terminally differentiated nucleated RBCs that we term “jkRBCs”. A screen of small molecule epigenetic regulators identified several bromodomain-specific inhibitors that promote differentiation, and enable production of synchronous populations of jkRBCs. Global surface proteomic profiling revealed that jkRBCs express all known P. falciparum host receptors in a similar fashion to cRBCs and multiple P. falciparum strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9 we deleted two host factors (BSG and CD44) for which no natural nulls exist. BSG interacts with the parasite ligand Rh5, a prominent vaccine candidate. A BSG knockout was completely refractory to parasite invasion in a strain-transcendent manner, confirming the essential role for BSG during invasion. CD44 was recently identified in an RNAi screen of blood group genes as a host factor for invasion, and we show that a CD44 knockout results in strain-transcendent reduction in invasion. Furthermore we demonstrate a functional interaction between these two determinants in mediating P. falciparum erythrocyte invasion.
Erythrocytes, Hematopoietic Stem Cells, Cell Line, Tumor, K562 Cells, Humans, Plasmodium falciparum, Malaria, Malaria, Falciparum, Leukemia, Erythroblastic, Acute, Carrier Proteins, Protozoan Proteins, Antigens, Protozoan, Ligands, Proteomics, Cell Differentiation, Epigenesis, Genetic, Host-Parasite Interactions, Gene Knockout Techniques, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Hyaluronan Receptors, Basigin
WELLCOME TRUST (108070/Z/15/Z)
External DOI: https://doi.org/10.1073/pnas.1711310114
This record's URL: https://www.repository.cam.ac.uk/handle/1810/276127