Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 44, Pages E9356-E9365Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1711310114
Keywords
BSG; CD44; CRISPR/Cas9; Plasmodium falciparum; parasite invasion
Categories
Funding
- NIH [R01AI091787, R01HL139337]
- Wellcome Trust Senior Clinical Research Fellowship [108070/Z/15/Z]
- Swiss National Science Foundation Postdoctoral Fellowship
- Canadian Institutes of Health Research Postdoctoral Fellowship
- Bill and Melinda Gates Foundation [OPP1023594]
- Bill and Melinda Gates Foundation [OPP1023594] Funding Source: Bill and Melinda Gates Foundation
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During malaria blood-stage infections, Plasmodium parasites inter act with the 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 the gen eration 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 that multiple P. falciparum strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9, we deleted two host factors, basigin (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 CD44 knockout results in strain-transcendent reduction in invasion. Furthermore, we demonstrate a functional interaction between these two determinants in mediating P. faldparum erythrocyte invasion.
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