Designing a Robust Platform for the In vitro Propagation of Babesia Microti in Human RBCs - The overall goal of the R61 phase of our proposal is to establish a continuous in vitro culture system of B. microti in human RBCs to enable a platform amenable for future experimental investigation, specifically the identification and characterization of human RBC receptor-B. microti ligand interactions that facilitate parasite invasion which will be explored in the R33 phase. We hypothesize that B. microti like other erythrocyte seeking protozoans have preferential cellular invasion and proliferation needs. To establish if this is true, we will in the R61 phase, Milestone1, analyze the parasite-hosting capabilities of sub-populations of RBCs using separation and purification protocols from peripheral blood, umbilical cord blood and hemochromatosis blood draws, using parasite inoculum from both B. microti infected mice and infected human sources. We will also analyze pure populations from erythroid progenitor cells that have been FACS sorted for the ability to be infected with B. microti. Once a specific host RBC has been identified along with the inoculum, we will assay culture media along with serum/lipid additives and vitamin/mineral additives, and ideal gas conditions, to achieve Milestone 2. Working with the highest infection achieving media, host RBC and parasite source, we will monitor parasite egress and re-invasion after ensuring adequate host RBCs that the parasite requires, in culture, in Milestone 3. FACS and Giemsa estimation of parasitemia and overall culture dynamics will be constantly monitored, paying particular attention to sub-population structure of the cultures, which will inform us of success of various Milestones. As part of Milestone 4, we will cryopreserve and revive stocks of culture-adapted parasites. Comprehensive Bio-Protocols will be written up detailing specific aspects of each step in the culturing process. Once an in vitro culture system is in place critical aspects of human host-parasite relationships can be explored and we will investigate B. microti invasion of the human RBC, in the R33 phase. To identify and characterize RBC surface molecules that function in invasion, we will use enzymatically treated RBCs, clinically defined RBCs deficient in specific receptor, inhibition of invasion assays using antibodies/peptides and forward genetic approaches using shRNA mediated knockdown of specific RBC receptors. Cognate B. microti ligands will then be identified using gel overlay and pull-down assays. Validation of these receptor-ligand interactions will be carried out via AVEXIS and BIAcore assays. Functional validation assays will include inhibition of invasion assays with anti-ligand antibodies and recombinant ligand proteins. We believe that these innovative approaches will greatly enhance the opportunities and capabilities for investigations into B. microti biology.
