In vitro culture of Babesia microti - Risk of human babesiosis due to infection by Babesia microti has greatly increased in the northeastern United States within the last decade. We badly need new drugs because relapsing babesiosis secondary to drug resistance is an increasingly encountered and is very difficult to treat. In part, our progress in the development new treatment regimens has been hindered by our inability to culture B. microti. We propose to develop methods for continuous propagation of B. microti in vitro. Our proposed project comprises new approaches that focus on the tick stages, question the need for intact erythrocytes, as well as use predictions from phylogenetic metabolomics. We leverage unique expertise in the transmission biology of B. microti, from tick biology to laboratory maintenance of the parasite, to population genetics and metabolomics, as well as clinical experience. Specifically, for Phase I, we shall (1) propagate B. microti within tick cells. We shall determine whether sporozoites or kinetes may be propagated within existing tick cell lines. Although erythrocyte cultures are the classical objective, continuous propagation of parasites within tick cells would also allow for the development of genetic tools. (2) test the capacity for B. microti to replicate and be propagated on or within erythrocyte lysate substrates. The main obstacle in propagating B. microti has been the failure for merozoites to invade uninfected erythrocytes. It may be that intraerythrocytic invasion is not required for asexual parasite replication (merogony). (3) Use a phylogenetic approach to metabolomics and transcriptomics to predict and use physiologic requirements for in vitro propagation of B. microti within erythrocytes. This parasite has life cycle characteristics that are more like Theileria spp. than the Babesia spp. that have been successfully cultivated; the predicted metabolomes of Theileria parva; B. microti; and the cultured B. duncani, may comprise shared or unique cell invasion or nutritional pathways and these could be used to guide efforts for tick cell or extracellular culture conditions. If successful in Phase I, we propose two aims for Phase II: (1) develop and validate methods for genetic manipulation for vertebrate (erythrocyte or extraerythrocytic) parasites, as well as tick stages so that they express a phenotypic marker; and (2) develop and test methods of generating chimeric parasites, modifying those that are easily culturable (e.g., B. duncani) with homologous genes from B. microti to allow probing the molecular basis for phenotypic traits. This high risk-high payoff aim would serve as proof of concept of a general strategy for modifying parasitic protozoa that are refractory to continuous propagation in vitro. Cultures of B. microti in erythrocytes, in cell free biphasic cultures, or tick cells would allow for the development of tools for genetic manipulation. The tasks that we propose will also provide additional details for life cycle facets that remain poorly explored, thereby improving our general knowledge of the biology of B. microti.
