Iron sourcing strategies and adaptive protein responses to iron deprivation in the intraerythrocytic parasite Babesia divergens

Iron is indispensable for the survival and pathogenicity of Babesia parasites, yet the mechanisms underlying its acquisition remain largely unresolved. In contrast to other apicomplexan parasites, tick-borne Babesia species rapidly lose the parasitophorous vacuole following erythrocyte invasion and do not form hemozoin, suggesting the evolution of distinct strategies for iron metabolism. Here, we demonstrate that Babesia divergens utilizes a sequential, multi-route iron acquisition system to meet its metabolic demands within host erythrocytes. Kinetic uptake analyses reveal a pronounced preference for ferrous iron (Fe 2+ ), which is imported through an active, energy-dependent process supported by extracellular ferric reductase activity that converts host-derived ferric iron (Fe 3+ ) into a bioavailable form prior to uptake. Using immunogold transmission electron microscopy, we provide evidence for the active internalization of host transferrin and show that parasite proliferation is dose-dependently inhibited by apo-transferrin, establishing iron as a limiting factor for parasite growth. Quantitative proteomic profiling revealed a limited, stage-specific response to deferoxamine-induced iron deprivation. Three proteins showed statistically significant changes in abundance in free merozoites: the conserved multipass membrane protein Bdiv_019230 increased, whereas the hypothetical protein Bdiv_002300 and the putative serine hydroxymethyltransferase Bdiv_027330 decreased. No proteins reached the statistical significance threshold in intraerythrocytic parasites. These results identify a small set of statistically supported candidates for investigating the response of extracellular B. divergens merozoites to iron-restricted conditions.