Structural and Functional Alteration of host RBCs by Babesia

Babesia spp., the etiologic agents of babesiosis in animals and humans, are intraerythrocytic protozoan parasites transmitted to hosts by Ixodes ticks. Long known to cause disease in domestic animals, B. microti and B. divergens have emerged as a growing public health concern for humans, causing fulminant disease in immuno- compromised and asplenic populations and an alarmingly high rate of hospitalizations and sometimes fatality in these individuals. Most clinical complications are due to the ability of the parasites to strikingly alter properties of host RBCs. I hypothesize that structural, morphological, rheological, and functional changes occurring in infected RBCs (iRBCs) are the result of the export of specific Babesia proteins, including proteases, which interact with the cytoplasmic, membrane skeletal and membrane components of the RBCs. The overarching goal of this proposal is to elucidate the alterations to the host RBCs and uncover the mechanisms underlying these changes by identifying and characterizing the molecular players-both impacted RBC cytoskeletal proteins and the key parasite molecules responsible for the changes, to build a comprehensive picture of host RBC remodeling during infection. Aim 1 will conduct morphological, rheological, and biophysical analyses to define alterations of iRBCs using Image flow cytometry along with machine learning analysis, ektacytometry and Stimulated Emission Depletion microscopy. Changes in 5 key RBC cytoskeletal proteins on infection-Band 3, spectrin, ankyrin, Proteins 4.1 and 4.2, will be mapped. Specific antibodies will be used in immuno-chemical methods to track changes in these cytoskeletal elements up on infection and mass spectrometry performed to identify specific phosphorylation and cleavage modifications. Aim 2 will identify and characterize 2 parasite cysteine proteases and 2 aspartic proteases including their processing, sub-cellular localization, identification of export sequence signatures and develop molecular tools for downstream functional analysis. The specific interactions of these proteases with the host RBC cytoskeleton proteins will be studied using inside-out RBC vesicle assays, immunoprecipitation, and Surface Plasmon Resonance to identify specific interactions and delineate the domains required for modification of host cytoskeletal proteins. Overall, these aims will provide insights into mechanisms of RBC remodeling by the parasite and its physiological relevance in babesiosis, opening doors for novel and targeted chemotherapeutics, in line with NIAID’s mission to tackle emerging diseases relevant to public health. A comprehensive mentorship team (Drs. Lobo, Narla, Hillyer, Yazdanbakhsh, Montero, Allred, Monetti and North) has been assembled to ensure that both scientific and career development goals are met. The immediate goal is to gain advanced training in a relatively understudied parasite using multipronged experimental approaches, along with acquiring soft skills needed for scientific independence. This K99/R00 grant would eventually provide data and molecular tools for R01-level funding, fulfilling long-term career goals for launching an independent career in studying host-parasite interaction in babesiosis.