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.

Grant Number: 5K99AI182422-02
NIH Institute/Center: NIH
Principal Investigator: Divya Beri