Elucidating parasite and host-cell factors enabling Babesia infection in sickle red cells under hypoxic/hyperoxic conditions

Abstract

Sickle RBCs (SS) represent a naturally existing host-cell resistance mechanism to hemoparasite infections. Here, we investigate the basis of this resistance using Babesia divergens grown in sickle and sickle trait cells. We found that oxygenation and its corresponding effect on RBC-sickling, frequency of fetal hemoglobin (HbF+) cells, the cellular redox environment and parasite proliferation dynamics, all play a role in supporting or inhibiting Babesia proliferation. To identify cellular determinants that supported infection, an image-flow-cytometric tool was developed which could identify sickled-cells and constituent hemoglobin. We showed that hypoxic conditions impaired parasite growth in both HbSS and HbAS cells. Further, cell-sickling was alleviated by oxygenation which in turn relieved the inhibition of parasite proliferation in SS cells under hypoxic conditions. Interestingly, our tool identified HbF+-sickle cells as host-cells of choice under both hypoxia and hyperoxia, which was confirmed using cord RBCs which contain high amounts of HbF. In contrast to AA and AS cells, uninfected HbSS cells showed a higher ROS-containing environment, which was further perturbed on infection. We investigated the parasite's response in hostile SS cells and found that it alters its sub-population structure, with 1N dominance under hypoxic conditions yielding to equivalent ratios of all parasite forms at hyperoxic conditions favorable for its growth. Multiple factors, including oxygenation and its impact on cell-shape, HbF positivity, redox status, and parasite pleiotropy allow Babesia propagation in sickle RBCs. Our studies provide a cellular and molecular basis of natural resistance to Babesia which will aid in defining novel therapies against human babesiosis.

Global Metabolomic Profiling of Host Red Blood Cells Infected with Babesia divergens Reveals Novel Antiparasitic Target Pathways

“…As parasite growth continued, there was an increased reliance on energy pathways like glycolysis and the TCA cycle to fuel the rapid proliferation, and these changes were reflected in the metabolome of the infected host cells. Additionally, as expected and as previously shown by us using redox dyes ( 32 ), there were significant changes in the redox regulatory pathways in the infected red cells ( Fig. 3f ).…”

“…Strikingly, the key metabolites in the γ-glutamyl cycle and glutathione metabolism were downregulated at D2 (low parasitemia), but as infection progressed, several of these intermediate γ-glutamyl-amino acid metabolites, N -acetylcysteine, S -lactoyl-glutathione, cysteine, glutamate, and 5-oxoproline, were upregulated severalfold, with significant reductions in cysteinyl-glycine and GSH (reduced glutathione). This suggests that with increased proliferation of the parasite, there is an increased reliance on this redox regulatory pathway, as has also been implicated in a recent research study from our laboratory ( 32 ). These findings also largely agree with studies on the metabolomics of Plasmodium falciparum -infected RBCs, where it has been shown that with the progress of infection, these redox pathway intermediates are elevated severalfold ( 29 , 38 ).…”

“…Other interesting metabolites uniquely present in iRBCs were those belonging to glutathione metabolism and redox metabolism ( N -acetylcysteine and cysteine-glutathione disulfide), cAMP, folate, and derivatives of thiamine. Redox metabolism has been implicated as playing a major role in protozoan parasites like Plasmodium ( 28 – 31 ) and, more recently by us, in Babesia ( 32 ). cAMP has also been implicated as playing an essential role in signaling in other parasites ( 33 ).…”

Identification and characterization of extracellular vesicles from red cells infected with Babesia divergens and Babesia microti

“…Interestingly, miR-4463 is associated with apoptosis and oxidative stress in endothelial cells. Elevated oxidative stress in the host cells has been linked to intracellular parasite pathogens like Mycobacterium tuberculosis (Chawla et al, 2012) and P. falciparum (Beri et al, 2017;Beri et al, 2019), and our previous work had shown that B. divergensinfected red cells experience disturbed redox homeostasis (Beri et al, 2022). Thus, miRNAs may play a role in causing parasiterelated oxidative stress and could be investigated in future transduction experiments.…”

Babesia divergens Shows Equal Predilection for Human ABO Blood Types in an In Vitro Erythrocyte Preference Assay

“…Peak parasitemia was also significantly reduced among RhD+ individuals than RhD- individuals among babesiosis-hospitalized patients. Generally, immune status, age, sickle cell phenotype and asplenia are some of the other known factors that can affect the severity of babesiosis [ 2 , 4 , 27 , 28 , 29 ].…”