Cross-Linking of Erythrocyte CD44 Promotes Plasmodium Falciparum Invasion

Malaria remains a global threat to public health, as drug resistance continues to emerge and vaccines have limited efficacy. Malaria symptoms include the classical fever and chills, which correlate with the asexual life cycle of Plasmodium falciparum parasites in human red blood cells (RBCs). P. falciparum invasion of RBCs requires several host-parasite interactions, some of which have potential as therapeutic or vaccine targets. The roles of host RBC factors involved in malaria parasite invasion remain underexplored, in part because mature enucleated RBCs are refractory to genetic manipulation, and thus are challenging to study. Previous work from our lab identified the RBC membrane protein CD44 as important for P. falciparum invasion, likely acting as a co-receptor to facilitate signaling to the RBC cytoskeleton (Baro et al., 2023). Here, we sought to investigate the therapeutic potential of targeting CD44 to disrupt P. falciparum invasion. We performed parasite invasion assays in the presence of anti-CD44 monoclonal antibodies that target different epitopes. Unexpectedly, we observed that none of the anti-CD44 antibodies inhibited invasion. Instead, one antibody, BRIC 222, significantly promoted invasion, ranging from 10%-80% increase, in a dose-dependent manner. To determine whether the increased invasion was due to the cross-linking of CD44 by BRIC 222, we performed invasion assays with BRIC 222 or just its antigen binding fragment, BRIC 222 F(ab). BRIC 222 F(ab) had no effect on parasite invasion, confirming that the enhanced invasion observed with BRIC 222 is due to CD44 cross-linking. In other cells, CD44 cross-linking has been shown to induce cellular changes including altered expression or localization of surface proteins (Nakano et al., 2007), but little is known about the function of CD44 in human RBCs. To determine how cross-linking CD44 may affect the RBC membrane, we performed a flow cytometry-based assay to quantify the detection of specific RBC surface proteins in the presence or absence of BRIC 222. We discovered that BRIC 222, but not BRIC 222 F(ab), increased the detection of several RBC membrane proteins, including basigin (CD147), an essential receptor for P. falciparum, which co-localizes with CD44. We previously demonstrated that the P. falciparum invasion ligand EBA-175 can interact with CD44 in addition to glycophorin A (GYPA), its primary receptor, and that these interactions induced altered phosphorylation of RBC cytoskeletal proteins (Baro et al., 2023). Moreover, recombinant EBA-175 has been shown to promote P. falciparum growth and RBC agglutination (Paing et al., 2018), similar to what we observed for BRIC 222. To further explore the connection between EBA-175 and CD44, we performed phosphoprotein profiling of RBC ghosts stimulated with BRIC 222 or BRIC 222 F(ab). We found that BRIC 222-induced cross-linking was associated with altered phosphorylation of RBC cytoskeletal proteins. Together, our findings support a model where CD44 cross-linking promotes invasion by altering the RBC membrane, including increasing basigin accessibility and facilitating signaling to the RBC cytoskeleton, potentially via EBA-175. These studies on the role of RBC CD44 advance new insights into the mechanisms underlying P. falciparum invasion, with the potential to inform new therapeutic approaches to combat malaria.