The αI domain of Lymphocyte Function-Associated Antigen-1 (LFA-1) binds to Plasmodium falciparum–infected erythrocytes.

(A) Schematic representation of full-length of CD11a, a subunit of LFA-1 and the recombinant construct encoding the C-terminal Fc-tagged αI domain of LFA-1. (B) Expression and purification of recombinant LFA-1 αI-Fc fusion protein. The αI domain of LFA-1 was cloned into the pFUSE-hIgG1-Fc2 vector and expressed in CHO-K1 cells. The fusion protein was purified from culture supernatants using Protein-A affinity chromatography. (i) SDS-PAGE analysis of the purified protein revealed a prominent Coomassie-stained band at ∼45 kDa, corresponding to the expected molecular weight of the LFA-1 αI-Fc fusion protein. (ii) Western blot analysis using anti-human IgG antibody confirmed the identity of the fusion protein. (C) Binding of LFA-1 αI-Fc (250nM) fusion protein to P. falciparum–infected red blood cells. Flow cytometry analysis using PE-Texas Red–conjugated anti-human IgG antibody demonstrated specific binding of the LFA-1 αI-Fc protein to ring, trophozoite, and schizont stages of iRBCs, indicating interaction across all major asexual blood stages.

PfGBP-130 on the surface of Plasmodium falciparum–infected erythrocytes bind the LFA-1 αI domain.

(A) Identification of PfGBP-130 as an interacting partner of LFA-1 αI-Fc. LC-MS/MS analysis of immunoprecipitates from P. falciparum–infected erythrocyte lysates pulled down with LFA-1 αI-Fc fusion protein revealed PfGBP-130 (PF3D7_1016300) as a major interacting protein. The table summarizes proteins specifically enriched in the LFA-1 αI-Fc pull-down in all the three biological replicates, showing high peptide coverage and spectral abundance, indicating a specific and robust interaction. (B) Characterization and localization of PfGBP-130. (i) Schematic representation of the domain organization of PfGBP-130 and the N-terminal fragment (amino acids 69–270) that was expressed in E. coli (termed PfGBP-130-N). (ii) SDS-PAGE and western blot analysis of purified PfGBP-130-N using anti-rabbit PfGBP-130 antibodies. A prominent band at ∼30 kDa corresponds to the expected molecular weight of the recombinant fragment. (iii) Immunofluorescence assay (IFA) demonstrating surface localization of PfGBP-130 on trophozoite-stage iRBCs using anti-PfGBP-130 antibodies. PfGBP-130 (green) partially colocalizes with PfGARP (red), a well-established iRBC surface protein with an extracellular domain. Nuclei were stained with DAPI (blue), confirming surface expression. (iv) Western blot analysis of iRBC lysate and of immunoprecipitate of LFA1 αI-Fc bound iRBC (figure 2A) using anti-rabbit GBP-130 antibody. Lane1, shows the presence of PfGBP-130 in the immunoprecipitate. The ∼130kDa band is notably absent in the control IP eluate where no LFA-1 αI-Fc, was bound to iRBC, demonstrating the specificity of the LFA-1 αI-Fc and PfGBP-130 interaction. Lane 2 shows the detection of native PfGBP-130 as a ∼110 kDa protein in trophozoite stage P. falciparum lysate, consistent with its predicted molecular weight. (C) Biophysical and computational validation of PfGBP-130 and LFA-1 αI interaction. (i) Biolayer interferometry (BLI) was used to assess real-time binding between PfGBP-130-N and LFA-1 αI-Fc. Sensorgrams at increasing concentrations of PfGBP-130-N demonstrate dose-dependent binding kinetics. (ii) In silico docking analysis showing the energy-minimized complex of PfGBP-130 (salmon) and LFA-1 αI domain (green) generated using ClusPro 2.0. Representative hydrogen bonds and interacting residues are shown as sticks. Visualizations were prepared using PyMOL. (iii) Molecular dynamics (MD) simulation of the PfGBP-130/LFA-1 αI complex. The graph depicts the root mean square deviation (RMSD) over time, confirming structural stability of the protein–protein complex.

Specificity of interaction between LFA-1 on Primary NK cells and THP-1 cells with PfGBP-130.

Primary NK and THP-1 cells treated with LFA-1 siRNAs showed reduced binding to recombinant extracellular domain of PfGBP-130 expressed in CHO K1 cells. (A) Schematics representation of PfGBP-130 and its extracellular domain cloned in pFUSE-hIgG1-Fc2 vector for the expression in CHO K1 cells. The construct comprises the extracellular domain of PfGBP-130 (including putative LFA-1 binding sites) fused to the Fc region of human IgG1. The Fc fusion provides stability, solubility, and facilitates detection. (B) Interaction of PfGBP-130 ECD-Fc with THP-1 cells. (i) Flow cytometric analysis shows strong binding of PfGBP-130 ECD-Fc to THP-1 cells, compared to an hIgG1 isotype control, indicating specific interaction. LFA-1 knockdown in THP-1 cells via siRNA treatment significantly reduced PfGBP-130 ECD-Fc binding, thus confirming the specificity of interaction between PfGBP130 ECD-Fc and LFA-1 on THP cells. (ii) Western blot analysis using anti-CD11a antibody confirmed the siRNA-mediated knockdown of CD11a subunit of LFA-1 protein on THP-1 cells. (C) Interaction of PfGBP-130 ECD-Fc with primary human NK cells. (i) Flow cytometry revealed a marked increase in PfGBP-130 ECD-Fc binding to NK cells over the isotype control. siRNA-mediated knockdown of LFA-1 in NK cells led to a notable reduction in PfGBP-130 ECD-Fc binding, confirming that LFA-1 is essential for this interaction. (ii) Western blot analysis using anti-CD11a antibody confirmed the siRNA-mediated knockdown of CD11a subunit of LFA-1 protein in NK cells, confirming LFA-1 as a critical receptor for PfGBP130 ECD-Fc binding to both NK as well as THP-1 cells. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

NK cells activated in the presence of PfGBP-130.

(A). (i) Expression of PfGBP-130 ECD fused with Transferrin membrane domain on the membrane of CHO K1 cells by infecting the lentiviral vector; pMSCV Puro and its immunofluorescence analysis using anti-rabbit PfGBP antibody. (ii) CHO K1 cells expressing PfGBP-130 ECD bind LFA-1 αI-Fc. Binding of purified LFA-1 αI-Fc to PfGBP-130 ECD expressing CHO cells was assessed by FACS using an PE-Texas red anti-human IgG antibody. (B) NK cells activation in the presence of CHO K1 cells expressing PfGBP ECD. Human NK cells were purified (>95%) from fresh PBMC and co-cultured with CHO K1 cells expressing PfGBP ECD in 2: 1 ratio (20,000 CHO-K1 cells:10,000 NK cells) and these cells were stimulated with (Poly I:C/lipofectamine 2000) for 24 h. NK cells were separated from adherent CHO K1 cells and NK cells activation was assessed by assaying the expression of activation markers (CD69, CD25) and a degranulation marker; CD107a . NK cells co-cultured with CHO K1 cells expressing PfGBP-ECD protein showed significant increase in the expression of CD25 and CD69, as well as CD107a in comparison to the NK cells co-cultured with mock CHO cells. Addition of anti-CD11a (HI111 clone) antibodies reduced the expression of both activation and degranulation markers. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

NK cells activated in the presence of iRBCs, controls parasite infection

(A) Activated human NK cells eliminate iRBCs in vitro. Human NK cells when co-cultured with iRBCs reduce parasitemia significantly after 96h and in the presence of anti-PfGBP-130 antibodies this reduction in parasitemia was blocked. Presence of anti-GBP-130 abs resulted in parasitemia simialr to the control when NK cells were incubated with iRBCs alone. (B) NK cells activation in the presence of iRBCs. Human NK cells were were purified (>95%) from fresh PBMC and co-cultured with synchronized schizont stage iRBCs at a parasitemia of 0.5% in 10:1 ratio (NK: iRBC) for 48h. Quantification of activation and degranulation markers was performed after 48 hours. NK cells co-cultured with iRBCs showed significant increase in the expression of CD25 and CD69, the two activation markers as well as for the expression of CD107a, a degranulation marker in comparison to the NK cells co-cultured with RBCs alone. Addition of anti-rabbit PfGBP-130 antibodies reduced the expression of both activation and degranulation markers in these NK cells in comparison to rabbit IgG isotype control. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.