LFA-1 interaction with GBP-130 on Plasmodium falciparum-infected red blood cells mediates NK cell activation and parasite control
- Malaria Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, India
- Cellular Immunology Group, International Centre for Genetic Engineering and Biotechnology, India
- Amity Institute of Biotechnology (AIB), Amity University, Kant Kalwar, NH-11C, India
- Department of Bioinformatics, Amity School of Biological science, Amity University, India
- Parasite Cell Biology Group, International Centre for Genetic Engineering and Biotechnology, India
Figures
Figure 1 with 1 supplement
The αI domain of lymphocyte function-associated antigen-1 (LFA-1) binds to P. falciparum-infected erythrocytes.
(A) Schematic representation of full-length 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 Chinese hamster ovary (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 (250 nM) fusion protein to P. falciparum-infected red blood cells (iRBCs). 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.
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Figure 1—source data 1
PDF file containing original uncropped Coomassie Blue-stained SDS-PAGE gel corresponding to Figure 1Bi and original uncropped western blot image corresponding to Figure 1Bii, with relevant bands and experimental treatments indicated.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig1-data1-v1.zip
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Figure 1—source data 2
Original uncropped Coomassie Blue-stained SDS-PAGE gel corresponding to Figure 1Bi and original uncropped western blot image corresponding to Figure 1Bii.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig1-data2-v1.zip
Figure 1—figure supplement 1
Sequence conservation of the LFA-1 αI domain and validation of its specific binding to P. falciparum- infected erythrocytes.
(A) Amino acid sequence alignments of ‘αI domain’ of lymphocyte function-associated antigen-1 (LFA-1) with similar von Willebrand factor (vWA) domains of human and Mac-1 I-domain showing the similarity and differences among these proteins. (B) Specificity of LFA-1 αI-Fc binding to infected erythrocytes. Importantly, no significant difference in binding to uRBCs was detected between LFA-1 αI-Fc and the isotype control, indicating minimal non-specific interaction with the erythrocyte surface.
Figure 2 with 1 supplement
PfGBP-130 on the surface of P. falciparum-infected erythrocytes binds 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 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 Escherichia 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 co-localizes 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 (A) using anti-rabbit GBP-130 antibody. Lane 1 shows the presence of PfGBP-130 in the immunoprecipitate. The ~130 kDa 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 an ~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) Bio-layer interferometry (BLI) analysis of real-time binding between PfGBP-130-N and LFA-1 αI domain. Averaged sensorgrams across independent experiments (n≥3) demonstrate concentration-dependent association and dissociation kinetics. The calculated equilibrium dissociation constant (KD) was (1.7±0.22)×10–8 M, indicating high-affinity binding. (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.
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Figure 2—source data 1
PDF file containing original uncropped Coomassie Blue-stained SDS-PAGE gel corresponding to Figure 2Bii, and original uncropped western blot images corresponding to Figure 2Bii and Figure 2Biv, with relevant bands and experimental treatments indicated.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig2-data1-v1.zip
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Figure 2—source data 2
Original uncropped Coomassie Blue-stained SDS-PAGE gel corresponding to Figure 2Bii, and original uncropped western blot images corresponding to Figure 2Bii and Figure 2Biv.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig2-data2-v1.zip
Figure 2—figure supplement 1
Cloning and molecular validation of LFA αI-Fc and PfGBP-130 constructs.
(A) Schematic showing vector map of pFUSE-hIgG1-Fc2 and sites used for cloning the LFA αI domain gene used for the expression of LFA αI-Fc fusion protein. (B) Agarose gel electrophoresis showing PCR amplification of DNA corresponding to the LFA αI domain and restriction analysis of the cloned plasmid, pFUSE-hIgG1-LFA αI-Fc2. (C) (i) Agarose gels showing the PCR amplification of 600 bp fragment of PfGBP-130 gene (Pf3D7_1016300). (ii and iii) Agarose gel electrophoresis showing restriction analysis of cloned PfGBP-130 gene fragment in pGEM-T and pET-28b vectors, respectively.
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Figure 2—figure supplement 1—source data 1
PDF file containing original uncropped agarose gel electrophoresis images corresponding to Figure 2—figure supplement 1B and C, with relevant bands and experimental treatments indicated.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original uncropped agarose gel electrophoresis images corresponding to Figure 2—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig2-figsupp1-data2-v1.zip
Figure 3 with 2 supplements
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 Chinese hamster ovary (CHO) K1 cells. (A) Schematic 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 PfGBP-130 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 PfGBP-130 ECD-Fc binding to both NK and THP-1 cells. * Denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001. Representative flow plots depict the percentage of cells within a predefined positive gate, whereas the accompanying summary graph quantifies fluorescence intensity across the analyzed population. These two metrics report distinct properties of the distribution and are therefore not expected to be numerically identical.
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Figure 3—source data 1
PDF file containing original uncropped western blot images corresponding to Figure 3Bii and Cii, with relevant bands and experimental treatments indicated.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig3-data1-v1.zip
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Figure 3—source data 2
Original uncropped western blot images corresponding to Figure 3Bii and Cii.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig3-data2-v1.zip
Figure 3—figure supplement 1
Expression and validation of recombinant PfGBP-130-Fc protein and siRNA-mediated inhibition of LFA-1 (CD11a) expression in immune cells.
(A) SDS-PAGE and (B) western blot analysis showing the purified PfGBP-130-Fc fusion protein expressed in Chinese hamster ovary (CHO) K1 line. Western blot analysis was performed using anti-IgG abs. (C) FACS analysis showing expression of LFA-1 subunit CD11a using anti-CD11a antibody on primary NK cells and its expression inhibition on the same cells using SmartPool Accel siRNA corresponding to CD11a of LFA-1. (D) FACS analysis showing expression of CD11a subunit of LFA-1 on THP-1 cells and its expression inhibition on the same cells using SmartPool Accel siRNA corresponding to CD11a of LFA-1. Secondary anti-mouse PE-Texas Red was used for detection.
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Figure 3—figure supplement 1—source data 1
PDF file containing original uncropped Coomassie Blue-stained SDS-PAGE gel corresponding to Figure 3—figure supplement 1A and original uncropped western blot image corresponding to Figure 3—figure supplement 1B, with relevant bands and experimental treatments indicated.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original uncropped Coomassie Blue-stained SDS-PAGE gel corresponding to Figure 3—figure supplement 1A and original uncropped western blot image corresponding to Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/110942/elife-110942-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
PfGBP-130-Fc binds specifically to LFA-1 expressing cells but not to negative control cell lines.
(A–D) To confirm ligand specificity and exclude non-specific binding, PfGBP-130-Fc binding was assessed on multiple cell types by flow cytometry. THP-1 cells, which express LFA-1, were used as a positive control, while non-immune cell lines (HEK293T, HepG2) and stem cells (low/negative basal LFA-1 expression) were included as negative controls. Robust PfGBP-130-Fc binding was observed on THP-1 cells, whereas HEK293T, HepG2, and stem cells showed minimal to negligible binding comparable to the hIgG isotype control.
Figure 4 with 1 supplement
Natural killer (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 Chinese hamster ovary (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 a PE-Texas Red anti-human IgG antibody. (B) NK cell activation in the presence of CHO K1 cells expressing PfGBP ECD. Human NK cells were purified (>95%) from fresh peripheral blood mononuclear cell (PBMC) and co-cultured with CHO K1 cells expressing PfGBP ECD in a 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 hr. NK cells were separated from adherent CHO K1 cells, and NK cell 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.
Figure 4—figure supplement 1
Schematics of the PfGBP-130-ECD lentiviral expression vector and characterization of primary NK cell purity.
(A) Schematics of cloned PfGBP-130-ECD fused to transmembrane domain of transferrin receptor (TfR-TM) in pMSCV-puro plasmid to produce lentiviral vector. (B) Staining of purified primary natural killer (NK) cells using anti-CD3 and anti-CD56 showing purity of NK cells.
Figure 5
Natural killer (NK) cells activated in the presence of infected red blood cells (iRBCs) control parasite infection.
(A) Activated human NK cells eliminate iRBCs in vitro. Human NK cells when co-cultured with iRBCs reduce parasitemia significantly after 96 hr, and in the presence of anti-PfGBP-130 antibodies, this reduction in parasitemia was blocked. Presence of anti-GBP-130 antibodies resulted in parasitemia similar to the control when NK cells were incubated with iRBCs alone. (B) NK cell activation in the presence of iRBCs. Human NK cells were purified (>95%) from fresh peripheral blood mononuclear cells (PBMCs) and co-cultured with synchronized schizont-stage iRBCs at a parasitemia of 0.5% in a 10:1 ratio (NK:iRBC) for 48 hr. Quantification of activation and degranulation markers was performed after 48 hr. 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.
Author response image 1
Additional files
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Supplementary file 1
Table A.
Table of MS/MS hits of the beads+hIgG control. Table B. Docking scores and binding energy calculated using various computational tools to assess the quality of the GBP-LFA1 docked complex.
- https://cdn.elifesciences.org/articles/110942/elife-110942-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110942/elife-110942-mdarchecklist1-v1.pdf
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LFA-1 interaction with GBP-130 on Plasmodium falciparum-infected red blood cells mediates NK cell activation and parasite control
eLife 15:RP110942.
https://doi.org/10.7554/eLife.110942.3
