Additional file 1 of A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host–pathogen interactions

Additional file 1: Figure S1. Sporozoite candidate selection strategy and western blots of sporozoite protein bait library. Flow chart explaining the candidate selection process for sporozoite AVEXIS library. Western blots were performed after concentration adjustment by dilution or spin-filter concentration, with detection using anti-Ctag antibody. As described in the text and Supplementary Table 1, expression of 45 of these proteins from related constructs has previously been reported and, in these cases, this figure is intended to demonstrate quality rather than imply novelty. The band seen at c. 55 kDa in blots of pre-concentrated baits is believed to represent reactivity of the anti-Ctag antibody with a HEK293-cell protein (rather than degraded bait) as it was also seen in supernatant from cells transfected with irrelevant constructs. Figure S2. Hepatocyte candidate selection strategy and western blots of hepatocyte surface protein prey library. Flow chart explaining the candidate selection process for hepatocyte AVEXIS library. Proteins are arranged approximately in decreasing order of concentration as quantified by luminescence measurement. Western blots were performed after concentration adjustment by dilution or spin-filter concentration, with detection using anti-FLAG-tag antibody. As described in the text and Supplementary Table 1, expression of 127 of these proteins from related constructs has previously been reported and, in these cases, this figure is intended to demonstrate quality rather than imply novelty. Figure S3. (a) Murine FGFR4 does not interact with Pb34 or Py34. Absence of AVEXIS-detectable interaction of P. berghei and P. yoelii orthologs of Pf34 (‘Pb34’ [PBANKA_0721800] and ‘Py34’ [PY17X_0721800], in bait format) with murine FGFR4 (mFGFR4, in prey format). (b) Quality of purified proteins used for SPR. Purified monomeric Pf34-CD4-Bio-His (~62 kDa) and FGFR4-CD4-Bio-His protein (~64 kDa) used for SPR, on SDS-PAGE gel stained with Coomassie Blue. (c) Quality of PfRON6 C-terminal fragment bait on SDS-PAGE gel (a) and western blot (b) – no purification of this protein was undertaken, as it was captured specifically on the flow cell by anti-Fc antibody. Legend indicates expected molecular weight of each construct in kDa, including tags but excluding post-translational modifications. Figure S4. Kinetics of Pf34-FGFR4 and Pf34-PfRON6 interactions. (a) and (b) show SPR sensorgrams during application of FGFR4 analyte onto PfRH5 (reference, Fc1) and Pf34 (active, Fc2) coated flow cells respectively, in the experiment shown in Fig. 3C, D. (c) – (e) show Fc1, Fc2, and double-reference-subtracted curves (Fc2-1 after subtraction of Fc2-1 with buffer-only analyte) in a replicate SPR assay. (f) and (g) show SPR sensorgrams during application of Pf34 analyte onto PfRH5 (reference, Fc1) and PfRON6 C-terminal fragment (active, Fc2) coated flow cells respectively, in the experiment shown in Fig. 4C. (h) tabulates results of 1:1 binding model fitting to SPR kinetic measurement of Pf34-FGFR4 and Pf34-PfRON6 intercations. The equilibrium dissociation constant, KD, is expressed in both molar units and micrograms per millilitre. Half-life (t1/2) on seconds is calculated using formula 0.693/kd. Figure S5. Effect of antibodies on NF54 sporozoite invasion and 3D7 blood-stage growth. (a) – (b) shows the effect of anti-FGFR4 antibodies in ISI using NF54 parasite line (graph details are similar to Fig 5 Panel (d) – (f)). The experiment shown in panel A and B was performed in singlicate. Separate red and black lines in panel B represent control and active antibodies respectively form two independent rabbits. (c) Blood stage growth inhibitory activity of anti-Pf34 antibodies. Anti-Pf34 rabbit polyclonal IgG were tested in vitro GIA at a range of concentrations against 3D7 clone P. falciparum. Error bars indicated the interwell SEM triplicate wells and separate lines represent IgG from independent rabbit serum samples. Figure S6. Attempted generation of Pf34-disrupted parasites. (a) Schematic diagram illustrating the strategy used for attempted conditional silencing of Pf34 using CRISPR/Cas9 system. Parasite genomic locus is shown with Pf34 CDS, 5’ and 3’ UTRs and location of primers are indicated with arrows. Repair plasmid is shown with homology regions (HR1 and HR2), re-codonized Pf34 (RC-Pf34) with the insertion of loxP sites (see materials and methods). Modified locus is shown if the integration of repair plasmid into NF54-DiCre parasite genome occurs following transfection and after rapamycin treatment. (b) Strategy used for attempted generation of Pf34-3XHA-GFP cKO parasites. A similar repair plasmid that is shown in Fig 6A with the addition of 3XHA after signal peptide, and eGFP after silent loxP cassette was used for transfection. No drug resistant parasites were observed for Pf34 cKO and Pf34-3XHA-GFP cKO parasites. Figure S7. Generation of PfRON6-disrupted parasites. (a) Generation of PfRON6 cKO parasites. Parasite genomic locus is shown with PfRON6 CDS, 5’ and 3’ UTRs and location of primers are indicated with arrows. Repair plasmid is shown with homology regions (HR1 and HR2), re-codonized PfRON6 (RC-PfRON6) with the insertion of loxP sites (see Methods). Modified locus is shown after the integration of repair plasmid into NF54-DiCre parasite genome following transfection and after rapamycin treatment. Diagnostic PCR using the primer sets P1/P5 and P6/P4 (805 bp and 719 bp respectively) is showing the integration of PfRON6 cKO repair plasmid and the absence of wild type contamination is shown using primer sets P1/P2 and P3/P4 in PfRON6 cKO clone B4 and F7 (that produce 864 bp and 636 bp amplicons respectively using wild type genomic DNA). (b) Generation of PfRON6-3XHA-GFP cKO parasites. A similar repair plasmid that is shown in Fig 6C with the addition of 3XHA and GFP was used for transfection. Diagnostic PCRs are shown for the correct integration of PfRON6-3XHA-GFP repair plasmid in the endogenous locus using the primer sets P1/P5 and P6/P4 (805 bp and 1541 bp, respectively) and the absence of non-transfectants in PfRON6-3XHA-GFP cKO clone B3 and E10 using primer sets P1/P2 and P3/P4 as above.