Fig 1.
Bioinformatics analysis of the BmP53 sequence.
(A) The DNA and translated amino acid sequences of BmP53 are shown, with the underlined segment of the sequence representing the TSP1 domain. The CDS was from genomic region LN871599 join(1510300..1511012, 1511060..1511239, 1511261..1511438, 1511689..1511869, 1511945..1512132). The consensus sequence deduced from sequencing of the PCR amplicons from the Gray strain DNA is shaded in grey (accession number KX174293). The penultimate glutamine (Q) residue is framed. (B) Overview of the domain structure for BmP53, showing elected truncates, in addition to hydrophilicity and antigenicity indices.
Fig 2.
Western blot analysis to detect native BmP53 within the lysate of parasitized host erythrocytes.
Lane M: Pre-stained protein marker. Lanes 1 and 3: Lysates of B. microti infected hamster erythrocytes. Lanes 2 and 4: Lysates of non-infected hamster erythrocytes. Lanes 1 and 2 were probed with mouse anti-rGST-BmP53tr1-TSP1 serum. Lanes 3 and 4 were probed with mouse pre-immune sera. Black arrow point to specific 53.7-kDa band for native BmP53.
Fig 3.
Cellular localization of the native BmP53 protein via Immunofluorescence Antibody Assay (IFA).
(A) Thin blood smear of B. microti-parasitized hamster erythrocytes showing reactivity of parasite erythrocytic and extra-erythrocytic stages with mouse anti-rGST-BmP53tr1-TSP1 serum (green), nuclear staining (blue). Scale bar: 7.5µm. (B) A Co-localization study of BmP53 and BmSA1 on thin blood smear of B. microti-parasitized hamster erythrocytes showing reactivity of parasite erythrocytic stage with mouse anti-rGST-BmP53tr1-TSP1 serum (red), rabbit anti-BmSA1 serum (green), nuclear staining (blue).
Fig 4.
Immunofluorescence Antibody Assay (IFA) showing non-nucleated cross-reactant hamster cells.
Thin blood smear of B. microti-infected hamster erythrocytes showing reactivity of mouse anti-rGST-BmP53tr1-TSP1 serum (green) with the parasite (a) and non-nucleated hamster cells (b). Nuclear staining was performed with Hoechst DNA specific dye (blue).
Fig 5.
Detection of cross-reacting shared epitopes between BmP53 and hamster platelets.
(A) Phase contrast image of the thin smear of isolated platelets from whole blood of non-infected SPF hamster. Black arrows indicate platelets. Scale bar: 50µm. (B) Immunofluorescence antibody assay (IFA) on isolated platelets from non-infected blood of SPF hamster, showing specific reactivity of platelets surface proteins with mouse anti-rGST-BmP53tr1-TSP1 (green), nuclear staining (blue).
Fig 6.
Multiple sequence alignment of TSP1 domains from apicomplexan proteins and human or mouse thrombospondin.
Domains are labelled by the Uniprot ID of the protein and coordinates in the amino acid sequence. The percent of sequence identity with B. microti BmP53-TSP1 domain is given for each TSP1 domains and residues conserved in more than 80% of the sequences in the alignment were highlighted in grey.
Fig 7.
Close phylogenetic relationship of TSP1 domains from B. microti P53 and its orthologues with Thrombospondin TSP1 domains.
Comparison is performed with TSP1 domains from TRAP and TRAP-like proteins of B. microti and P. falciparum. Phylogenetic analysis was performed using the Phylogeny.fr server using “à la carte” options, with clustal W for the multiple alignment, BioNJ for the phylogenetic analysis with a Gamma parameter of 1 and the Jones-Taylor-Thornton substation matrix. TSP1 domains of thrombospondin and TRAP protein are identified by their coordinates and UNIPROT ID. UNIPROT species ID: BABMR, Babesia microti; PLAF7, Plasmodium falciparum (isolate 3D7); HUMAN, Homo sapiens and MOUSE, Mus musculus. The scale bottom left-hand corner indicates the number of substitutions between sequences. The scale in the legend indicate the length of the protein that are schematically represented on the right-hand part of the figure.
Fig 8.
Disorder regions and trans-membrane domain predictions for BmP53 and TSP1 domain structure predictions.
(A) Disorder regions prediction for BmP53. DISOclust protein disorder prediction is shown. The SMART domain definitions are superimposed to highlight the TSP1 and Trans-membrane domain locations. (B) Trans-membrane domain prediction for BmP53 using TOPCONS server. (C) Comparing TSP1 domain structure from B. microti P53 and Homo sapiens. [PDBID 1LSL]. (i) Model of the TSP1 domain from the BmP53 protein (green), built using the IntFOLD server. (ii) Crystal structure of the Thrombospondin-1 Type 1 (TSP1-1) domain from Homo Sapiens [PDBID 1LSL] (Blue). (iii) Superposition of TSP1 domains from BmP53 protein (green) and PDBID 1LSL (blue), which have a TM-score of 0.5936, thus both structures have the same fold.
Fig 9.
Evaluation of host immune response against the non-mimic, TSP1 domain-free truncate (rGST-BmP53tr2) using SDS-PAGE and Western blot analysis.
Lane M: Low molecular weight marker. Lanes 1, 3 and 5: E. coli pellet contained rGST-BmP53tr2. Lanes 2, 4 and 6: Purified rGST. Lanes M, 1 and 2 were stained by amide black stain. Lanes 3 and 4 were probed with experimentally infected hamster serum (12-day post infection). Lanes 5 and 6 were probed with non-infected hamster serum. Black arrow point to a 43.4-kDa band revealing the presence of host immune reactivity against rGST-BmP53tr2.