Fig 1.
High-throughput discovery of antigenic MASPs.
A) Pie charts showing the total MASP content and representation of subgroups (MEMEs, Trypomastigote) included in the initial pool (left) and the resulting pool after Chagas-chip serological evaluation (right). B) Column chart showing the cumulative reactivity values for each of the 69 positive MASPs. The original affiliation of each MASP (either to the MEMEs or Trypomastigote subgroup) is indicated with a color code as in panel A. C) Box-and-whiskers chart comparing the Chagas-chip average reactivity for MEMEs and Trypomastigote subgroups. D) Box-and-whiskers chart comparing the Chagas-chip average reactivity for positive MASPs, TcMUC, gp85/TS-like molecules, and the overall positive proteins in the Chagas-chip (Chip).
Fig 2.
Reactivity and clustering analysis of positive MASP peptides.
A) Column chart showing Chagas-chip mean reactivity values for the most reactive peptide (MRP) within each positive peak (n = 86). The Chagas-chip prevalence calculated for each MRP is also indicated in the column chart of the right. B) Simplified cladogram showing the clustering of MRPs. Clusters numbers and n values are indicated to the left. C) Consensus antigenic motifs corresponding to clusters with n ≥ 3 (color-coded as in panel A) are shown as WebLogo graphics derived from the alignment of constituent sequences. Clusters with n < 3 (in white in panel A) are shown in black. D) Dispersion chart showing relative reactivity values for every MRP within each cluster. E) Analysis of most relevant antigenic positions from cluster 1. Relative antigenicity of peptides is plotted as function of sequence identity (%) relative to the most reactive peptide within this group. ClustalW alignments between two subgroups (a,b) of peptides with similar % identity displaying great dispersion in their reactivity. Divergent positions in relation to the first sequence are boxed.
Fig 3.
Genomic features of MASP motifs.
A) Pie chart accounting for the representation (in %) of each cluster to the total number of MASP-derived MRPs identified in the Chagas-chip (n = 86). B) Pie charts depicting the representation of each cluster in the total augmented list of MASP proteins derived from the in silico motif homology-based search (n = 332, upper chart), in the CL Brener MASP collection of genes (n = 301, middle chart) or pseudogenes (n = 31, bottom chart). C) The graphic depicts the co-occurrence profile of each motif within a single MASP polypeptide (co-occurrence with 0 (i.e. itself or motif alone), 1, 2 or 3 additional motifs is represented with light blue, green, blue and red boxes, respectively). Total n values for each motif are indicated. The number of motif co-occurrence events of each type is indicated in the corresponding box and their % representation is proportional to the box size. Co-occurring motifs are linked by lines at right. D) Correlation analysis of the genomic prevalence as a function of the relative average reactivity (in %) of MASPs motifs.
Fig 4.
MASP reactivity is focused to the mature C-terminal region.
A) Lack of reactivity against MASPs SP and GPI-anchoring sequences. Multiple alignments results of MASPs SP and GPI-anchoring sequences included in the Chagas-chip (n = 232) are depicted as WebLogo graphics (above) along with their individual reactivity (below). For SP sequences, the predicted initial Meth is denoted as residue 1 whereas for GPI sequences amino acid positions are indicated with negative numbers and the STOP codon is denoted as residue 0. The negative (cutoff) line is indicated with a dashed line. Consensus residues for SP cleavage and GPI addition are denoted by asterisks. B) Relative position (RP) of the most reacting peptides (MRPs) corresponding to each MASP antigenic motif. C) Correlation analysis of the relative prevalence of MASP motifs as a function of their RP. D) Correlation analysis of the relative average reactivity of MASP motifs as a function of their RP. For every correlation, Pearson’s r coefficient and the p-value are indicated.
Fig 5.
Serological validation of prioritized MASP motifs.
A) Complete profile of recognition of our panel of positive serum samples (n = 58) towards MASP antigenic motifs. Cutoff-defined true positive and negative results are indicated in green and red, respectively. TSSA (Trypomastigote Small Surface Antigen) was used as positive control whereas a scrambled TSSA peptide and GST (Glutathione S-transferase) were used as negative controls. Not evaluated serum/motif combinations are indicated by empty dots. B) Box-and-whiskers charts of reactivity values of true positive and negative sera (expressed as % of a reference serum) for each antigenic motif and controls. The level of statistical significance (* and p-values) of Mann-Whitney non parametric test are indicated. C) Column chart showing % sensitivity (red columns) and specificity (green columns) for each prioritized motif and controls. D) Correlation analysis of ELISA (red columns) and Chagas-chip (green columns) reactivity. Pearson´s r coefficient (parametric), p-value and Spearman’s r coefficient (non- parametric) are indicated.
Fig 6.
Prioritized antigenic motifs drive MASP reactivity.
A) Schematic representation of GST- fusion constructs (green) derived upon MASP TcCLB.511173.64 (173, 173C and 173ΔC) or TcCLB.507959.280 (959) generated to perform validation assays. Antigenic motifs (1, 16 and 28) are indicated as red lines within 173 and 173C. B) Relative reactivity (left) and one-to-one schematic profiling (right) of Chagasic sera (n = 20) against each GST-fusion protein is indicated as in legend to Fig 5. C) Box-and-whiskers charts of reactivity values of true positive and negative sera (expressed as % of a reference serum) for the indicated GST-fusion proteins. The level of statistical significance (* and p-values) of Mann-Whitney non parametric test are indicated. D) ROC curves (%Sensitivity vs. 100%- %Specificity) analysis of 173 recombinant constructs and controls. The area under the curve (AUC) and the 95% confidence interval (C.I.) are indicated. E) Reactivity values (expressed as the % of reactivity of PBS-added sera) of a Chagasic serum reactive against the GST-fusion protein spanning motif 2 treated with increasing amounts (0.1, 1.0 or 10 μg) of the specific (motif 2 peptide) or non-related (‘scrambled’) peptides.