TCLP 1 co-localizes with markers of the endocytic but not degradative pathway.

<p>IIF assays of TCLP 1::3XFLAG-transfected epimastigotes or wild type Adriana amastigotes (lower panels) were probed with monoclonal anti-FLAG (αFLAG) or anti-TCLP 1 (αTCLP 1) antibody (green) and co-stained either with ConA-rhodamine (ConA), anti-cruzipain (αCZP) or monoclonal FK-2 (αFK2) antibody (all in red). DAPI signals are shown in blue and enlarged rectangular regions are shown in merged images.</p

TCLP 1 belongs to a novel, multi-domain family of proteins conserved in trypanosomatids.

<p>(A) <i>In silico</i> predictions and annotated motifs are underlined in the TCLP 1 deduced sequence from the CL Brener clone of <i>T</i>. <i>cruzi</i>. Asterisk denotes the actual initial Methionine residue identified in this work for TCLP 1. Abbreviations: SP, Signal Peptide; UBL, Ubiquitin-Like domain; CEST, CesT-like domain; PDZ, <u>P</u>SD95/<u>D</u>lg1/<u>z</u>o-1 domain; NLS, Nuclear Localization Signal; NES, Nuclear Export Signal. The cognate peptide recognized by the αTCLP 1 antibody is boxed. (B) A simplified Clustal-W alignment of TCLP 1 CEST motif and most relevant bacterial CEST molecules. Conserved residues are shaded, and overall amino acid identity with TCLP 1 (in %) is indicated to the right. (C) A phylogeny tree of "CEST-like" molecules identified in trypanosomatids. The tree was rooted with the lowest branch as outgroup (<i>Escherichia coli</i>). Trypanosomatid “CEST-like” proteins are named with their corresponding Gene ID. The domain architecture of Class I (UBL-CEST-PDZ) and Class II (UBL-CEST) trypanosomatid “CEST-like” proteins and bacterial CEST molecules is indicated by a color code. (D) 100 bootstrap tree derived of a Clustal-W alignment of selected trypanosomatid "CEST-like" proteins. Bootstrap support values (in %) for the main branches are indicated. The scale indicates the amino acid substitution distance along the branches. Abbreviations: TcCLB, <i>T</i>. <i>cruzi</i> CL Brener clone; MOQ, <i>T</i>. <i>cruzi marinkellei</i>; TCSYLVIO, <i>T</i>. <i>cruzi</i> Sylvio X-10 strain; Lbr, <i>Leishmania braziliensis;</i> Lta, <i>L</i>. <i>tarentolae;</i> Lmx, <i>L</i>. <i>mexicana;</i> Lmj, <i>L</i>. <i>major;</i> Lin, <i>L</i>. <i>infantum;</i> LDBPK, <i>L</i>. <i>donovani;</i> Tb, <i>T</i>. <i>brucei;</i> Tbg, <i>T</i>. <i>brucei gambiense;</i> TcIL, <i>Trypanosoma congolense;</i> TvY, <i>Trypanosoma vivax</i>.</p

Structural conservation between TCLP 1 and <i>Salmonella</i> TIIISS chaperones:

<p>(A) The sequences of the TCLP 1-derived CEST motif, 3epuA and 1jyoD were aligned with the program PROMALSD3D. Secondary structure motifs predicted for the consensus sequence obtained for both bacterial molecules are depicted below as solid (α helices) or empty (ß sheets) black boxes. Secondary structure motifs independently predicted for the TCLP 1 CEST motif using Jpred3 analysis are shown below as solid (α helices) or empty (ß sheets) grey boxes. (B) Model superimposition of the TCLP 1 CEST motif with templates 3epuA and 1jyoD. <i>Left</i>, CEST motif model I (3epuA-based, red) and 3epuA structure (green). <i>Middle</i>, CEST motif model II (1jyoD-based, red) superimposed with 1jyoD structure (blue). <i>Right</i>, Model of 1jyoD, 3epuA-based (green), superimposed with 1jyoD structure (blue). QMEAN values are shown below each panel.</p

TCLP 1-transfected parasites show diminished endocytic capacity.

<p>(A) ConA-rhodamine (above) or Tf-Alexa 488 (below) internalization by either Adriana epimastigotes transfected with TCLP 1::3xFLAG (TCLP 1, red) or Adriana WT (Ad WT, green) parasites at the indicated time points was monitored by flow cytometry. Unmarked (no probe) parasites are shown in grey. (B) Representative epifluorescence images of Ad WT (upper panels) and TCLP 1 (lower panels) epimastigotes upon different times of incubation with ConA-rhodamine or Tf-Alexa 488 as indicated under Materials and Methods. C) Fluorescence quantification of dye labeling at the FP area (indicated with asterisks in (B)). Mean values of fluorescence (in Arbitrary Units) and standard deviation are plotted for TCLP 1 (dark grey) and Ad WT (light grey) parasites upon incubation with either dye. Asterisks denote significant differences (***, <i>p</i><0.001; **, <i>p</i><0.01) between Ad WT and TCLP 1 epimastigotes as evaluated by Student’s T-test.</p

Fine sub-cellular localization analysis of TCLP 1 in <i>T</i>. <i>cruzi</i>.

<p>(A) IIF assays of TCLP 1::3XFLAG-transfected epimastigotes were probed with monoclonal anti-FLAG antibody (αFLAG, green) and co-stained either with anti-Aquaporin 1 (αAqp, upper panels) or anti-GRASP (αGRASP, middle panels) antibody (both in red). Lower panels, TcBilbo-1::mCherry-transfected parasites (BILBO, red) were stained with anti-TCLP 1 (αTCLP 1) antibody (green). DAPI signals are shown in blue and enlarged rectangular regions are shown in merged images. (B) Immunoelectron microscopy of wild type CL Brener epimastigotes (upper panels) and amastigotes (lower panels) labeled with αTCLP 1 (5-nm gold particles). Longitudinal sections of the parasite forms highlighting the flagellar pocket (FP), Nucleus (N) and Kinetoplast (K). Enlarged areas showing αTCLP 1 reactivity in the FP are indicated with rectangles in the original images. Asterisks highlight gold particles. Bar scale, 0.2 μm.</p

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of <i>Trypanosoma cruzi</i> during human infections

<div><p>Background</p><p>The <i>Trypanosoma cruzi</i> genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a ‘mosaic’ structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of <i>T</i>. <i>cruzi</i> and may be involved in parasite-host interactions and/or in diverting the immune response.</p><p>Methods/Principle findings</p><p>High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the <i>T</i>. <i>cruzi</i> seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in <i>in vitro</i> and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection.</p><p>Conclusions/Significance</p><p>In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from <i>T</i>. <i>cruzi</i> MASPs during human infection.</p></div

High-throughput discovery of antigenic MASPs.

<p><b>A)</b> 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>B)</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. <b>C)</b> Box-and-whiskers chart comparing the Chagas-chip average reactivity for MEMEs and Trypomastigote subgroups. <b>D)</b> 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).</p

Serological validation of prioritized MASP motifs.

<p><b>A)</b> 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 <i>S</i>-transferase) were used as negative controls. Not evaluated serum/motif combinations are indicated by empty dots. <b>B)</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 <i>p</i>-values) of Mann-Whitney non parametric test are indicated. <b>C)</b> Column chart showing % sensitivity (red columns) and specificity (green columns) for each prioritized motif and controls. <b>D)</b> Correlation analysis of ELISA (red columns) and Chagas-chip (green columns) reactivity. Pearson´s <i>r</i> coefficient (parametric), <i>p</i>-value and Spearman’s <i>r</i> coefficient (non- parametric) are indicated.</p

Genomic features of MASP motifs.

<p><b>A)</b> Pie chart accounting for the representation (in %) of each cluster to the total number of MASP-derived MRPs identified in the Chagas-chip (<i>n</i> = 86). <b>B)</b> Pie charts depicting the representation of each cluster in the total augmented list of MASP proteins derived from the <i>in silico</i> motif homology-based search (<i>n</i> = 332, upper chart), in the CL Brener MASP collection of genes (<i>n</i> = 301, middle chart) or pseudogenes (<i>n</i> = 31, bottom chart). <b>C)</b> 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 <i>n</i> 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. <b>D)</b> Correlation analysis of the genomic prevalence as a function of the relative average reactivity (in %) of MASPs motifs.</p

Reactivity and clustering analysis of positive MASP peptides.

<p><b>A)</b> 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>B)</b> Simplified cladogram showing the clustering of MRPs. Clusters numbers and <i>n</i> values are indicated to the left. <b>C)</b> Consensus antigenic motifs corresponding to clusters with <i>n</i> ≥ 3 (color-coded as in panel A) are shown as <i>WebLogo</i> graphics derived from the alignment of constituent sequences. Clusters with <i>n</i> < 3 (in white in panel A) are shown in black. <b>D)</b> Dispersion chart showing relative reactivity values for every MRP within each cluster. <b>E)</b> 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.</p