Structural Basis of the Interaction of a <em>Trypanosoma cruzi</em> Surface Molecule Implicated in Oral Infection with Host Cells and Gastric Mucin

<div><p>Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogenic microorganisms. As concerns <em>Trypanosoma cruzi</em>, which causes Chagas disease, the insect vector-derived metacyclic trypomastigotes (MT) initiate infection by invading host cells, and later blood trypomastigotes disseminate to diverse organs and tissues. Studies with MT generated in vitro and tissue culture-derived trypomastigotes (TCT), as counterparts of insect-borne and bloodstream parasites, have implicated members of the gp85/trans-sialidase superfamily, MT gp82 and TCT Tc85-11, in cell invasion and interaction with host factors. Here we analyzed the gp82 structure/function characteristics and compared them with those previously reported for Tc85-11. One of the gp82 sequences identified as a cell binding site consisted of an α-helix, which connects the N-terminal β-propeller domain to the C-terminal β-sandwich domain where the second binding site is nested. In the gp82 structure model, both sites were exposed at the surface. Unlike gp82, the Tc85-11 cell adhesion sites are located in the N-terminal β-propeller region. The gp82 sequence corresponding to the epitope for a monoclonal antibody that inhibits MT entry into target cells was exposed on the surface, upstream and contiguous to the α-helix. Located downstream and close to the α-helix was the gp82 gastric mucin binding site, which plays a central role in oral <em>T. cruzi</em> infection. The sequences equivalent to Tc85-11 laminin-binding sites, which have been associated with the parasite ability to overcome extracellular matrices and basal laminae, was poorly conserved in gp82, compatible with its reduced capacity to bind laminin. Our study indicates that gp82 is structurally suited for MT to initiate infection by the oral route, whereas Tc85-11, with its affinity for laminin, would facilitate the parasite dissemination through diverse organs and tissues.</p> </div

Comparison of gp82 sequences associated with recognition by mAb 3F6 or binding to gastric mucin with the equivalent sequences in Tc85-11.

<p>A) The gp82 sequence identified as the epitope for mAb 3F6 (P3) was aligned with the equivalent Tc85-11 sequence, with the differences highlighted in red. B) Soluble extracts of MT and TCT were analyzed by Western blot using mAb 3F6. C) The gp82 sequence corresponding to the gastric mucin-binding site (P7) was aligned with the equivalent Tc85-11 sequence, with the changed residues indicated in red. D) Transwell filters coated with gastric mucin were placed onto 24-well plates containing MT or TCT. After 30 or 60 min incubation, samples from the filter chamber were collected and the number of parasites counted. Values are the means ± SD of three independent experiments. E) Assays were performed as in (D) using transwell filters coated with gastric mucin alone, or mixed with the recombinant protein J18 or GST. The difference between the filter containing J18 and the control was significant (*P<0.05, **P<0.01). F) Assays were performed as in (D) using transwell filters coated with gastric mucin alone, or mixed with the synthetic peptide P7 or P7*. The difference between the filter containing P7 and the control was significant (*P<0.05, **P<0.0005).</p

The solvent accessibility of the residues from peptides P4 and P8, measured in water exposed surface in Ų.

<p>The solvent accessibility of the residues from peptides P4 and P8, measured in water exposed surface in Ų.</p

The structural model of gp82.

<p>A) Cartoon representation highlighting the cell binding sites P4 (magenta) and P8 (blue). B) Surface representation of sites P4 and P8. C) The epitope for mAb 3F6 (P3) is highlighted (green). D) The portion of P3 that overlaps with P4 is indicated (yellow).</p

Comparative analysis of Tc85-11 sequences mapped as laminin-binding sites and the equivalent sequences in gp82.

<p>A) The Tc85-11 sequences N-17 and N-21, corresponding to laminin-binding sites, were aligned with the equivalent sequences in gp82, with the differences highlighted in red. B) Microtiter plates were coated with laminin or gastric mucin (10 µg/well), and ELISA assay was performed using anti-laminin or anti-gastric mucin antisera, at the indicated dilutions. C) Laminin- or gastric mucin-coated plates were incubated with J18, the recombinant protein containing the full length gp82 sequence, at the indicated concentrations. Binding of J18 was revealed by anti-J18 antibodies. Values are the means ± SD of triplicates of a representative experiment.</p

Sequences of <i>T. cruzi</i> surface proteins gp82 and Tc85-11.

<p>Shown are the aminoacid sequences deduced from cDNA clone J18, containing the full-length metacyclic stage gp82 (GenBank L14824), and from the cDNA insert containing Tc85-11 open reading frame (GenBank AF085686). In gp82, the sequences identified as P4 and P8 represent the host cell binding sites, P3 corresponds to the epitope for mAb 3F6, and P7 constitutes the main gastric mucin-binding site. In Tc85-11, the sequences corresponding to cell adhesion sites are identified as N-17, N-20 and N-21 and overlap with laminin-binding sites N17 and N-21 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042153#pone.0042153-MarroquinQuelopana1" target="_blank">[34]</a>. Points represent residues that are conserved in the two proteins, nonconserved amino acids are indicated, and dashes represent residues that are lacking.</p

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Delta-amastin is more abundant in less infective <i>T. cruzi</i> extracellular amastigotes. A.

<p><b>mRNAs in EAs of the G strain are less abundant when compared to EAs of the CL-Brener clone </b><b><i>T. cruzi</i></b><b>.</b> Transcript levels were determined by quantitative real-time PCR using SYBR® Green I chemistry. qRT-PCR was performed on RNA samples from EAs of G and CL strains. The comparative mRNA levels were determined after normalization with GAPDH amplicons. Standard deviations are derived from three replicates. *p<0.05 <b>B. mRNA corresponding to amastin is preferentially expressed in amastigotes from CL Brener clone.</b> Northern blot analyses of total RNA (10 µg) from <i>T. cruzi</i> epimastigotes (E), trypomastigotes (T) and amastigotes (A) from CL-Brener clone or the G <i>T. cruzi</i> strain was submitted to electrophoresis and blotted on nylon membranes by standard procedures. Each blot was hybridized with amastin probe previously labeled with [α-^32P]-dCTP. To determine equal loading of RNA, the 1.2% agarose/MOPS/formaldehyde gel was stained with ethidium bromide (bottom panel).</p

GST-δ-AmastinH specifically binds to HeLa cells in a dose-dependent saturable manner.

<p><b>A. SDS-PAGE showing the purity of recombinant GST-δ-amastinH:</b> 1; Molecular weigh markes, in kiloDaltons; 2: GST alone; 3: purified recombinant GST-δ-AmastinH; 4 total extract of induced <i>E. coli</i>. <b>B: Recombinant GST-δ-AmastinH binds to HeLa cells</b>. Increasing concentrations of GST-AmastinH or GST (negative control) were added to wells in ELISA plates containing adhered and fixed HeLa cells. After washing, cells were sequentially incubated with anti-GST antibodies and anti-rabbit IgG conjugated to peroxidase. The bound enzyme was revealed by <i>o-</i>phenylenediamine as a substrate. Representative results of two independent experiments are shown. *p<0.05.</p

Schematic proposed model for the role of δ-amastin in <i>T. cruzi</i> virulence.

<p>The model indicates two distinct parasites, EAs expressing high levels of amastin was represented by dotted line membrane of amastigotes (left cell side) whereas the low amastin expression was represented by spaced dots in the amastigotes membrane (right cell side), during EA invasion and differentiation processes of <i>T. cruzi</i> virulence in a hypothetical host cell. <b>1.</b> EAs expressing more amastin (left) show a lower infectivity rate when compared with parasites expressing lower levels of the protein. <b>2.</b> High levels of amastin accelerate the transformation of amastigotes into TCTs (left).</p