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

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

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

A Parametric Framework for Reversible pi-Calculi

This paper presents a study of causality in a reversible, concurrent setting. There exist various notions of causality in pi-calculus, which differ in the treatment of parallel extrusions of the same name. In this paper we present a uniform framework for reversible pi-calculi that is parametric with respect to a data structure that stores information about an extrusion of a name. Different data structures yield different approaches to the parallel extrusion problem. We map three well-known causal semantics into our framework. We show that the (parametric) reversibility induced by our framework is causally-consistent and prove a causal correspondence between an appropriate instance of the framework and Boreale and Sangiorgi's causal semantics

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

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

Surface Molecules Released by <i>Trypanosoma cruzi</i> Metacyclic Forms Downregulate Host Cell Invasion

<div><p>Background</p><p>The question whether metacylic trypomastigote (MT) forms of different <i>T</i>. <i>cruzi</i> strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using <i>T</i>. <i>cruzi</i> strains that differ widely in the ability to invade cells.</p><p>Methodology/Principal Findings</p><p>Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca²⁺ and Mg²⁺ (PBS⁺⁺). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS⁺⁺, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS⁺⁺ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion.</p><p>Conclusion/Significance</p><p>Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.</p></div

Change in the expression of surface gp90 and gp82 proteins in <i>T</i>. <i>cruzi</i> G strain metacyclic forms after incubation in different media.

<p>Parasites were incubated or not in D10 or PBS⁺⁺ for 1h at 37°C. After centrifugation, the supernatant was discarded and the parasites were incubated for 1 h with monoclonal antibody directed to gp90 or gp82. Following fixation and reaction with Alexa Fluor 488-conjugated anti-IgG, the parasites were analyzed by flow cytometry. Controls consisted of parasites incubated with the second antibody only. Shown in the lower panel are images of parasites visualized by epifluorescence microscope, with 100X objective.</p

Reversal of the inhibitory effect of G-CM on PBS⁺⁺-induced lysosome spreading by specific antibodies.

<p>HeLa cells were incubated for 30 min in PBS⁺⁺ in absence (-) or in the presence of CL-CM, G-CM, G-CM preincubated with mAb 5E7, mAb 2C2, anti-J18 or anti-C03 antibody and processed for confocal immunofluorescence analysis using anti-LAMP2 antibody, Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, with 63X objective. Scale bar: 15 μm. HeLa cells incubated in PBS⁺⁺ in the presence of r-gp90 served as control for inhibition of lysosome spreading.</p