Role of the gp85/trans-sialidases in Trypanosoma cruzi tissue tropism: preferential binding of a conserved peptide motif to the vasculature in vivo

Abstract

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature.

Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied.

Findings and conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Figure 1. The FLY phage mimics the VTVTNVFLYNRPLN peptide.

(a) Binding of FLY phage and fd-tet to immobilized CK18 and to (b) LLC-MK₂ cells. (c) Binding of FLY phage to CK18 in the presence of increasing concentrations of VTVTNVFLYNRPLN synthetic peptide (FLY, black squares) or the alanine mutagenized version VTVTNVFAYNRPLN synthetic peptide (FAY, open triangles). Results are show as percentage of binding relative to FLY phage in the absence of peptides. Shown are standard error of the mean (SEM) of two biological replicates performed in triplicate.

Figure 2. FLY phage binding to organ-derived endothelial cells.

Binding of FLY phage to bone marrow, bladder, heart or lung-derived endothelial cells; fd-tet and FAY phage were used as control. Phage binding was normalized to endothelial cell DNA, quantified using ribosomal RNA specific probes. The error bars are standard error of the mean (SEM) of experiments performed in triplicate. Where indicated, * denotes P<0.05.

Figure 3. FLY binding to the vasculature of the mouse.

FLY and FAY phage homing in vivo was evaluated following intravenous phage administration into mice. The number of phage particles that accumulated in the vasculature of select organs was quantified by qPhage. Results are shown relative to the amount of mouse genomic DNA (quantified using ribosomal RNA specific probes) with the SEM of experiments performed in triplicate. Where indicated, * denotes P<0.05.

Figure 4. FLY phage interaction with intermediate filament proteins.

Phage binding to immobilized cytokeratin-8 (CK8), -18 (CK18) and -20 (CK20), and to vimentin. The FAY and fd-tet phage were used as control. Shown are SEM of experiments performed in triplicate. Where indicated, * denotes P<0.05.

Figure 5. Vimentin and cytokeratin are present on the cell surface.

Different organ derived endothelial cells were incubated with anti-pan-CK (red) or anti-vimentin (green) specific antibodies. In (a), antibodies were incubated with live cells and in (b), the antibodies were incubated with p-formaldehyde fixed and detergent permeabilized cells. DAPI staining (blue), merged fluorescence images and the corresponding DIC images are also shown.