Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion

Abstract

Successful infection by Trypanosoma cruzi, the agent of Chagas' disease, is critically dependent on host cell invasion by metacyclic trypomastigote (MT) forms. Two main metacyclic stage-specific surface molecules, gp82 and gp90, play determinant roles in target cell invasion in vitro and in oral T. cruzi infection in mice. The structure and properties of gp82, which is highly conserved among T. cruzi strains, are well known. Information on gp90 is still rather sparse. Here, we attempted to fill that gap. gp90, purified from poorly invasive G strain MT and expressing gp90 at high levels, inhibited HeLa cell lysosome spreading and the gp82-mediated internalization of a highly invasive CL strain MT expressing low levels of a diverse gp90 molecule. A recombinant protein containing the conserved C-terminal domain of gp90 exhibited the same properties as the native G strain gp90: it counteracted the host cell lysosome spreading induced by recombinant gp82 and exhibited an inhibitory effect on HeLa cell invasion by CL strain MT. Assays to identify the gp90 sequence associated with the property of downregulating MT invasion, using synthetic peptides spanning the gp90 C-terminal domain, revealed the sequence GVLYTADKEW. These data, plus the findings that lysosome spreading was induced upon HeLa cell interaction with CL strain MT, but not with G strain MT, and that in mixed infection CL strain MT internalization was inhibited by G strain MT, suggest that the inhibition of target cell lysosome spreading is the mechanism by which the gp90 molecule exerts its downregulatory role.

Keywords: Trypanosoma cruzi; host cell invasion.

FIG 1

Host cell lysosome spreading is induced by MT of highly invasive T. cruzi CL strain but not by poorly invasive G strain. (A) HeLa cells were incubated for 1 h with MT of CL or G strain, along with control cells, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody, Alexa Fluor 488-conjugated anti-mouse IgG (green), and DAPI (blue) for DNA, with a 63× objective. Scale bar, 30 μm. Note the lysosome scattering induced by CL strain MT and parasites associated with lysosome markers (red arrows), whereas lysosomes remained concentrated at the perinuclear area upon interaction with G strain MT. The framed region indicates the position of the magnified field shown in the lower right image. Scale bar, 10 μm. (B) HeLa cells were incubated for 1 h with MT of CL or G strain and then processed for intracellular parasite counting. Values are the means ± standard deviations (SD) from four independent assays performed in duplicate. (C) HeLa cells were incubated for 1 h with MT of CL or G strain alone or with both strains. Values are the means ± SD from three independent assays performed in duplicate. CL strain internalization was significantly inhibited in mixed infection (*, P = 0.0001).

FIG 2

CL and G strain MT express distinct gp90 molecules that share conserved structural features. (A) Live parasites were incubated for 1 h with MAb 1G7. Following fixation and reaction with Alexa Fluor 488-conjugated anti-IgG (green) and DAPI (blue), the parasites were visualized with a confocal microscope with a 100× objective. Alternatively, parasites were fixed before reaction with MAb 5E7 and then processed as described above. Scale bar, 10 μm. Note the lack of reaction of CL strain MT with MAb 1G7. (B) Western blots of detergent-soluble MT extracts revealed by the indicated monoclonal antibodies. Note the lower intensity of the CL strain gp90 band recognized by MAb 5E7. (C) Protein A/G magnetic beads, cross-linked to MAb 1G7, were incubated for 1 h with G strain MT lysates. After washings, the proteins bound to beads were eluted and subjected to SDS-PAGE and Western blotting, along with unbound proteins. Eluates from MAb 1G7 beads (lanes 1 and 3) and unbound proteins (lanes 2 and 4) were revealed with MAb 1G7 or MAb 5E7. (D) Sequences of peptides spanning the C-terminal domain of g90. (E) Microtiter plates coated with a recombinant protein containing the C-terminal portion of gp90 (r-gp90C) were incubated with MAb 5E7 in the absence or in the presence of 100 μg/ml individual peptides shown in panel D. Binding of MAb 5E7 was measured by ELISA. Values are the means ± SD from triplicates. The sequences shared by peptides 12 and 13, which inhibited antibody binding, are highlighted in red in panel D. *, P = 0.0005.

FIG 3

Host cell lysosome spreading and CL strain MT invasion are inhibited by gp90 molecule from G strain MT. HeLa cells were incubated for 30 min in complete medium (control) or in PBS⁺⁺, in the absence or in the presence of 20 μg/ml gp90 purified from G strain MT, and processed for confocal fluorescence analysis using anti-LAMP2 antibodies as described for Fig. 1A. Scale bar, 30 μm. Note the lysosome spreading upon incubation in PBS⁺⁺, with accumulation of lysosome markers at the cell borders (red arrows), and the inhibitory effect of gp90. The graph shows the rate of CL strain MT invasion upon 1 h of incubation of parasites with HeLa cells in the absence or in the presence of gp90 purified from G strain MT at the indicated concentrations. Values are the means ± SD from three independent assays performed in duplicate. gp90 significantly inhibited MT internalization at all concentrations (*, P < 0.005; **, P ≤ 0.0005).

FIG 4

Recombinant protein r-gp90C counteracts host cell lysosome spreading induced by recombinant protein r-gp82 and CL strain MT internalization. (A) HeLa cells were incubated for 30 min with r-gp82 alone or mixed with r-gp90C and processed for immunofluorescence as described for Fig. 1A. Note the spreading of lysosomes induced by r-gp82, with accumulation at the cell edges (red arrows) and the lysosome retention at the perinuclear region in the presence of r-gp90C. The graph shows the rate of CL strain MT invasion upon a 1-h incubation of parasites with HeLa cells in the absence or in the presence of r-gp90C at the indicated concentrations. Values are the means ± SD from three independent assays performed in duplicate. MT internalization was significantly inhibited at all concentrations (*, P < 0.001). (B) Sequences of peptides comprising the gp90 C-terminal domain. (C) HeLa cells incubated for 1 h with CL strain MT in the absence or in the presence of 50 μg/ml individual peptides shown in panel B and processed for intracellular parasite counting. Values are the means ± SD from three assays performed in duplicate. The sequences shared by peptides 12 and 13, which inhibited MT internalization, are highlighted in red in panel B. *, P < 0.005; **, P < 0.0005.

FIG 5

G strain MT internalization is inhibited by r-gp82 and r-gp90C, which have opposite effects on LAMP2 expression. (A) HeLa cells were incubated for 1 h with G strain MT in the absence or in the presence of r-gp90C or r-gp82, at various concentrations, and then processed for intracellular parasite counting. Values are the means ± SD from three independent assays performed in duplicate. MT invasion was significantly inhibited by r-gp90C and r-gp82 at all concentrations (*, P < 0.005; **, P < 0.001). (B) HeLa cells were incubated for 30 min in the absence or in the presence of r-gp82 alone or r-gp82 plus r-gp90C, and the detergent extract was analyzed by Western blotting using anti-LAMP-2 and anti-β-tubulin antibodies. The band intensities were measured with ImageJ software. LAMP2 intensity was normalized to the untreated control value, which was set equal to 100%. A representative result out of three is shown.