Thrombospondin-1 Plays an Essential Role in Yes-Associated Protein Nuclear Translocation during the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells

Abstract

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.

Keywords: Chagas heart disease; Trypanosoma cruzi; heart endothelial cells; hippo signaling; transcriptional enhancer factor (TEF) family/TEA domain (TEAD) family; yes-associated protein1.

Figure 1

TSP-1 is essential for activation of hippo signaling cascade during the early phase of T. cruzi infection. Lysates (20 µg) from WT or TSP-1KO MHEC challenged with T. cruzi at different time points were resolved by SDS-PAGE, blotted, and probed with antibodies against (A) SAV1, (B) MOB1A in WT MHEC and (C) SAV1, (D) MOB1A in TSP-1KO MHEC, and developed as described. The blots were stripped, reprobed with antibodies against housekeeping GAPDH and developed with the corresponding IRDye conjugated secondary antibody. The developed blots were scanned using the infrared fluorescence detection Odyssey Imaging Systems. The normalized fold change in the level of each unphosphorylated protein was determined and plotted in the bar graph for WT MHEC (A, lower panel) SAV1, (B, lower panel) MOB1A, respectively and for TSP-1KO MHEC (C, lower panel) SAV1 and (D, lower panel) MOB1A, respectively. The bar graphs represent mean values ± SE from three independent biological replicates. The value of p < 0.05 was considered significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

YAP is translocated into the nuclei of WT MHEC early during T. cruzi infection. WT MHEC grown on coverslips coated with 1% gelatin in 6-well culture plates were challenged with T. cruzi at different time points, washed, fixed, perforated with 0.1% Triton-X100, blocked with 3% BSA-PBS, and incubated at 4 °C overnight in solutions containing phalloidin and (A) mouse anti YAP antibodies. The cells were washed and reprobed with goat anti mouse Alexa Fluor 488 conjugated secondary antibody. The washed cells were mounted with mounting media containing DAPI to stain the nuclei. (B) Stained slides were analyzed by confocal microscopy and the mean nuclear fluorescence intensity (MNFI) values were plotted for YAP. Images were captured at 60× at scale bar 10 μm. Each confocal microscopy image is a representative of three independent biological replicates. The bar graphs represent MNFI values ± SE from three independent biological replicates. The value of p < 0.05 was considered significant. ** p < 0.01.

Figure 3

T. cruzi induces significant translocation of YAP into the nuclei of TSP-1KO MHEC early during T. cruzi infection. TSP-1KO MHEC grown on coverslips coated with 1% gelatin in 6 well culture plates were challenged with T. cruzi at different time points, washed, fixed, perforated with 0.1% Triton-X100, blocked with 3% BSA-PBS, and incubated at 4 °C overnight, in solutions containing phalloidin and (A) mouse anti YAP antibodies. The cells were washed and reprobed with goat anti mouse Alexa Fluor 488 conjugated secondary antibody. The washed cells were mounted with mounting media containing DAPI to stain the nuclei. (B) Stained slides were analyzed by confocal microscopy and the mean nuclear fluorescence intensity (MNFI) values were plotted for YAP. Images were captured at 60× at scale bar 10 μm. Each confocal microscopy image is a representative of three independent biological replicates. The bar graphs represent MNFI values ± SE from three independent biological replicates. The value of p < 0.05 was considered to be significant. ** p < 0.01.

Figure 4

YAP and pan-TEAD are colocalized in the nuclei of WT MHEC during the early phase T. cruzi infection. WT MHEC grown on coverslips coated with 1% gelatin in 6 well culture plates were challenged with T. cruzi at different time points, washed, fixed, perforated with 0.1% Triton-X100, blocked with 3% BSA-PBS, and incubated at 4 °C overnight, in solutions containing phalloidin and (A) mouse anti YAP and rabbit anti pan-TEAD antibodies. The washed cells were reprobed with a cocktail of goat anti mouse Alexa Fluor 488 and goat anti rabbit Alexa Fluor 647 conjugated secondary antibodies. The cells were washed and mounted with mounting media containing DAPI to stain the nuclei. Stained slides were analyzed by confocal microscopy and images were captured at 60× at scale bar 10 μm. The mean fluorescence intensities of the merged signals were analyzed using confocal microscopy software to generate Pearson’s correlation coefficients. (B) The bar graphs represent Pearson’s correlation coefficients values ± SE from three independent biological replicates. Each confocal microscopy image is a representative of three independent biological replicates. The value of p < 0.05 was considered significant. * p < 0.05.

Figure 5

Transcriptional co-activators YAP and pan-TEAD are colocalized in the nuclei of TSP-1KO MHEC during the early phase of T. cruzi infection. TSP-1KO MHEC grown on coverslips coated with 1% gelatin in 6 well culture plates were challenged with T. cruzi at different time points, washed, fixed, perforated with 0.1% Triton-X100, blocked with 3% BSA-PBS, and incubated at 4 °C overnight in solutions containing phalloidin, and the following antibodies; (A) mouse anti YAP and rabbit anti pan-TEAD antibodies. The cells were washed and reprobed with a cocktail of goat anti mouse Alexa Fluor 488 and goat anti rabbit Alexa Fluor 647 conjugated secondary antibodies. The cells were washed and mounted with mounting media containing DAPI to stain the nuclei. Stained slides were analyzed by confocal microscopy and images were captured at 60× at scale bar 10 μm. Each confocal microscopy image is a representative of three independent biological replicates. The mean fluorescence intensities of the merged signals were analyzed using confocal microscopy software to generate Pearson’s correlation coefficients. (B) The bar graphs represent Pearson’s correlation coefficients values ± SE from three independent biological replicates. The value of p < 0.05 was considered to be significant. * p < 0.05.