Entamoeba histolytica-induced dephosphorylation in host cells

Abstract

Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation.

FIG. 1.

Western blot analysis of protein tyrosine phosphorylation in CHO, Jurkat cells, and E. histolytica. Jurkat (A) or CHO (B) cells (5 × 10⁵) were mixed with amebae (5 × 10⁴) at a 1:10 target cell-E. histolytica ratio, incubated for 15 min at 37°C, and lysed with boiling sample buffer. Proteins from lysates were separated by SDS-PAGE on 10% (A) or 12% (B) polyacrylamide gels, transferred to PVDF membrane, and incubated with HRP-pTyr-Ab RC20. The black dots indicate proteins that were completely dephosphorylated in target cells after incubation with amebae. Molecular masses of the standards, in kilodaltons, are shown on the left.

FIG. 2.

FACS analysis of E. histolytica-induced protein dephosphorylation in Jurkat cells. (A) Jurkat cells were incubated with E. histolytica trophozoites at a 1:10 Jurkat cell-E. histolytica ratio for 15 min at 37°C, formaldehyde fixed, permeabilized, and stained with FITC-pTyr-MAb PT-66. The data express the fluorescence intensities from Jurkat cells only. (A) FACS profile of the Jurkat cell population in medium alone (dotted line) or after incubation with amebae (continuous line, shaded area). (B) Phosphotyrosine protein levels in Jurkat cell after incubation with E. histolytica at different cell ratios. Cells were incubated for 15 min at 37°C prior to pTyr-FACS analysis. The data are expressed as the percentage of fluorescence intensity of each sample compared to the basal phosphotyrosine level from Jurkat cells alone (100%). (P < 0.03 at a 100:1 cell ratio and P < 0.001 at 50:1, 10:1, 2:1, and 1:1 cell ratios compared to Jurkat cells alone).

FIG. 3.

Time course analysis of E. histolytica-induced protein dephosphorylation in Jurkat cells. E. histolytica trophozoites incubated with Jurkat cells at 37°C were evaluated at different time points (0, 2.5, 10, 20, and 30 min) and then fixed with formaldehyde. Cells were permeabilized, stained with FITC-pTyr-MAb PT-66, and analyzed by FACS. Symbols: •, Jurkat cells alone; ○, Jurkat cells incubated with E. histolytica (P < 0.03 at 10 min and P < 0.005 at 20 and 30 min compared to Jurkat cells alone). The mean fluorescence intensity for the entire Jurkat cell population is expressed in arbitrary units (a.u.).

FIG. 4.

Effect of galactose on E. histolytica-induced protein dephosphorylation in Jurkat cells. E. histolytica trophozoites were incubated with Jurkat cells (at a 10:1 Jurkat cell-E. histolytica ratio) for 15 min at 37°C in control medium (open bar) or in the presence of either galactose (25 mg/ml) (solid bar) or amebic lysates (gray bar). Cells were formaldehyde fixed, permeabilized, and stained with FITC-pTyr-MAb PT-66 for FACS analysis. Data are expressed as the percentage of fluorescence intensity of each sample compared to the basal phosphotyrosine level from Jurkat cells alone (100%). ★, P < 0.03 compared to the control medium.

FIG. 5.

Phosphorylation recovering in Jurkat cells. (A) E. histolytica trophozoites were incubated with Jurkat cells (at a 10:1 Jurkat cell-E. histolytica ratio) for 5 min at 37°C. After incubation, galactose (25 mg/ml) was added, and the cells were further incubated for 15, 30, and 60 min at 37°C (solid bars). ★, P < 0.03 compared to control (open bar). (B) E. histolytica and Jurkat cells were incubated for 5 min, and then galactose (25 mg/ml) was added alone (solid bar) or in conjunction with genistein (200 μM) (gray bar). Cells were further incubated for 10 min at 37°C, followed by pTyr-FACS analysis. ★, P < 0.01 compared to the control (open bar). Data are expressed as the percentage of fluorescence intensity of each sample compared to basal phosphotyrosine level for Jurkat cells alone (100%).

FIG. 6.

Effect of PAO on E. histolytica-induced protein dephosphorylation in Jurkat cells. (A) E. histolytica trophozoites were incubated with Jurkat cells (at a 10:1 Jurkat cell-E. histolytica ratio) for 15 min at 37°C in medium alone (open bar) or in the presence of PAO at 0.5, 0.8, or 1.0 mM (solid bars). Cells were formaldehyde fixed, permeabilized, and stained with FITC-pTyr-MAb PT-66 for FACS analysis. P < 0.02 at 0.5 mM, P < 0.04 at 0.8 mM, and P < 0.01 at 1.0 mM compared to the control (open bar). (B) Effect of selective exposure of either Jurkat cells or E. histolytica to PAO on ameba-induced dephosphorylation. Jurkat cells (solid bar) or E. histolytica (gray bar) were preincubated with PAO (0.8 mM) for 15 min at 37°C, washed, and then incubated (15 min at 37°C) with E. histolytica or Jurkat cells (at a 10:1 cell ratio), respectively. Cells were formaldehyde fixed, permeabilized, and stained with FITC-pTyr-MAb PT-66 for FACS analysis. ★, P < 0.02 compared to the control (open bar). The data are expressed as a percentage of fluorescence intensity of each sample compared to basal phosphotyrosine level from Jurkat cells alone (100%).

FIG. 7.

Effect of calpeptin on E. histolytica-induced protein dephosphorylation in Jurkat cells. E. histolytica trophozoites were incubated with Jurkat cells at different cell ratios (i.e., 10:1, 50:1, or 100:1 Jurkat cell-E. histolytica ratios) for 15 min at 37°C in medium alone or in the presence of calpeptin (1.0 mM). Cells were formaldehyde fixed, permeabilized, and stained with FITC-pTyr-MAb PT-66 for FACS analysis. ★, P < 0.05 compared to Jurkat cells plus E. histolytica in medium alone. The y axis represents the percentage of inhibition of ameba-induced dephosphorylation.

FIG. 8.

Detection of PTP1B cleavage by Western blot analysis. Jurkat cells were incubated with E. histolytica (at a 10:1 Jurkat cell-E. histolytica ratio) for 15 min at 37°C in medium alone or in the presence of galactose (25 mg/ml), EGTA (5 mM), or calpeptin (1 mM) and lysed with boiling sample buffer. Proteins from lysates were separated by SDS-10% PAGE, transferred to PVDF membrane, and incubated with anti-PTP1B MAb, clone FG6-1G. The relative migration of 50-, 42-, 40-, and 36-kDa PTP1B proteins are depicted with arrows and are based upon estimates of molecular standards.

FIG. 9.

Effects of PAO and calpeptin on E. histolytica-induced Jurkat cell DNA fragmentation. Jurkat cells were pretreated with either 1.0 mM PAO (A) or 1.0 mM calpeptin (B) for 15 min at 37 C. The cells were washed, resuspended with E. histolytica at a 10:1, 50:1, or 100:1 cell ratio (Jurkat cells to E. histolytica), and incubated for 60 min at 37°C. DNA fragmentation was analyzed by 2% agarose gel electrophoresis. An equal number of amebae and Jurkat cells were incubated in medium alone as negative controls. Lane M is a 100-bp marker.