Entamoeba histolytica-Induced Dephosphorylation in Host Cells

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.

宿主细胞蛋白酪氨酸磷酸酶（protein tyrosine phosphatases, PTPases）的激活与蛋白质去磷酸化，是多种微生物用以灭活或杀伤宿主防御细胞的重要机制。为明确蛋白酪氨酸去磷酸化是否在影响溶组织内阿米巴（Entamoeba histolytica）介导的宿主细胞杀伤的信号通路中发挥作用，我们探究了溶组织内阿米巴黏附靶细胞过程中PTPases的参与情况。我们通过抗磷酸酪氨酸单克隆抗体检测发现，Jurkat细胞黏附溶组织内阿米巴后，其细胞内蛋白酪氨酸水平快速下降。阿米巴诱导的蛋白质去磷酸化具有接触依赖性，且需要完整的虫体：因为用半乳糖阻断阿米巴黏附可抑制酪氨酸去磷酸化，而阿米巴裂解液对磷酸酪氨酸水平无影响。此外，通过半乳糖干扰阿米巴黏附可促进Jurkat细胞的磷酸化恢复，这表明去磷酸化发生在靶细胞死亡之前。相关证据表明，阿米巴诱导的去磷酸化由宿主细胞磷酸酶所介导。用PTPases抑制剂苯胂化氧（phenylarsine oxide）预处理Jurkat细胞，可抑制阿米巴诱导的去磷酸化。我们还发现，Jurkat细胞与阿米巴接触后，会发生蛋白酪氨酸磷酸酶1B（PTPase 1B, PTP1B）的蛋白水解切割。钙依赖性蛋白酶钙蛋白酶（calpain）负责PTP1B的切割及其酶活激活。用钙蛋白酶抑制剂卡肽素（calpeptin）预处理Jurkat细胞，可阻断PTP1B的切割并抑制阿米巴诱导的去磷酸化。此外，用苯胂化氧抑制Jurkat细胞的PTPases，可阻断溶组织内阿米巴诱导的Jurkat细胞凋亡。上述结果表明，溶组织内阿米巴介导的宿主细胞死亡，通过一条涉及PTPases激活的机制实现。