Laboratory of Biochemistry of Parasites, Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Department of Clinical Pathology, State University of Campinas, Campinas, São Paulo, Brazil.
Front Cell Infect Microbiol. 2021 Oct 4;11:731372. doi: 10.3389/fcimb.2021.731372. eCollection 2021.
, the etiological agent of Chagas disease in humans, infects a wide variety of vertebrates. Trypomastigotes, the parasite infective forms, invade mammalian cells by a still poorly understood mechanism. Adhesion of tissue culture- derived trypomastigotes to the extracellular matrix (ECM) prior to cell invasion has been shown to be a relevant part of the process. Changes in phosphorylation, S-nitrosylation, and nitration levels of proteins, in the late phase of the interaction (2 h), leading to the reprogramming of both trypomastigotes metabolism and the DNA binding profile of modified histones, were described by our group. Here, the involvement of calcium signaling at a very early phase of parasite interaction with ECM is described. Increments in the intracellular calcium concentrations during trypomastigotes-ECM interaction depends on the Ca uptake from the extracellular medium, since it is inhibited by EGTA or Nifedipine, an inhibitor of the L-type voltage gated Ca channels and sphingosine-dependent plasma membrane Ca channel, but not by Vanadate, an inhibitor of the plasma membrane Ca-ATPase. Furthermore, Nifedipine inhibits the invasion of host cells by tissue culture- derived trypomastigotes in a dose-dependent manner, reaching 95% inhibition at 100 µM Nifedipine. These data indicate the importance of both Ca uptake from the medium and parasite-ECM interaction for host-cell invasion. Previous treatment of ECM with protease abolishes the Ca uptake, further reinforcing the possibility that these events may be connected. The mitochondrion plays a relevant role in Ca homeostasis in trypomastigotes during their interaction with ECM, as shown by the increment of the intracellular Ca concentration in the presence of Antimycin A, in contrast to other calcium homeostasis disruptors, such as Cyclopiazonic acid for endoplasmic reticulum and Bafilomycin A for acidocalcisome. Total phosphatase activity in the parasite decreases in the presence of Nifedipine, EGTA, and Okadaic acid, implying a role of calcium in the phosphorylation level of proteins that are interacting with the ECM in tissue culture- derived trypomastigotes. In summary, we describe here the increment of Ca at an early phase of the trypomastigotes interaction with ECM, implicating both nifedipine-sensitive Ca channels in the influx of Ca and the mitochondrion as the relevant organelle in Ca homeostasis. The data unravel a complex sequence of events prior to host cell invasion itself.
克氏锥虫是人类恰加斯病的病原体，能感染多种脊椎动物。游走体是寄生虫的感染形式，通过一种仍未被充分了解的机制侵入哺乳动物细胞。在细胞侵入之前，组织培养衍生的游走体与细胞外基质（ECM）的黏附已被证明是该过程的一个相关部分。在相互作用的晚期（2 小时），我们的研究小组描述了蛋白质磷酸化、S-亚硝基化和硝化水平的变化，导致了游走体代谢和修饰组蛋白的 DNA 结合图谱的重新编程。在这里，描述了寄生虫与 ECM 相互作用的早期阶段钙信号的参与。在 ECM 中，当基质结合后，钙从细胞外进入细胞内，而当细胞外液中的 EGTA 或 Nifedipine（一种 L 型电压门控钙通道和鞘氨醇依赖性质膜钙通道的抑制剂）存在时，会增加细胞内钙浓度，但当质膜 Ca-ATPase 的抑制剂 Vanadate 存在时，细胞内钙浓度不会增加。此外，Nifedipine 以剂量依赖性方式抑制组织培养衍生的游走体对宿主细胞的侵袭，在 100µM Nifedipine 时达到 95%的抑制作用。这些数据表明，从中摄取钙和寄生虫与 ECM 的相互作用对宿主细胞的入侵都很重要。用蛋白酶预先处理 ECM 会消除钙摄取，进一步强化了这些事件可能相关的可能性。在与 ECM 相互作用期间，线粒体在锥虫的钙稳态中发挥重要作用，这表现在 Antimycin A 的存在下，细胞内钙浓度增加，而在其他钙稳态破坏剂（如内质网的 Cyclopiazonic acid 和酸钙体的 Bafilomycin A）中则没有这种情况。在 Nifedipine、EGTA 和 Okadaic acid 的存在下，寄生虫的总磷酸酶活性降低，这意味着钙在与 ECM 相互作用的组织培养衍生的锥虫中与 ECM 相互作用的蛋白质磷酸化水平中发挥作用。综上所述，我们在这里描述了在锥虫与 ECM 相互作用的早期阶段钙的增加，这暗示了尼非地平敏感的钙通道在钙内流中的作用，以及线粒体作为钙稳态中的相关细胞器。这些数据揭示了宿主细胞入侵本身之前的一系列复杂事件。
