Interaction With the Extracellular Matrix Triggers Calcium Signaling in Trypanosoma cruzi Prior to Cell Invasion

Trypanosoma cruzi, 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 Ca²⁺ uptake from the extracellular medium, since it is inhibited by EGTA or Nifedipine, an inhibitor of the L-type voltage gated Ca²⁺ channels and sphingosine-dependent plasma membrane Ca²⁺ channel, but not by Vanadate, an inhibitor of the plasma membrane Ca²⁺-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 Ca²⁺ uptake from the medium and parasite-ECM interaction for host-cell invasion. Previous treatment of ECM with protease abolishes the Ca²⁺ uptake, further reinforcing the possibility that these events may be connected. The mitochondrion plays a relevant role in Ca²⁺ homeostasis in trypomastigotes during their interaction with ECM, as shown by the increment of the intracellular Ca²⁺ 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 Ca²⁺ at an early phase of the trypomastigotes interaction with ECM, implicating both nifedipine-sensitive Ca²⁺ channels in the influx of Ca²⁺ and the mitochondrion as the relevant organelle in Ca²⁺ homeostasis. The data unravel a complex sequence of events prior to host cell invasion itself.