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

Abstract

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.

The Kinetoplastid-Specific Protein TcCAL1 Plays Different Roles During In Vitro Differentiation and Host-Cell Invasion in Trypanosoma cruzi

“…The key early events in the invasion of mammalian cells by T. cruzi involve the recognition and adhesion to the target-plasma membrane, the parasite internalization and maturation inside a recently-assembled parasitophorous vacuole, and the release from this organelle-like-structure to differentiate into replicative amastigotes in the host cytosol ( Andrews and Colli, 1982 ; Maeda et al., 2012 ; Rodríguez-Bejarano et al., 2021 ). Prior to cell invasion, the interaction of membrane surface molecules in T. cruzi and their ligands expressed in the host extracellular matrix initiates bidirectional signaling cascades which include Ca 2+ mobilization in the parasite ( Manchola Varón et al., 2021 ). Then, T. cruzi harnesses several mechanisms for its internalization into the host cell, including lysosome-dependent exocytosis, endocytosis, or phagocytosis ( Andrade and Andrews, 2004 ; Barrias et al., 2013 ; Batista et al., 2020 ).…”

“…Also, during the invasion of mammalian cells in vitro , measurements with fluorescent indicators showed that the [Ca 2+ ] c in trypomastigotes transiently increases as a consequence of parasite adhesion to the outer membrane of the host cell ( Moreno et al., 1994 ; Yakubu et al., 1994 ). Furthermore, the [Ca 2+ ] c increase observed in tissue culture-derived trypomastigotes is also triggered by interaction with mammalian extracellular matrix proteins ( Manchola Varón et al., 2021 ). Regarding this, the main intracellular reservoirs studied to date for Ca 2+ storage and mobilization are the acidocalcisomes, the endoplasmic reticulum and the mitochondria ( Lu et al., 1998 ; Maeda et al., 2012 ; Chiurillo et al., 2020 ).…”

Down the membrane hole: Ion channels in protozoan parasites

“…L-type nifedipine-sensitive Ca 2+ conductances have been described in T . cruzi [ 109 – 111 ], Leishmania [ 105 , 110 , 112 ], and T . gondii [ 4 ].…”