Down Regulation of NO Signaling in Trypanosoma cruzi upon Parasite-Extracellular Matrix Interaction: Changes in Protein Modification by Nitrosylation and Nitration

Abstract

BackgroundAdhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas' disease in humans, to components of the extracellular matrix (ECM) is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding •NO signaling.Methodology/Principal FindingsTrypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, •NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins) or by the biotin switch method (S-nitrosylated proteins) and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation.Conclusions/SignificanceFor the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of •NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of •NO signaling in the parasite, probably an essential move for the ensuing invasion step.

Redox Balance Keepers and Possible Cell Functions Managed by Redox Homeostasis in Trypanosoma cruzi

“…Alternatively, selfprotein oxidative modifications could be exploited by the parasite for signaling various pathways. Indeed, it was shown that in presence of extracellular matrix that constitute the cell surrounding layer crossed by the parasite for invasion of host cells, pathogen undergoes selective S-nitrosylation and tyrosine nitration (Pereira et al, 2015). These authors proposed that T. cruzi actively modulates its nitrosylation allowing its internalization into host cells.…”

Proteomics and Leishmaniasis: Potential Clinical Applications

“…Protein S‐nitrosylation involves the addition of a NO to cysteine thiols and regulates diverse cellular functions and signaling events including phosphorylation, acetylation, ubiquitylation and redox‐based cellular signaling . In T. cruzi , this PTM has been mapped by nLC‐MS/MS based detection of S‐nitrosylated peptides including enolase and histones after incubation of trypomastigotes with extracellular matrix . Currently, no MS‐based studies on Leishmania have mapped protein S‐nitrosylation of the parasites in different physiological and stress conditions.…”

Protein cysteine S-nitrosylation provides reducing power by enhancing lactate dehydrogenase activity in Trichomonas vaginalis under iron deficiency

“…nitrate and nitrite. Recent pro les of S-nitrosylated and nitrated proteomes in protists showed that glycolytic enzymes and ribosomal proteins are the general targets for SNO modi cation [18][19][20][21]. It has been reported that protein nitrosylation enhances the activity of glycolytic enzymes in mice [22].…”