Old Yellow Enzyme from Trypanosoma cruzi Exhibits In Vivo Prostaglandin F2α Synthase Activity and Has a Key Role in Parasite Infection and Drug Susceptibility

Abstract

The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F₂α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F_2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF₂α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host-parasite interaction.

Keywords: Old Yellow Enzyme; Trypanosoma cruzi; benznidazol and nifurtimox activation; differentially expressed gene; prostaglandin F2α synthase.

Figure 1

Evolutionary relationship of Old Yellow Enzyme proteins. The structure-based sequence alignment was performed using the accurate mode of T-Coffee software. The tree was built with the Maximum Likelihood method with WAG as best-fit model. Species name, protein ID, and KEGG/NCBI accession numbers of these sequences are shown in Table S1 in Supplementary Material.

Figure 2

TcOYE presents citosolic localization in epimastigotes. Immunolocalization of TcOYE, TccTXNPx, TcCZP, and TcmTXNPx in epimastigotes using rabbit α-TcOYE (1/3,000) and mouse α-TccTXNPx (1/100), α-TcCZP (1/50), and α-TcmTXNPx (1/100) polyclonal antibodies. Bar: 5 µm. DAPI was used as nucleus and kinetoplast marker.

Figure 3

TcOYE is expressed in replicative stages of Trypanosoma cruzi lifecycle. (A) TcOYE expression during the parasite lifecycle was evaluated by western blot with total protein extracts from different parasite stages and Vero cells (since trypomastigotes and amastigotes are derived from infected Vero cells, they were used as a cross-reactivity control), using rabbit TcOYE antiserum. Relative expression was estimated by densitometry normalized by tubulin expression. V, Vero cells, T, trypomastigotes, A, intracellular amastigotes, E, epimastigotes. (B) Kinetics of TcOYE expression during mammalian cells infective cycle. Parasites were followed by inmunocitolocalization with rabbit α-TcOYE polyclonal antibodies. Bar 10 µm (except 1–9 h 5 µm). (C) Confocal microphotographs showing TcOYE and TccTXNPx immunolocalization in different stages of Vero cells infection. Bar 10 µm.

Figure 4

TcOYE overexpressing parasites presents higher levels of PGF_2α than control parasites. (A) Western blot analysis of total protein extracts from epimastigotes transfected with pTREX-n (empty vector) and pTREX-n TcOYE (includes complete TcOYE coding sequence) using rabbit α-TcOYE polyclonal antibodies. The relative expression was estimated by image densitometry analysis normalized by tubulin expression. TcOYE overexpression in epimastigotes was 3.4 times more than controls. (B) Trypomastigotes and extracellular amastigotes overexpression analyzed by IIF using rabbit α-TcOYE polyclonal antibodies. (C) Growth curves of TcOYE overexpressing parasites (pTREX-n TcOYE) vs. empty vector containing parasites (pTREX-n). (D,E) Determination of PGF_2α in epimastigotes and trypomastigotes. These results correspond to concentration values of PGF_2α from two replicates. Asterisks represent statistical significance (two tailed Student’s t-test).

Figure 5

TcOYE overexpressing parasites present differential viability to benznidazole (Bzn), nifurtimox (Nfx), or hydrogen peroxide (H₂O₂). (A) Viability percentages of transfected parasites challenged with different concentrations of Bzn. (B) Viability percentages of transfected parasites challenged with different concentrations of Nfx. (C) Viability percentages of transfected parasites challenged with different concentrations of hydrogen peroxide. The percentages of cell viability are normalized against parasites without treatment. Values are the means of three independent assays performed in quadruplicate. Asterisks represent statistical significance (two tailed Student’s t-test).

Figure 6

TcOYE overexpression modulates parasite infective cycle. (A) Trypanosoma cruzi invasion capacity was not affected by TcOYE overexpression. HeLa cells were incubated for 4 h with trypomastigotes and then the number of infected cells vs. total cells was evaluated. At least 200 cells per replicate were evaluated. No statistically significant differences were observed between the means of three independent assays performed in triplicates. (B) Intracellular amastigotes replication was not affected by TcOYE overexpression. HeLa cells were incubated for 4 h with trypomastigotes and the number of internal parasites was counted 48 h post infection in at least 100 cells. No statistically significant differences were observed between the means of three independent assays performed in triplicates. (C,D) TcOYE overexpression reduces the capacity of parasites to complete the infective cycle in different cell types. Number of trypomastigotes per milllilter of supernatant of HeLa cell or J774 macrophages infected with pTREX-n control parasites or pTREX-n TcOYE transfected parasites. Asterisks represent statistical significance (two tailed Student’s t-test).

Figure 7

TcOYE overexpression increased cardiac tissue parasite load in infected mice. (A) Parasitemia progression in BALB/c mice infected with TcOYE overexpressing and control parasites. Parasitemia was measured every 3 days by microscopic examination of thin tail-blood smears. (B) Parasite load in cardiac tissue of mice infected with transfected Trypanosoma cruzi euthanized at the 30^th dpi. N = 2–5. Asterisks represent statistical significance (two tailed Student’s t-test).