Thioredoxin reductase fromToxoplasma gondii: an essential virulence effector with antioxidant function

Abstract

Thioredoxin reductase (TR) can help pathogens resist oxidative-burst injury from host immune cells by maintaining a thioredoxin-reduction state during NADPH consumption. TR is a necessary virulence factor that enables the persistent infection of some parasites. We performed bioinformatics analyses and biochemical assays to characterize the activity, subcellular localization, and genetic ablation of Toxoplasma gondii TR (TgTR), to shed light on its biologic function. We expressed the TgTR protein with an Escherichia coli expression system and analyzed its enzyme activity, reporting a K m for the recombinant TgTR of 11.47-15.57 mM, using NADPH as a substrate, and 130.48-151.09 mM with dithio-bis-nitrobenzoic acid as a substrate. The TgTR sequence shared homology with that of TR, but lacked a selenocysteine residue in the C-terminal region and was thought to contain 2 flavin adenine dinucleotide (FAD) domains and 1 NADPH domain. In addition, immunoelectron microscopy results showed that TgTR was widely dispersed in the cytoplasm, and we observed that parasite antioxidant capacity, invasion efficiency, and proliferation were decreased in TR-knockout (TR-KO) strains in vitro, although this strain still stimulated the release of reactive oxygen species release in mouse macrophages while being more sensitive to H 2 O 2 toxicity in vitro. Furthermore, our in vivo results revealed that the survival time of mice infected with the TR-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. These results suggest that TgTR plays an important role in resistance to oxidative damage and can be considered a virulence factor associated with T. gondii

The Nrf2 pathway is required for intracellular replication of Toxoplasma gondii in activated macrophages

“…Under oxidative stress conditions, manipulation of host cells to reduce intracellular ROS levels is an effective strategy for parasite survival. Although we did not provide evidence that parasites drive regulation of ROS in host cells, other reports suggest that reducing ROS levels is required for parasite proliferation . Therefore, it will be interesting to determine whether parasite‐derived factors are involved in p62‐mediated regulation of ROS.…”

“…Growing evidence suggests that accumulation of intracellular ROS activates many effector molecules or adaptor proteins in host cells infected with T gondii to eliminate the intracellular parasites . More recent studies provide direct evidence that ROS is toxic to T gondii . Antioxidative genes expressed by T gondii are important for overcoming oxidative stress; this also requires down‐regulation of ROS‐producing enzymes in host cells.…”

“…[30][31][32] More recent studies provide direct evidence that ROS is toxic to T gondii. 10,13 Antioxidative genes expressed by T gondii are important for overcoming oxidative stress; this also requires downregulation of ROS-producing enzymes in host cells. However, it is still not known whether the cytoprotective pathway contributes to parasite survival.…”

“…Although we did not provide evidence that parasites drive regulation of ROS in host cells, other reports suggest that reducing ROS levels is required for parasite proliferation. 13 Therefore, it will be interesting to determine whether parasite-derived factors are involved in p62-mediated regulation of ROS. However, it is still necessary to determine whether the p62regulated Nrf pathway is required for parasite infection in vivo considering the role of ROS as a secondary messenger during innate immune responses.…”

“…Higher levels of ROS increase inherent cellular oxidative stress, thereby contributing to resistance against invading T gondii . To overcome the effects of oxidative stress, T gondii has developed its own antioxidant machinery, which neutralizes excess ROS . A previous report suggests that regulating ROS‐producing pathways in T gondii ‐ infected cells is required for intracellular redox homeostasis .…”