Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness

Zhang, Zhi-Wei; Wang, Meng; Sun, Li-Xiu; Elsheikha, Hany M.; Lei, Cheng-Lin; Wang, Jin-Lei; Fu, Bao-Quan; Luo, Jian-Xun; Zhu, Xing-Quan; Li, Ting-Ting

Abstract

Background To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. Methods We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. Results Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. Conclusions Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Citation

Zhang, Z., Wang, M., Sun, L., Elsheikha, H. M., Lei, C., Wang, J., …Li, T. (2024). Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness. Parasites and Vectors, 17(1), Article 178. https://doi.org/10.1186/s13071-024-06259-9

• Journal Article Type: Article
• Acceptance Date: Mar 21, 2024
• Online Publication Date: Apr 4, 2024
• Publication Date: Apr 4, 2024
• Deposit Date: Apr 4, 2024
• Publicly Available Date: Apr 5, 2024
• Journal: Parasites and Vectors
• Electronic ISSN: 1756-3305
• Publisher: Springer Nature
• Peer Reviewed: Peer Reviewed
• Volume: 17
• Issue: 1
• Article Number: 178
• DOI: https://doi.org/10.1186/s13071-024-06259-9
• Keywords: Toxoplasma gondii; Thioredoxin; Parasitophorous vacuole; Virulence; TurboID
• Public URL: https://nottingham-repository.worktribe.com/output/33291077
• Publisher URL: https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-024-06259-9
• Additional Information: Received: 26 February 2024; Accepted: 21 March 2024; First Online: 4 April 2024; : ; : All animal experimentation was reviewed and approved by the Animals Research Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. 2020-009).; : Not applicable.; : The authors declare that they have no competing interests. The corresponding author Prof. Xing-Quan Zhu serves as the Subject Editor for the section “Parasite genetics, genomics and proteomics” of Parasites & Vectors.