Identification and Function of Apicoplast Glutaredoxins in Neospora caninum

Glutaredoxins (GRXs), important components of the intracellular thiol redox system, are involved in multiple cellular processes. In a previous study, we identified five GRXs in the apicomplexan parasite, Neospora caninum. In the present study, we confirmed that the GRXs S14 and C5 are located in the apicoplast, which suggests unique functions for these proteins. Although single-gene deficiency did not affect the growth of parasites, a double knockout (Δgrx S14Δgrx C5) significantly reduced their reproductive capacity. However, there were no significant changes in redox indices (GSH/GSSG ratio, reactive oxygen species and hydroxyl radical levels) in double-knockout parasites, indicating that grx S14 and grx C5 are not essential for maintaining the redox balance in parasite cells. Key amino acid mutations confirmed that the Cys²⁰³ of grx S14 and Cys^253/256 of grx C5 are important for parasite growth. Based on comparative proteomics, 79 proteins were significantly downregulated in double-knockout parasites, including proteins mainly involved in the electron transport chain, the tricarboxylic acid cycle and protein translation. Collectively, GRX S14 and GRX C5 coordinate the growth of parasites. However, considering their special localization, the unique functions of GRX S14 and GRX C5 need to be further studied.