Functional characterization of the orphan hypothetical protein TGGT1_245740, a transmembrane protein required for Toxoplasma gondii growth and virulence II

Numerous hypothetical proteins in Toxoplasma gondii (T. gondii) are predicted to participate in diverse biological processes. TGGT1_245740, characterized in this study, is annotated as a hypothetical protein lacking identifiable orthologs, conserved domains, and intrinsically disordered regions (IDRs), suggesting that it represents an orphan protein in T. gondii, a class that remains largely unexplored. In this study, the biological function of TGGT1_245740 was investigated through bioinformatic analysis, monoclonal antibody production, expression profiling, gene tagging, gene knockout, phenotypic assays, and transcriptomic analysis. TGGT1_245740 was found to be diffusely distributed in the inner membrane complex (IMC). The knockout of TGGT1_245740 gene could reduce the intracellular proliferation and virulence of the parasite. The host transcriptome suggested that infection with the knockout strain enhanced the host immune response, and the two sets of data together further revealed the potential role of the protein in regulating parasite metabolic homeostasis and host immune recognition. This study provides the first functional characterization of this orphan protein, highlighting its potential role in the physiological metabolism and host–parasite interaction of T. gondii.