The SV40 T-ag nuclear localization signal affects Toxoplasma gondii viability by targeting importin α

Nucleocytoplasmic transport is essential in eukaryotes and mediated by importin (Imp) /{beta} receptors that recognize nuclear localization signals (NLSs) on cargo proteins. The SV40 large T-antigen NLS (SV40-NLS), studied extensively, binds importin with high affinity in the nanomolar range, while modified versions, the Bimax peptides, bind in the picomolar range. Bimax peptides impede nuclear import and reduce viability in yeast and human cells, highlighting the potential of NLS peptides as inhibitors of nuclear transport. In this study, we investigated the potential of the SV40-NLS to target Toxoplasma gondii importin (TgImp). Expression of the SV40-NLS fused to a GFP reporter led to cytotoxicity in T. gondii tachyzoites; this depended on the SV40-NLS sequence and its position within the protein. Over-expression of TgImp rescued parasites from the SV40-NLS-induced cytotoxicity, confirming that the mechanism of action involves disruption of nuclear import. Importantly, the same construct did not affect cell viability in mammalian cells, suggesting a selective vulnerability in importin -mediated nuclear transport in the parasite. The SV40-NLS peptide offers an advantage over small-molecule inhibitors by targeting large interaction surfaces of importin with high specificity, minimizing off-target effects. This study lays the groundwork for a novel peptide-based therapeutic strategy employing NLS motifs to selectively inhibit nuclear import in T. gondii.