Comparative proteomic analysis of Toxoplasma gondii Me49-derived strains adapted to in vitro treatments with thiosemicarbazones

Thiosemicarbazones and their metal complexes have been studied widely for their diverse biological activities, including against bacteria, cancer cells and protozoan parasites ^1,2. We have identified one gold (III) complex (C3) and its respective ligand (C4) that profoundly inhibit the proliferation of T. gondii tachyzoites in vitro (IC50 = 0.103 μM and 0.030 μM, respectively). Proteins potentially interacting with these two compounds were identified using differential affinity chromatography coupled with mass spectrometry (DAC-MS). Moreover, long-term in vitro treatment with C3 and C4 was also performed to investigate whether the compounds have parasitostatic or parasiticidal activity.

DAC-MS showed that among the most abundant T. gondii proteins binding both compounds were 18 ribosomal proteins, suggesting that they interfere in protein synthesis. The study also revealed that prolonged exposure to these compounds resulted in parasitostatic activity, and that the parasites adapted to the treatment within 5-6 days. Thus, we explored the underlying mechanisms of adaptation using comparative shotgun proteomics of 3 clones of adapted parasites for each compound comparatively to the respective wild-type (WT) Tg Me49.

Whole-cell shotgun analysis in C3 and C4 adapted clones identified a total of 3860 proteins, with only 31 proteins being differentially expressed. Of these, 23 were found in clones adapted to the gold compound C3, and 8 in clones adapted to compound C4. Clone P1E5 (adapted to C3) exhibited the highest number of differentially expressed proteins (19), followed by clone P2F3 (adapted to compound C4) (6). Notably, two proteins, a transporter encoded by TGME49_258700 and a putative transmembrane protein encoded by TGME4_248140, were upregulated in clones adapted to both C3 and C4. These results suggest that parasites become less susceptible to these drugs by potentially reducing the intake or increasing the excretion of the toxic compounds. The next step will be to calculate the IC₅₀ of the compounds on the single clones of adapted parasites, to confirm (or not) resistance.

Pelosi, G. Thiosemicarbazone Metal Complexes: From Structure to Activity. The Open Crystallography Journal vol. 3(2010) Beraldo H. & Gambino D. The Wide Pharmacological Versatility of Semicarbazones, Thiosemicarbazones and Their Metal Complexes. Mini-Reviews in Medicinal Chemistry 4,(2004)