Cloning, expression, and purification of an α-carbonic anhydrase from Toxoplasma gondii to unveil its kinetic parameters and anion inhibition profile.

Abstract

Toxoplasmosis, induced by the intracellular parasite Toxoplasma gondii, holds considerable implications for global health. While treatment options primarily focusing on folate pathway enzymes have notable limitations, current research endeavours concentrate on pinpointing specific metabolic pathways vital for parasite survival. Carbonic anhydrases (CAs, EC 4.2.1.1) have emerged as potential drug targets due to their role in fundamental reactions critical for various protozoan metabolic processes. Within T. gondii, the Carbonic Anhydrase-Related Protein (TgCA_RP) plays a pivotal role in rhoptry biogenesis. Notably, α-CA (TcCA) from another protozoan, Trypanosoma cruzi, exhibited considerable susceptibility to classical CA inhibitors (CAIs) such as anions, sulphonamides, thiols, and hydroxamates. Here, the recombinant DNA technology was employed to synthesise and clone the identified gene in the T. gondii genome, which encodes an α-CA protein (Tg_CA), with the purpose of heterologously overexpressing its corresponding protein. Tg_CA kinetic constants were determined, and its inhibition patterns explored with inorganic metal-complexing compounds, which are relevant for rational compound design. The significance of this study lies in the potential development of innovative therapeutic strategies that disrupt the vital metabolic pathways crucial for T. gondii survival and virulence. This research may lead to the development of targeted treatments, offering new approaches to manage toxoplasmosis.

Keywords:

• carbonic anhydrase
• anion inhibitors
• Toxoplasmosis
• enzyme kinetics

Acknowledgements

We are grateful to Chiara Nobile and Marco Petruzziello for technical assistance.

Authors' contributions

Conceptualisation, C.C. and C.S.; methodology, V. De L., S.G., C.C.;; investigation, C.C., V. De L, S.G.; data curation, C.C., V. De L. and S. G.; writing—original draft preparation, C.C.; writing—review and editing, C.C.; C.S.; supervision, C.C. and C.S. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

CT Supuran is Editor-in-Chief and Clemente Capasso is an Associate Editor of the Journal of Enzyme Inhibition and Medicinal Chemistry. They were not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Data availability statement

We will provide access to the data upon readers' request.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.