Optogenetic dissection of cyclic nucleotide signaling in a model parasite Toxoplasma gondii

• Applicant: Dr. Nishith Gupta
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 275905048

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all warm-blooded organisms. Its fast-replicating tachyzoite stage causes a rapid tissue necrosis. Upon immune and physicochemical stress, tachyzoites differentiate into dormant, tissue-dwelling bradyzoites, which usually persist for the life of infected host. Cyclic nucleotides (cAMP, cGMP) are universal and essential regulators of stress signaling. The physiological role and mechanism of cyclic nucleotide signaling during the asexual development of T. gondii remain speculative, however. This has been primarily due to lack of a precise method to modulate cyclic nucleotide levels within the parasite without perturbing host cells. We have resolved this issue by adapting an optogenetic approach to photo-induce cAMP and cGMP pools in T. gondii. The work also indicated a defined necessity of parasite-derived cAMP for stage differentiation and lytic cycle. We will now establish knockdown of parasite cNMPs by expressing cAMP- and cGMP-specific photo-regulated phosphodiesterases. It offers several much-desired advantages compared to our former proof-of-principle study using light-activated cyclases, e.g. conditional repression of endogenous signaling in parasite cultures, and prevent unwarranted effects due to ATP/GTP depletion and overriding of natural cNMP level. Such optogenetic lines will be used to examine the importance of cNMP signaling for the asexual cycle of T. gondii, which will then be endorsed using the mutants of cAMP- and cGMP-dependent kinases. In brief, while advancing our current knowledge, proposed work pledges the groundwork for eventual mechanistic studies and pioneers the application of optogenetics in infection research.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Peter Hegemann