Signaling and adaptive mechanisms in trypanosomatids: a possible Achilles heel in the fight against trypanosomiasis

SALOMÉ CATALINA VILCHEZ LARREA; ALEJANDRA CECILIA SCHOIJET; ALEJO FACUNDO PREGO; GUILLERMO DANIEL ALONSO

Medellín

Simposio; IX Simposio de Biología Molecular de la Enfermedad de Chagas; 2022

Trypanosoma cruzi is the etiological agent of Chagas disease or American trypanosomiasis, a neglected tropical condition historically endemic to Latin America that, due to human migration, has spread beyond its traditional boundaries. It is now estimated that 6 to 8 million people suffer from this disease worldwide. T. cruzi is a protozoan parasite that presents a complex life cycle, during which it must cope with sudden changes in the environment to which it must respond immediately to survive. Cyclic nucleotide phosphodiesterases have been implicated in the proliferation, differentiation and osmotic regulation of trypanosomatids. In some trypanosomatid species they have been validated as molecular targets for the development of new therapeuticagents. It has also been shown that many inhibitors of mammalian PDEs lack an inhibitory effect on trypanosomatid enzymes, indicating that there are differences in the substrate specificity of parasite and host enzymes, foreshadowing the possibility of identifying compounds that selectively inhibit the parasite enzyme. The limited possibilities to edit T. cruzi genome have made it difficult to carry out knockout and knockdown experiments, and drug target validation has been limited to biochemical evaluations. Recently, the development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to the study of Trypanosoma cruzi. According to the most often applied protocols, epimastigotes are co-transfected with a single plasmid bearing both the gene for Cas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointly with a lineal donor DNA encompassing a selection marker flanked by sequences homologous to thetarget gene. Here, we tested an alternative approach for the generation of Phosphodiesterase (PDE) knockout parasites.We obtained epimastigotes from Tul II strain stably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects on epimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to the intended gene target. sgRNA were obtained by in vitro transcription using a template DNA bearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA we designed a pre-donor formed by a sequence including several restriction enzyme recognition sites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This pre-donor allowed to easily generate a variety of donor DNAsby cloning alternative selection markers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated by PCR using mixed primer pairs: while one of the primers annealed to the target gene, the second primer annealed to a sequence in the donor DNA, allowing assessment of its correct insertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include its versatility for choosing and switching between alternative selection markers and a quick and affordable generation of the components of the system and analysis of the transfected cultures, while possibly facilitating complementation assays on the KO lines. Finally, our results indicate that loss of PDEs (even partial) is incompatible with normal parasite function/parasite viability.