Reduced levels of inositol hexakisphosphate kinase (IP6K) impair life-cycle transitions and the intracellular development of Trypanosoma cruzi within human cardiomyocytes
Trypanosoma cruzi is a protozoan parasite etiological agent of the vector-borne disease American trypanosomiasis, also known as Chagas disease. During its life cycle, T. cruzi undergoes some fundamental processes: metacyclogenesis (transition from epimastigote to metacyclic trypomastigote form) – occurring in the invertebrate host, amastigogenesis (transition from trypomastigote to amastigote form) and trypomastigogenesis (transition from amastigote to trypomastigote form) – both occurring within nucleated cells of the mammalian host, and finally epimastigogenesis (transition from trypomastigote to epimastigote forms) – which occurs as soon as the invertebrate host ingests trypomastigote forms during the bloodmeal. The precise mechanisms that regulate these processes remain elusive but certainly depend on kinases. The canonical function of the inositol hexakisphosphate kinases (IP6Ks) is to phosphorylate the phosphate groups from inositol hexaphosphate (IP6), leading to the formation of inositol pyrophosphates (PP-IPs). However, recent studies have been describing non-canonical functions for this kinase family. Here, after disrupting a single IP6K allele of T. cruzi and confirming its downregulation, we observed that the life cycle of this parasite was profoundly impaired. Epimastigote forms of T. cruzi IP6K-deficient showed morphological alterations, increased population presenting a dormant-like state (quiescence), and a reduced differentiation capacity during metacyclogenesis. The restricted metacyclic forms of T. cruzi IP6K-deficient that were able to differentiate had reduced infective potential (invasion rate) in human cardiomyocytes. Interestingly, 120 h after infection, the amastigote forms of T. cruzi IP6K-deficient showed an impaired ability to transform into trypomastigotes, with most of the population egressing from human cardiomyocytes without completing trypomastigogenesis. Transcriptomic data show that the levels of surface proteins (mucins and trans-sialidases) were altered in both epimastigote and trypomastigote forms in response to the low levels of IP6K, which helps explain our findings. Finally, we observed that the few trypomastigote forms of T. cruzi IP6K-deficient that egressed from human cardiomyocytes could not complete the epimastigogenesis. Together, our findings strongly suggest that IP6K is critical to sustain the T. cruzi life cycle. Thus, as the disruption of both IP6K alleles did not generate viable parasites and the similarity relative to its human homolog is ~15%, this kinase could be a potential target for drug development against Chagas disease. Overall design: RNA-seq profiling of control T. cruzi (CL Brener) lineage (parental T7/Cas9) and its IP6K single-knockout lineage (using epimastigotes and trypomastigotes forms).