Intracellular DNA replication and differentiation ofTrypanosoma cruziis asynchronous within individual host cellsin vivoat all stages of infection

Abstract

ABSTRACTInvestigations into intracellular replication and differentiation ofTrypanosoma cruziwithin the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combiningin vivoimaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms ofT. cruziin the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle ofT. cruzi in vivois more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.AUTHOR SUMMARYChagas disease, caused by the protozoan parasite Trypanosoma cruzi, is becoming an emerging threat in non-endemic countries and establishing new foci in endemic countries. The treatment available has not changed significantly in over 40 years. Therefore, there is an urgent need for a greater understanding of parasite biology and disease pathogenesis to identify new therapeutic targets and to maximise the efficient use of existing drugs. We have used genetically modified strains ofT. cruzicarrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replicationin vivoduring both acute and chronic infections in a mouse model. Utilising TUNEL assays for mitochondrial DNA replication and EdU incorporation for total DNA replication, we have found that parasite division within infected cells is asynchronous in all phases of infection. Differentiation also appears to be uncoordinated, with replicating amastigotes co-existing with non-dividing trypomastigotes in the same host cell.