This has not, as yet, been demonstrated for the metacyclic trypomastigote which initiates natural mammalian infection. post contamination) C3H/HeN mice revealed by EdU-labelling. Replication of parasite DNA within mice infected by clone CL-Luc::Neon (Costa et al., 2018) was assessed after inoculating two EdU pulses 18 CCND3 and 28 hours prior to tissue sampling (Experimental procedures). Parasites were located in histological sections by fluorescence (mNeon, green). a) DNA replication (red) in a CPPHA chronic phase parasite nest (colon). The combined DAPI/EdU image illustrates the heterogeneity of parasite replication within the nest. Bar = 10 m. b) Section from colon of mouse showing parasite nest. Upper panels show individual channels and a merged image. The lower panel shows DAPI and EdU channels only, allowing visualisation of the interspersed nature of EdU+ve amongst EdU-ve parasites. (a) and (b) are from different mice. CPPHA Bars indicate 10 m.(PPTX) pntd.0008007.s004.pptx (2.9M) GUID:?A54B8F60-675D-47B0-920E-793CED9B60D0 S5 Fig: Multiple morphological forms within single infected cells. Each image shows an MA104 cell (blue, nucleus) 6 days after contamination with (green) showing amastigotes (arrow a) dividing amastigotes (arrow da), epimastigote-like forms (arrow e) and trypomastigotes (arrow t) within the same cell. (a-d) sequential still images from S1 Movie, (e-h) sequential still images from S2 Movie. Bars indicate 20 m.(TIF) pntd.0008007.s005.tif (7.5M) GUID:?5F484C75-2E80-4837-83E4-F3D287A804B6 S1 Movie: Multiple morphological forms within a single infected cell. Live cell imaging of an CPPHA MA104 cell 6 days after contamination with showing dividing amastigotes, epimastigote-like forms and trypomastigotes within the same cell. See S5ACS5D Fig for locations of representative parasites for each morphotype.(MP4) pntd.0008007.s006.mp4 (2.3M) GUID:?9FD15752-7887-4F00-949D-BA7520B2F619 S2 Movie: Another example of multiple morphological forms within a single infected cell. Live cell imaging of an MA104 cell 6 days after contamination with showing amastigotes, epimastigote-like forms and trypomastigotes within the same cell. See S5ECS5H Fig for locations of representative CPPHA parasites for each morphotype.(MP4) pntd.0008007.s007.mp4 (1.0M) GUID:?257180F6-2009-4A88-8FFF-122D85363426 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract Investigations into intracellular replication and differentiation of within 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 altered parasites that express a bioluminescent/fluorescent fusion protein. By combining imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies uncover 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 populace, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence CPPHA of distinct non-canonical morphological forms of in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of is usually 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 summary Chagas disease, caused by the protozoan parasite Tcarrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replication during both acute and chronic.