Fig 1.
Kinetoplast replication of T. cruzi amastigotes is asynchronous in vitro.
(a) Epimastigote at early stage of kDNA replication with TUNEL labelling of antipodal sites. (b) Epimastigotes at late stage of kDNA replication showing TUNEL labelling of entire kDNA disk. (c) MA104 cells infected with T. cruzi CL-Luc::Neon amastigotes for 72 hours then fixed and labelled with the TUNEL reagent. Left hand panel: cell containing 11 amastigotes with non-replicating kDNA (all TUNEL-ve); central panel: cell with parasites in which kDNA replication is asynchronous (mix of TUNEL+ve and TUNEL-ve); right hand panel: cell where all amastigotes are TUNEL+ve, but at different stages of kDNA replication (7 of 8 amastigotes display bright antipodal staining, the eighth is faintly TUNEL+ve, as shown by white arrows in the inset). (d) TUNEL data from 200 infected cells pooled from 3 replicate wells. The red line represents the number of infected cells assessed that contained the specified number of resident amastigotes. The black bars represent the percentage of amastigotes per cell that label as TUNEL+ve. Bar = 5 μm.
Fig 2.
Asynchronous replication of parasite mitochondrial DNA within single infected host cells in vivo revealed by TUNEL assays.
(a) Asynchronous replication of kDNA in intracellular parasites infecting mouse spleen cells during an acute stage infection (day 19). BALB/c mice were infected with T. cruzi CL-Luc::Neon and histological sections prepared from bioluminescent tissue (Experimental procedures). Parasites were detected by green fluorescence (mNeon), and the tissue sections subjected to TUNEL assays to highlight replicating kDNA (red). (b) Asynchronous replication of kDNA in an amastigote nest detected in the smooth muscle layer of stomach tissue during a chronic stage infection (day 117). Bar = 10 μm.
Fig 3.
Quantification of TUNEL in BALB/c mice during the acute stage of infection with T. cruzi CL-Luc::Neon. Tissue sections from mice sacrificed on day 19 post-infection were processed for imaging and subjected to TUNEL staining (Experimental procedures).
The graphs show the number of amastigotes that were TUNEL+ve (red) or TUNEL-ve (blue) in individual infected cells within the specified tissues. The x-axis refers to individual host cells. Bars containing both TUNEL-ve and +ve amastigotes were present in all tissues examined. Note that the level of TUNEL signal may vary between amastigotes within a given cell, so even bars that are red only may represent parasites at different stages of kDNA replication (c.f. differential levels of TUNEL staining in Fig 2A and 2B, DAPI/TUNEL panels).
Fig 4.
Asynchronous parasite DNA replication within single infected host cells in vivo revealed by EdU-labelling.
Replication of parasite DNA within mice infected by T. cruzi clone CL-Luc::Neon was assessed after inoculating EdU (for (a) and (b), one pulse 6 hours prior to tissue sampling; for (c), two pulses 18 and 28 hours prior to tissue sampling (Experimental procedures). Parasite location in histological sections was detected by green fluorescence (mNeon). (a) DNA replication (EdU, red) in a parasite nest during an acute stage infection (heart tissue, day 15 post-infection). In the DAPI stained image, the white arrow indicates parasite nest, and red arrow the host cell nucleus. The merged DAPI/EdU image, bottom left, illustrates the heterogeneity in the DNA replication status of parasites within the nest. (b) DNA replication in parasites within adipose tissue (day 15 post-infection). Red and white arrows in the DAPI image identify host and parasite DNA, respectively. Combined EdU and DAPI image shows replicating parasites interspersed with non-replicating parasites. (c) Section from GI tract of mouse, upper panel shows image at low magnification–note the presence of some EdU+ve mammalian cells within the mucosal layer due to epithelial cell replacement (indicated by white arrowheads). Lower panels show magnified view of parasite nest. EdU signal in magenta box is shown in higher magnification to the right; note a single amastigote with EdU labelling at antipodal sites of kDNA replication. All other parasites in this nest are negative. Bars = 10 μm.
Fig 5.
EdU labelling reveals that cells infected with small numbers of amastigotes have a lower percentage of actively replicating parasites in a chronic infection.
(a) Ex vivo imaging of organs. Bioluminescent foci were removed from the GI tract of three chronically infected C3H/HeN mice (day 211 post-infection) that had been injected with two pulses of EdU 18 and 28 hours prior to necropsy (Experimental procedures). (b) Each infected cell in the GI tract foci was imaged and the number of amastigotes that were positive or negative for EdU incorporation was quantified. The graphs show the total number of amastigotes in each cell (blue bars) and the number that were labelled with EdU (red bars). (c) Bioluminescent foci from the abdominal wall muscle were also dissected, stained for EdU and quantified as above. Asterisks above bars indicate cells were the number of parasites represents a minimum due to the infected nest being larger than the z-dimension of the section.
Fig 6.
Large nests are present in the chronic stage of infection (C3H/HeN mouse, day 211) and show asynchronous EdU incorporation throughout.
Images of the same nest taken from different sections through the tissue. The top row shows DAPI, EdU and mNeonGreen merged channels, whilst the lower row shows DAPI and EdU channels (for clarity). Bar = 10 μm. Note that sections are from the same infection focus but not all sections of this nest are included due to loss in processing.
Fig 7.
TUNEL assays indicate that amastigote replication and amastigote-to-trypomastigote differentiation can occur concurrently within single infected host cells in vitro.
(a) MA104 cells infected in vitro with T. cruzi. Two amastigotes (1 and 2) are visible within a cell full of trypomastigotes. The two lower right-hand panels show the two amastigotes at a higher magnification for clarity. (b) MA104 cells infected in vitro with T. cruzi. The cells were fixed 72 hours post-infection and subjected to a TUNEL assay. Two replicating amastigotes can be identified by antipodal TUNEL labelling on the kinetoplast, amongst a population of differentiated trypomastigotes. Bar = 10 μm.
Fig 8.
T. cruzi parasites display a wide range of morphologies during murine infections.
BALB/c mice were inoculated with parasites expressing a fluorescent/bioluminescent fusion protein and infected tissues identified by in vivo bioluminescence imaging (Experimental procedures). Fluorescent (green) flagellated “amastigote” forms detected in (a) adipose tissue (day 13 post-infection) (DNA stained red–appears yellow where mNeon fluorescence overlaps DNA), and (b) cardiac tissue (day 19 post-infection). (c) Parasite nests in the rectum (day 19 post-infection) containing a variety of morphological forms. Note that none of the flagellated forms displays the posterior rounded kinetoplast characteristic of trypomastigotes. Bar = 5 μm. (d and e) The flagellar length was measured in 100 amastigote-like cells from various tissue sites, where parasites were distinct enough to measure both flagellum and cell body. (d) Graph showing the flagellar length (μm) measured in each individual amastigote. (e) Graph showing the flagellar length (μm) plotted against parasite body length (μm).