In vivo analysis of Trypanosoma cruzi persistence foci at single cell resolution

Background:

Trypanosoma cruzi is a Eukaryotic parasite which causes American Trypanosomiasis (Chagas Disease), a neglected tropical infection endemic to Latin America. T. cruzi parasites are transmitted via the faeces of triatomine insects and, once in a host, they invade and live inside host cells. With no vaccine, Chagas disease is currently treated using drugs. Initially, symptoms of the infection are non-specific (e.g fever) however, long term (a chronic infection), this infection can seriously damage major organs including digestive organs and the heart resulting in severe morbidity or death (1). How T. cruzi parasites live in the host and avoid removal by the immune system is unclear, in part because it is difficult to find the exact locations of infected cells. The low number of parasites present in a chronic infection also suggests T. cruzi hides itself from immune detection leading to parasite reservoirs. Mouse models can be used to study Chagas disease, with bioluminescent parasites revealing parasite persistence in the digestive tract, and intermittent dissemination to the heart and other organs (2). To improve parasite detection and to identify individual host cells infected with T. cruzi, Ward and colleagues used a parasite cell line that expressed a fusion protein that was both bioluminescent and fluorescent (T. cruzi CL-Luc::Neon; 3). They show potential parasite reservoirs in colon circular muscle tissue, skeletal muscle and the skin.

 

Key Findings:

1. T. cruzi parasites are detected at high resolution using confocal laser scanning microscopy in mice

In a chronic infection, T. cruzi parasites can be found within the digestive tract however, we don’t know much about where and what cells become infected by T. cruzi at this site. By using a dual bioluminescent-fluorescent reporter cell line (T. cruzi CL-LUC::Neon; 3) and modifying mouse dissection techniques, Ward and colleagues use confocal laser scanning microscopy to track parasite bioluminescent and fluorescent signals generating a 3D picture of the mouse colon. By examining stacks of images, they were able to detect individual host cells infected with low numbers of parasites (<20). Typically, they found ~200-700 parasites in total persisting within the wall of the colon of each mouse.

 

2. Chronic infection is associated with the presence of T. cruzi ‘mega nests’

From their analysis of the colon wall, Ward and colleagues noticed some infected host cells contained large numbers of parasites (‘mega nests’). By looking at infected cell number and parasite burden in gut smooth muscle cells under acute and chronic infection conditions they show these ‘mega nests’ occur during chronic conditions, housing over 200 to upwards of 1000 parasites per ‘nest’. Interestingly, these parasites did not appear to be amastigotes rather than morphologically fully developed trypomastigote forms (the flagellated state of the trypanosome). Histological sectioning of the colon tissue under chronic conditions and using antibodies to determine the infected host cell type revealed that infected cells were mostly within the circular muscle layer, specifically smooth gut muscle myocytes likely acting as parasite reservoirs.

 

3. T. cruzi parasites are found in the skeletal muscle of chronically infected mice

Using the same approach as for the digestive tract, Ward and colleagues next chronically infected two different strains of mice (BALB/c and C3H/HeN) and examined for the localisation of T. cruzi CL-Luc::Neon parasites to ask if parasites could be found in skeletal muscle and adipose tissue. When parasite burden dynamics and infection foci were compared across the two different mouse strains, T. cruzi dissemination and burden were found to be greater in C3H/HeN than in BALB/c mice (as previously reported; 4), attributed by the authors to differences in the immune responses of these mice to the infection. Thus, in BALB/c mice, little bioluminescence signal in skeletal muscle and adipose tissue was observed (above background signal) however, in C3H/HeN mice, ‘mega nests’ of parasites could be detected within the skeletal muscle (specifically the muscle fibres) implicating these regions as sites of infection reservoir under chronic disease conditions.

 

4. The skin may act as an important reservoir of transmissible cruzi parasites

T. cruzi parasites have been seen in the skin of infected mice suggesting this location may host parasites suitable for transmission. To examine parasite dynamics in the skin, C3H/HeN and BALB/c mice were infected with parasites from one of two different T. cruzi strains (both bioluminescent and fluorescent) from two different genetic lineages (TcI and TcVI). The majority of the skin was dissected off the mouse intact, the adipose tissue underneath removed, and bioluminescent signal examined. Most of the mice (80-90%) had a focus of bioluminescent signal (parasites) in their skin. However, by looking for fluorescent parasites in thin skin sections, only one infected cell was seen in the epidermis of the skin (due to the technically challenging nature of this sectioning technique). However, given the discovery of parasites in this location, the authors propose these parasites might be more available for uptake by the triatomine insect.

 

What I liked about this preprint:

Addressing the chronic stage of Chagas disease is a clear barrier to the development of effective treatment for this serious infection. Here, Ward and colleagues took their previous approach and improved their technique to develop a higher resolution model to study chronic Chagas disease. I like their dual approach of locating sites of T. cruzi parasites, it is a clever idea to examine the overall ‘where’ then ask for more detailed information giving both a broad understanding of the infection dynamic and precise information on the types of cells which may act as parasite reservoirs. This approach could be used to investigate more targeted therapeutic agents perhaps allowing drug delivery to parasite reservoir cells.

 

Questions for the Authors:

1. Under chronic conditions, parasites found in ‘mega nests’ do not appear to be fully developed trypomastigotes. Could this be due to nutrient restriction/competition within the ‘mega nests’? Or is there another factor which could be restricting development of the cells?
2. Why do you think cells containing ‘mega nests’ of parasites are unable to be stained using the antibodies you used to determine infected cell type?
3. What are the immunological differences between the two strains of mice which could explain the higher dissemination rates in C3H/HeN mice vs BALB/c mice?
4. Your experiment to examine parasite burden in the skin uses two genetic lineages of cruzi (TcI and TcVI) showing very little difference in their behaviour in this context – would you expect the other genetic lineages to behave the same?

 

References:

1. https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis)
2. Lewis MD, Kelly JM. 2016. Putting Trypanosoma cruzi dynamics at the heart of Chagas disease. Trends Parasitol 32:899-911.
3. Costa FC, Francisco AF, Jayawardhana S, Calderano SG, Lewis MD, Olmo F, Beneke T, Gluenz E, Sunter J, Dean S, Kelly JM, Taylor MC. 2018. Expanding thetoolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescencefluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. PLoS Negl Trop Dis 12:e0006388.
4. Lewis MD, Fortes Francisco A, Taylor MC, Jayawardhana S, Kelly JM. 2016. Host and parasite genetics shape a link between Trypanosoma cruzi infection dynamics and chronic cardiomyopathy. Cell Microbiol 18:1429-1443.

 

Tags: 3d microscopy, bioluminescent, chagas disease, fluorescent, trypanosoma cruzi

Posted on: 3 June 2020

doi: https://doi.org/10.1242/prelights.21314

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