In Vivo Analysis of Trypanosoma cruzi Persistence Foci at Single-Cell Resolution

Abstract

Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we showed that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells each containing >200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development. IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

IL-10-Dependent and -Independent Mechanisms Are Involved in the Cardiac Pathology Modulation Mediated by Fenofibrate in an Experimental Model of Chagas Heart Disease

“…Infection with Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas disease, triggers both innate (1)(2)(3), and adaptive (4)(5)(6) immune responses that aim at the control of the parasite load both in tissues and peripheral blood [Reviewed in (7)]. However, these mechanisms do not succeed in the complete eradication of the parasite, which results in parasite persistence (8)(9)(10)(11)(12).…”

Trypanosoma cruziamastigotes have a reduced replication rate during chronic stage infections

“…(B) Percentage of parasites that were EdU+ve under each treatment regimen. Colonic tissue was extracted from mice post-mortem , 18 hours after the second injection (1-day treatment) or 4 hours after the final injection (3.5-days treatment), and infected cells detected by ex vivo imaging and confocal microscopy (Materials and methods) (Figure 3 – figure supplement 1) (Ward et al . 2020).…”

“…2014; Costa et al . 2018; Ward et al . 2020), we demonstrated that the gastrointestinal tract, specifically the colon and stomach, is a key site of T. cruzi persistence during chronic murine infections.…”

“…Persistent parasites are also frequently detected in the skin during chronic infections, and in C3H/CeN mice, the skeletal muscle (Lewis et al . 2016; Ward et al . 2020).…”

“…We have developed tissue processing protocols and imaging procedures that allow us to routinely detect T. cruzi persistence foci during chronic murine infections at single cell resolution (Costa et al . 2018; Ward et al . 2020).…”