Methods for the Investigation of Trypanosoma cruzi Amastigote Proliferation in Mammalian Host Cells.

In its mammalian host, the kinetoplastid protozoan parasite, Trypanosoma cruzi, is obliged to establish intracellular residence in order to replicate. This parasite can infect and replicate within a diverse array of cell and tissue types across many mammalian host species. The establishment of quantitative assays to assess the replicative capacity of intracellular T. cruzi amastigotes under different conditions is a critical facet to understanding this host-pathogen interaction. Several complementary methods are outlined here. Their strengths and deficiencies in quantifying intracellular amastigote growth and death are discussed. We describe three assays to assess growth/replication. (1) A high throughput multiplexed plate-based assay that quantifies both host cell and parasite abundance. This method allows for the rapid and simultaneous screening of many conditions (e.g., small molecule inhibitors, the impact of host gene knockdown or of altered environmental parameters). (2) Simple fluorescence microscopy-based enumeration of amastigotes within host cells and (3) flow cytometry-based quantification of amastigote proliferation following isolation from host cells. Each approach has advantages but none of these can assess lethal outcomes in a quantitative manner. For this, we describe a clonal outgrowth assay that identifies the proportion of parasites that succumb to a defined exposure. Even using these assays, it can be challenging to differentiate between direct (targeting the parasite) and/or indirect (targeting the host) effects of a given treatment on amastigote growth. Therefore, we also outline a method of purification of intracellular amastigotes that allows for downstream biochemical and metabolic investigations specifically on the isolated amastigote.