Monday, 20 September 2021

CyTRAK Orange - uncovering a drug MoA

The DNA and cytoplasmic dye CyTRAK Orange™ has aided comprehensive studies to elucidate the mechanism of action of a candidate therapeutic for cardiac complications of Chagas' Disease. Tc24-C4 is a modified recombinant protein vaccine derived from a calcium binding protein located in the flagellum of Trypanosoma cruzi.

Interestingly it was clear from the data that the w.t. Tc24 protein is expressed differentially in the different host stages of the parasite's life cycle and that it is expressed in the inner side of the flaggelar membrane.  This explains why Tc24-C4 does not offer protection against infection but operates as a vaccine to generate T cell mediated cell (CTL) killing of infected host cells and thereby ameliorate the effects of infection.

In one experiment cells were infected with T. cruzi and the progression from the infectious extracellular flagellate trypomastigote stage through to the intracellular, fully non-flagellate amastigote stage.  The host cells and parasites were stained with anti-Tc24 to detect stage-specific protein expression, Hoechst to label the nuclei, CyTRAK Orange to label the DNA of parasites and cells and DNA and cytoplasm of the host cells.  From this combination of three reagents it was possible to visualise the progress of the infection, from trypomastigote to amastigote and the latter's multiple divisions alongside the progressive loss of expression of Tc24.

Short of including its ability to cross the plasma membrane this application of CyTRAK Orange demonstrates its abilities to label DNA (here of host and parasite) and cytoplasmic compartment while being spectrally compatible with Hoechst and Alexa Fluor/GFP/FITC (and red-excited fluors - not used here).

Reference:

Versteeg L, Adhikari R, Poveda C, Villar-Mondragon MJ, Jones KM, Hotez PJ, et al. (2021) Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi. PLoS Negl Trop Dis 15(9): e0009689.

Read a recent review on CYTRAK Orange.