Figure 6.

TcHMGB levels affect cell infection, amastigote replication and metacyclogenesis. The infection performance of T. cruzi Dm28c/pTcINDEX-GW-TcHMGB(HA)₂ was analyzed in the absence (Tet−) or presence (Tet+) of 0.5 μg/ml tetracycline. To test the effect over the different phases of infection, we designed different induction protocols as follows: (−/−), Tet was never added to the medium, non-induced control; (+/−), trypomastigotes were pre-treated with Tet for 2 hours prior to infection, and during the invasion incubation period but not after; (−/+), trypomastigotes were not induced, Tet was only added after 48 hours post-infection to see the effect on the amastigote stage; (+/+), trypomastigotes were pre-treated and Tet was present during the whole infection assay, to see the overexpression effect both in trypomastigotes and amastigotes. The percentage of infected cells (A), the number of amastigotes per cell (B) and the number of trypomastigotes released 6 days post-infection (C) were determined by counting Giemsa-stained slides using an optical microscope. Results are expressed as mean ± SD of triplicates. Statistical analysis of the data was carried out using one-way ANOVA, *p < 0.05, **p < 0.001 and ***p < 0.0005. (D) In vitro metacyclogenesis using TAU medium of the pTcINDEX-GW-TcHMGB(HA)₂ parasites non-induced (−) or induced (+) with 0.5 µg/ml Tet for 96 h. The bar graph represents the mean ± SD from three independent experiments; ***p < 0.0005 (Student t test).