Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions

Abstract

We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.

Figure 1

SeCreEt parasites. (a) Schematic of the toxofilin-Cre fusion construct. Arrow indicates the transcription start. (b) Phase contrast and fluorescence images of SeCreEt parasites stained as indicated. Scale bar, 5 μm. (c) Western Blot of lysate from parental strain (RH) or SeCreEt strain probed anti-HA antibody. SAG-1 serves as the loading control.

Figure 2

SeCreEt parasites activate eGFP in reporter cell lines. (a) Schematic of the retroviral construct used to produce the Cre-reporter cell line. The DsRed and eGFP coding regions are driven by a promoter in the long terminal repeat (LTR) but have three stop codons separating them, thus preventing expression of eGFP. Arrow indicates transcription start. Blue triangles indicated loxP sites and their orientation. SD is splice donor, SA is splice acceptor, and Psi (ψ) is a retroviral packaging signal. (b,c) Cre-reporter cells 24 hours after infection with parental strain (b) or SeCreEt parasites (c). Panels from left to right: phase, red shows both DsRed expression and staining with Toxoplasma-specific anti-SAG1 antibody, green shows eGFP expression, merge. Scale bars, 5 μm. (d) The percentage of SeCreEt parasite-infected Cre-reporter cells that showed eGFP expression is plotted at the indicated time points.

Figure 3

Analysis of SeCreEt parasites in vivo. Bioluminescence imaging of Cre-reporter mice with a “floxed stop luciferase” after inoculation with the indicated numbers of parental (RH) or SeCreEt parasites. Ventral images from 4 to 9 days post-inoculation (dpi) are shown. An “X” indicates no further data could be obtained due to death of the animal.