Rapid control of protein level in the apicomplexan Toxoplasma gondii

Abstract

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

Figure 1

Rapid regulation of proteins fused to ddFKBP. (a) To examine upregulation, fluorescence micrographs were acquired after induction of intracellular parasites expressing ddFKBP-YFP with 1 μm Shld1 (top). To examine downregulation, infected cells treated with Shld1 were placed in medium without Shld1. Acquisition of fluoresence micrographs was started 80 min after removal of Shld1 (bottom). Scale bars, 10 μm. Fluorescence intensities for the indicated vacuoles (colored squares) were measured for the entire sequence and plotted for every time point (lines correspond to respectively colored squares; right). Black, background fluorescence. (b) Immunological analysis of intra- or extracellular parasites expressing either ddFKBP-YFP or GFP-ddFKBP induced with 1 μM Shld1 for the indicated amount of time before collection. The respective blot was simultaneously probed with polyclonal anti-FKBP12 (ABCAM) and monoclonal anti-MIC2 (6D10; ref. 9). The same lysates were probed with anti-TUB1 (ref. 10) in an independent blot. (c) Flow cytometry analysis of parasites expressing either ddFKBP-YFP (top) or GFP-ddFKBP (bottom) grown in the indicated concentration of Shld1. After egress parasites were subjected to flow cytometry analysis. We collected 100,000 data points for each sample.

Figure 2

Functional analysis of essential proteins using the ddFKBP system. (a) Immunological fluorescence analysis of T. gondii parasite strains expressing ddFKBP-MyoA, ddFKBP-YPT1_wt, ddFKBP-YPT1_DN or ddFKBP-Rab11A_DN for 4 h after induction using monoclonal anti-myc (9E10, Sigma) and polyclonal anti-IMC1 (ref. 11) or anti-GAP45 (ref. 12). Scale bar, 10 μm. (b) Immunological analysis on transgenic parasites shown in a. Parasites were grown in presence and absence of Shld1 for 48 h (ddFKBP-MyoA), for 8 h (ddFKBP-YPT1 and ddFKBP-YPT1_DN) or 2 h (ddFKBP-Rab11A_DN) before immunological analysis using the indicated antibodies. (c) Determination of plaque size after 5 d by parasite strain ddFKBP-YPT1 and ddFKBP-YPT1_DN (see Supplementary Fig. 3). Data represent mean values of 20 independent plaques ± s.d. Asterisks indicate significant difference in plaque sizes between ddFKBP-YPT1_DN grown in presence or absence of Shld1 (P < 0.001, two-tailed Student’s t-test). (d) Quantification of invasion and replication of parasite strain ddFKBP-Rab11A_DN (-/+, parasites treated with Shld1 after invasion; +/+, parasites treated with Shld1 before and after invasion). Total number of parasitophorous vacuoles and number of parasites per parasitophorous vacuole were determined. Mean values of 3 independent experiments ± s.d. are shown. Asterisks indicate significant difference in total invasion between parasite strain Rab11A_DN not treated with Shld1 and those treated with Shld1 before and after invasion (P < 0.01, two tailed Student’s t-test).