Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites

Abstract

The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro bradyzoites 4 days postinfection, and in vivo bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.

Fig. 1.

Scatterplots of average normalized expression values. Probe sets to Toxoplasma genes were normalized as described in Materials and Methods, average values for replicate arrays were plotted, and the correlation coefficient (R²) was determined using Prism software. Samples corresponded to tachyzoites grown for 2 days on HFF cells (2d Tachy), bradyzoites that had been grown on HFFs for 4 days in vitro (4d Brady), and tissue cysts harvested from infected mouse brains 21 days after oral infection with oocysts (21d Brady). The comparisons are 21d Brady versus 4d Brady (A), 21d Brady versus 2d Tachy (B), and 4d Brady versus 2d Tachy (C).

Fig. 2.

BPK1 and MCP4 are induced by bradyzoites. HFF monolayers were infected with the PRUΔhxgprt strain of parasites that had been engineered to express a BPK1 or MCP4 protein fused to a C-terminal HA tag (BPK-HA and MCP4-HA, respectively) for 2 days under tachyzoite and 2 days and 4 days under bradyzoite growth conditions. Parasites were released from cells by syringe lysis and counted before SDS-PAGE loading buffer was added. A total of 5 × 10⁵ parasite equivalents were added per lane and probed with antibodies specific for HA, SAG1, or SAG2X as described in Materials and Methods. Representative images from at least two independent experiments are shown. The values to the right are molecular sizes in kilodaltons.

Fig. 3.

BPK1 and MCP4 localize to the lumen and wall area of in vitro bradyzoite cysts. HFF monolayers on glass coverslips were infected with PRUΔhxgprt parasites expressing BPK1-HA (A to C) or MCP4-HA (D to F) for 2 days (A, D) under tachyzoite growth conditions and for 2 days (B, E) or 4 days (C, F) under bradyzoite growth conditions. Coverslips were fixed and stained, as indicated, with rat anti-HA (red) and DBA (green) as described in Materials and Methods. The corresponding merged and phase images are also shown. A capital T indicates tachyzoite vacuoles. Representative images from at least two independent experiments are shown. Scale bars represent 10 μm.

Fig. 4.

BPK1 and MCP4 localize to the cyst periphery in vivo. Forty-micrometer coronal brain sections from mice infected 40 days previously with parasites expressing BPK1-HA (A) or MCP4-HA (B) were probed for HA (green) and DBA (red) and examined by confocal microscopy as described in Materials and Methods. The corresponding merged images are also shown. Scale bars represent 10 μm.

Fig. 5.

BPK1 and MCP4 localize to the cyst wall. EM of tissue cysts stained for BPK1-HA (A and B) and for MCP4-HA (C) using an anti-HA antibody and visualized with 10-nm gold particles. (A) Low-power magnification of a small in vitro tissue cyst (5 dpi) containing bradyzoites (Br) showing numerous gold particles over the cyst wall (CW). Bar represents 1 μm. (B) Detail of the periphery of a 5-day in vitro tissue cyst labeled for BPK1 illustrating the numerous gold particles associated with the cyst wall. Bar represents 100 nm. (C) Detail of the periphery of an in vivo-derived tissue cyst (40 dpi) labeled with MCP4 showing the gold particles specifically associated with the cyst wall. Bar represents 100 nm.