The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation

Abstract

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

Figure 1. TgAP2XI-4 expression is regulated during bradyzoite differentiation and throughout the tachyzoite cell-cycle

(A) Wild type parasites from a T. gondii type II 76K strain were either used to produce bradyzoite tissue cysts (in vivo) or were cultured under normal conditions for tachyzoite growth (in vitro) (Dzierszinski et al., 2001). Total RNA was purified from all samples and analysed by quantitative RT-PCR to determine the relative levels of TgAP2 genes and Eno1 mRNA. The values are presented as the fold-changes in the bradyzoite samples relative to the corresponding tachyzoite samples. (B) Wild type parasites from a T. gondii type II strain were used to produce bradyzoite tissue cysts (in vivo) while wild type parasites from a type I strain were cultured under alkaline (pH 8.2) stress (in vitro) to induce the expression of bradyzoite genes. For comparison, type I and type II tachyzoite strains were grown in vitro under control conditions (pH 7.0). Total RNA was purified from all samples and analysed by quantitative RT-PCR to determine the relative levels of TgAP2XI-4 and Eno2 mRNA. The values are presented as the fold-change in the bradyzoite samples relative to the corresponding tachyzoite samples. (C) A whole-cell protein lysate from the TgAP2XI-4-HA mutant was fractionated on a 10% SDS-PAGE gel under reducing conditions. Western blot was carried out using a mouse monoclonal αHA antibody. An arrow indicates the band corresponding to TgAP2XI-4-HA. Molecular weight markers are in kDa. (D) Immunofluorescence assays were conducted on TgAP2XI-4 parasites fixed 24-hours after infection of HFF cells. The mouse monoclonal αHA antibody was used in combination with a rabbit αMORN1 antibody and detected with anti-mouse Alexa488 (green) and anti-rabbit Alexa594 (red), respectively. Daughter cell formation and mitosis were effectively monitored with αMORN1 and DAPI counterstaining (blue), respectively. TgAP2XI-4 protein expression peaks during the cytokinesis and early G1 phase. C, cytokinesis; G1, gap phase; S, synthesis phase; and M, mitosis. It should be noted that MORN1 localizes at ring structures at the apical and posterior ends of the inner membrane complex and to the centrocone. The centrocone-associated MORN1 concentrates at a focal point during G1 or as two focal points during S/M phase and is absent during C phase.

Figure 2. Sequence-specific DNA-binding of TgAP2XI-4

(A) The amino acid sequence of the TgAP2XI-4 AP2 region was aligned to the first of two AP2 domains in the P. falciparum homologue, PFD0985w. Three completely conserved β-sheets (β1, β2 and β3) are highlighted, along with a less conserved α-helix. (B) The solved PFD0985w DNA motif (Campbell et al., 2010) was used to design a 179 bp TgAP2XI-4 probe containing an extended version of the DNA motif. Specific and non-specific competitor probes were designed with the extended motif or a mutated motif, respectively. (C) An electrophoretic mobility shift assay was carried out using a recombinant protein spanning the TgAP2XI-4 AP2 domain. The shift caused by the binding of TgAP2XI-4 to the biotinylated probe is indicated by an arrow. TgAP2XI-4 binding was completely inhibited by a 500-fold excess of specific competitor but not by a non-specific competitor.

Figure 3. Construction of RH ΔTgAP2XI-4 using fusion PCR

(A) In the first PCR, the 5′ upstream and 3′ downstream flanking regions of the TgAP2XI-4 gene were amplified along with the first and second half of a pyrimethamine resistant DHFR cassette. Due to the introduction of complementary flanking regions, the TgAP2XI-4 5′ and 3′ products could be fused to the first and second halves of the DHFR products, respectively, in a second PCR. The two resulting PCR products were directly transfected into the RH ΔKu80 T. gondii type I strain. (B) PCR was used to confirm the correct integration of the fused ΔTgAP2XI-4 construct (using primer pairs ii and iii) and deletion of the TgAP2XI-4 coding sequence (primer pair i). The absence of TgAP2XI-4 transcripts was confirmed by RT-PCR (primer pair i). Primers for TgAP2XI-5 (TGME49_016220) were used as a control (con).

Figure 4. Microarray analysis of RH ΔKu80 ΔTgAP2XI-4 under control and pH 8.2-stress conditions

Total RNA purified from ΔTgAP2XI-4 and wild type parasites cultured either in control (pH 7) or stress (pH 8.2) conditions was analysed by microarray. Scatter plots represent the log fold-change (logFC) of T. gondii gene hits (plotted against average expression) in ΔTgAP2XI-4 vs wild type, at either pH 7 (A) or pH 8.2 (B). A broken green line surrounds an apparent sub-population of genes that show down-regulation shifts in ΔTgAP2XI-4 at pH 8.2. (C) A scatter plot compares the logFC due to pH 8.2 stress in the wild type and ΔTgAP2XI-4. Plotted points were limited to genes displaying logFC of >2.0 in the pH 8.2-stressed wild type (i.e. putative bradyzoite genes). The regression line is in green. All points below the broken black represent putative bradyzoite genes with reduced transcriptional activation in ΔTgAP2XI-4.

Figure 5. (A) Quantitative RT-PCR of bradyzoite transcripts from RH ΔKu80 ΔTgAP2XI-4

Total RNA purified from ΔTgAP2XI-4 and wild type parasites cultured either under control (pH 7) or stress (pH 8.2) conditions was analysed by quantitative RT-PCR. Genes coding for known bradyzoite proteins, including LDH2, P18, BAG1 and Eno1, were analysed alongside Eno2, which is not affected by alkaline stress. Values are presented as fold-change in pH8.2-stressed parasites relative to those under control conditions. An asterisk indicates a significant difference (P < 0.05) between the wild type and ΔTgAP2XI-4 mutant for individual genes. (B) Promoter assay in the parental and ΔTgAP2XI-4 strains.B-NTPase, SRS9 and SRS12D bradyzoite gene promoters were assayed in a luciferase promoter assay as well as the promoter of the TGME49_005250 gene as a negative control. After shifting the culture to alkaline pH, the Firefly luciferase activity was measured and normalised to the Renilla luciferase activity of the co-transfected tubulin promoter in the parental strain (empty bars) and in the ΔTgAP2XI-4 strain (black bars). Luciferase activity was measured in duplicate and is represented as a percentage of the activity produced by a given promoter in the parental strain.

Figure 6. Construction of Pru ΔKu80 ΔTgAP2XI-4

(A) Schematics representing the WT and recombinant TgAP2XI-4 locus. PCR was used to confirm the correct integration of the fused ΔTgAP2XI-4 construct (using primer pairs ii and iii) and deletion of the TgAP2XI-4 coding sequence (primer pair i). (B) Absence of TgAP2XI-4 transcripts was confirmed by RT-PCR (primer pair i). Primers for TgAP2XI-5 (TGME49_016220) were used as a control (con).

Figure 7. Phenotypic analysis of Pru ΔKu80 ΔTgAP2XI-4

(A) Quantitative RT-PCR of bradyzoite transcripts from Pru ΔKu80 ΔTgAP2XI-4. Total RNA purified from ΔTgAP2XI-4 and parental parasites cultured under either control (pH 7) or stress (pH 8.2) conditions were analysed by quantitative RT-PCR. Genes coding for known bradyzoite proteins including LDH2, P18, BAG1 and Eno1 were analysed alongside Eno2, which is not affected by alkaline stress. Values are presented as fold-change in pH 8.2-stressed parasites relative to those in control conditions. An asterisk indicates a significant difference (P < 0.05) between the wild type and the ΔTgAP2XI-4 mutant for individual genes. (B) Dolichos bifluorus lectin staining of parental and ΔTgAP2XI-4 strain after 2 days of treatment at pH 8.2. The percentage of vacuoles positive for lectin staining is represented. Data are means +/− SD. An asterisk indicates a significant difference (P < 0.05). (C) Cyst burden in mouse brain. Cysts were enumerated after Dolichos bifluorus lectin staining of the cyst wall. A minimum of five mice was used per group. Brains of mice infected with the Pru ΔTgAP2XI-4 strain are represented with circles. Brains of mice infected with the Pru strain are represented with triangles. The mean cyst burden for each group is represented by a horizontal bar.