ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst

Abstract

Cellular differentiation leading to formation of the bradyzoite tissue cyst stage is the underlying cause of chronic toxoplasmosis. Consequently, mechanisms responsible for controlling development in the Toxoplasma intermediate life cycle have long been sought. Here, we identified 15 Toxoplasma mRNAs induced in early bradyzoite development that encode proteins with apicomplexan AP2 (ApiAP2) DNA binding domains. Of these 15 mRNAs, the AP2IX-9 mRNA demonstrated the largest expression increase during alkaline-induced differentiation. At the protein level, we found that AP2IX-9 was restricted to the early bradyzoite nucleus and is repressed in tachyzoites and in mature bradyzoites from 30-d infected animals. Conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation and conferred alkaline pH-resistant growth, whereas disruption of the AP2IX-9 gene increased tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs, including the canonical bradyzoite marker, bradyzoite antigen 1 (BAG1). Using protein binding microarrays, we established the AP2 domain of AP2IX-9 binds a CAGTGT DNA sequence motif and is capable of binding cis-regulatory elements controlling the BAG1 and bradyzoite-specific nucleoside triphosphatase (B-NTPase) promoters. The effect of AP2IX-9 on BAG1 expression was direct because this factor inhibits expression of a firefly luciferase reporter under the control of the BAG1 promoter in vivo, and epitope-tagged AP2IX-9 can be immunoprecipitated with the BAG1 promoter in parasite chromatin. Altogether, these results indicate AP2IX-9 restricts Toxoplasma commitment to develop the mature bradyzoite tissue cyst.

Fig. 1.

Toxoplasma AP2IX-9 is a nuclear factor induced by alkaline-pH stress. The AP2IX-9 factor was C-terminally tagged with 3xHA via homologous recombination in PruΔku80 strain (designated PruQ). PruQ-AP2IX-9^HA parasites grown in human foreskin fibroblast (HFF) cell monolayers were costained with anti-HA antibody (red for AP2IX-9^HA expression), biotin-labeled D. biflorus agglutinin (DBA; green for the presence of cyst wall), and DAPI (blue for genomic DNA). After exposure to pH 8.2 media, AP2IX-9^HA protein was detected exclusively in the parasite nucleus (copositive for DAPI). Few vacuoles were positive for AP2IX-9^HA expression in parasites grown under tachyzoite conditions (12% pH 7.0 media). HA- and DBA-positive vacuoles (%) for each condition are indicated on the right. Note that AP2IX-9^HA staining declined after 2 d in both the number of vacuoles positive and the intensity of nuclear fluorescence.

Fig. 2.

AP2IX-9 binds functional sequence elements of the bradyzoite NTPase (B-NTPase) promoter. Recombinant GST-AP2IX-9 protein binds DNA (arrow) carrying the B-NTPase CRE in a dose-dependent manner and was inhibited by competition with unlabeled mut 4 DNA but not with mut 3 DNA. The probe and competitive DNA sequences used here duplicate key features of the CRE sequence controlling the B-NTPase promoter (probe) and two mutant promoters (mut 3 and 4) tested for activity in luciferase reporter assays (in vivo*, promoter mutations are underlined; see ref. for original luciferase results). Lane 1, biotin-labeled probe alone; lanes 2–4, 0.1, 0.3, or 0.9 μg of GST-IX-9; lanes 5–7, probe plus 0.3 μg of GST-IX-9 and 50-, 100-, or 300-fold unlabeled mutant 3 competitor DNA; lanes 8–10, probe plus 0.3 μg of GST-IX-9 and 50-, 100-, and 300-fold unlabeled mutant 4 DNA. Black triangles represent increasing amounts of protein or unlabeled competitor DNA fragments.

Fig. 3.

Overexpression of AP2IX-9 inhibits tissue cyst formation and promotes alkaline-resistant parasite growth. (A) Stimulation of tissue cyst formation in parental type III CTG and CTG-^DDAP2IX-9 transgenic parasites was compared 72 h after shift into pH 8.2 media with or without 250 nM Shield-1. The addition of Shield-1 reduced tissue cyst counts ∼fivefold in CTG-^DDAP2IX-9 parasites. Statistical significance was evaluated by using the unpaired t test between the parent and the transgenic strain (***P < 0.001; **P < 0.01; n = 5). (B) The effect of stabilizing ^DDAP2IX-9 with Shield-1 on parasite growth rates was determined by measuring the average vacuole size of CTG parent versus CTG-^DDAP2IX-9 parasites. Parental CTG is shown in white bars; no Shield-1 = light dots, plus 250 nM Shield-1 = open bars. CTG-^DDAP2IX-9 parasites; no Shield-1, gray bars; plus Shield-1, black bars. Because of host cell lysis the vacuole size for CTG-^DDAP2IX-9 parasites was not determined at 96 h (n/a). (Inset) ^DDAP2IX-9 was induced to detectable levels by pH 8.2 media alone and to higher levels when Shield-1 was added. Anti-PCNA1 antibody staining was included as a loading control.

Fig. 4.

AP2IX-9 regulates key features of the bradyzoite genetic program. (A) Nearly three dozen mRNAs (35) are significantly down-regulated (fold change shown) in CTG-^DDAP2IX-9 parasites compared with CTG parental controls under pH 8.2 media plus Shield-1 induction conditions. BAG1 mRNA was the most repressed along with other known bradyzoite mRNAs: LDH2, #4 mRNA; ENO1, #5; SAG2D, #8; SAG4.2, #23; BK1, #24; B-NTPase, #29 (see Dataset S1 for full gene list). Colored boxes indicate the number of AP2IX-9 motifs present in each promoter (2 kbp upstream of ATG start). (B) ^DDAP2IX-9 expression represses induction of a firefly luciferase transgene controlled by the BAG1 promoter (−1,195-bp region) (18). Luciferase activity in whole-cell lysates from PruIC2 parent and PruIC2-^DDAP2IX-9 parasites was determined at 72 h after shift to pH 8.2 media ±200 nM Shield-1 and compared with the appropriate pH 7.0 controls (data presented as the ratio pH 8.2/7.0, see SI Appendix, Methods for average light unit values). Gray bars, PruIC2-^DDAP2IX-9 parasites; black bars, PruIC2 parent (***P < 0.001) (C) An EMSA was performed with 0, 70, 200, and 600 ng (lanes 1–4) purified GST-IX-9 and 20 fmol of 59-bp 5′-biotin-labeled probe containing the BAG1 CRE sequence (5′-TACTGG-3′/3′ATGACC-5′) (18). A large complex (arrow) above the two probe-associated bands was competed with an unlabeled probe (300×, lane 5). See Dataset S1 for all oligonucleotide sequences. (D) ^DDAP2IX-9 occupies the BAG1 promoter in parasite chromatin. Specific binding was observed in all six target regions (1–6) tiled across the BAG1 promoter (−950 bp flanking the ATG indicated by arrow in diagram) with the central three regions showing the highest enrichment (see SI Appendix, Fig. S5 for full experimental details). Ten repeat motifs matching 5 of 6 bases in the AP2IX-9 DNA binding motif are indicated by black triangles above the diagram, whereas the BAG1 promoter CRE previously mapped by luciferase assay lies within region 4 and is marked by a triangle below the diagram (18).

Fig. 5.

Model for control of bradyzoite development by the AP2IX-9 transcriptional repressor. Four major transitions are thought to occur in the intermediate life cycle: following infections with sporozoites (or bradyzoites) parasites rapidly differentiate into replicating tachyzoites (A), limited tachyzoite proliferation leads to a slower growing prebradyzoite state (B), and (C) the timing of commitment to the dormant bradyzoite in the mature tissue cyst is asynchronous (C and D*). AP2IX-9 balances the competing interests of parasite expansion and efficient transmission through direct inhibition of commitment to the mature bradyzoite. This model is based on studies here and published results with the VEG strain (6, 7) that has been maintained in the Jitender P. Dubey laboratory (US Department of Agriculture, Beltsville, MD) by exclusive mouse-cat serial passage to preserve the native Toxoplasma developmental pathway (32).