Opposing transcriptional mechanisms regulateToxoplasmadevelopment

Abstract

The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst.IMPORTANCE Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

“…To control for nonspecific binding, we analyzed DD AP2IV-4 binding to the chromosome region (-950 bp) 5'flanking of the BAG1 bradyzoite gene. In contrast to stress-induced AP2IV-3, which we have recently reported activates BAG1 [26], AP2IV-4 does not regulate BAG1 (Dataset S1). No enrichment of DD AP2IV-4 binding was detected to the six regions tested within the BAG1 promoter ( Fig.…”

“…Notably, AP2VI-1 mRNA is expressed at high levels across the Toxoplasma intermediate and definitive life cycles (the only ApiAP2 with this profile) and AP2III-2 is highly expressed in unsporulated oocysts [26]. The three Group-of 12 ApiAP2 factors exclusively expressed in tachyzoites (AP2IV-4, AP2XII-2, AP2XII-9) [26] also failed knockout attempts in RHQ and had significant negative phenotype scores in the HFF/CRISPR study ( Fig. 1B).…”

“…Alternate developmental expression (and possible function), may help explain why half of the Group-of-12 ApiAP2s are not required for tachyzoite growth. AP2IX-4 (Huang et al, in preparation) and AP2XI-4 [21] are expressed in bradyzoites and recent studies indicate important roles for these factors in tissue cyst development; AP2III-2, AP2VI-1, and AP2XI-1 are also expressed in tachyzoites and bradyzoites [26]. Notably, AP2VI-1 mRNA is expressed at high levels across the Toxoplasma intermediate and definitive life cycles (the only ApiAP2 with this profile) and AP2III-2 is highly expressed in unsporulated oocysts [26].…”

“…AP2IX-4 (Huang et al, in preparation) and AP2XI-4 [21] are expressed in bradyzoites and recent studies indicate important roles for these factors in tissue cyst development; AP2III-2, AP2VI-1, and AP2XI-1 are also expressed in tachyzoites and bradyzoites [26]. Notably, AP2VI-1 mRNA is expressed at high levels across the Toxoplasma intermediate and definitive life cycles (the only ApiAP2 with this profile) and AP2III-2 is highly expressed in unsporulated oocysts [26]. The three Group-of 12 ApiAP2 factors exclusively expressed in tachyzoites (AP2IV-4, AP2XII-2, AP2XII-9) [26] also failed knockout attempts in RHQ and had significant negative phenotype scores in the HFF/CRISPR study ( Fig.…”

“…S3A and ref. [26]) and these spontaneously formed bradyzoites raise the population baseline expression of bradyzoite mRNAs. Nonetheless, microarray studies of PruQ-∆ap2IV-4 parasite mRNA expression clearly validated the conclusion that a major function of AP2IV-4 is to silence bradyzoite surface and cyst wall gene transcription in replicating tachyzoites.…”