Sex in a dish: high-efficiency Toxoplasma pre-sexual development

Abstract

Antunes et al. successfully grew cat-restricted stages of Toxoplasma gondii in cell culture by targeting parasite epigenetics and transcription factors. The highlight of this report is how efficiently parasites convert to these pre-sexual stages. Their work is an important step toward achieving feline-free recapitulation of the T. gondii sexual cycle.

Toxoplasma gondii is an obligate intracellular parasite that chronically infects around 20% of humans and causes severe disease in immunocompromised individuals. Like other eukaryotic parasites, T. gondii has multiple life stages that occur in different hosts (Figure 1). It propagates asexually in just about any warm-blooded animal, but it completes its sexual cycle only in felines. When ingested by a cat, haploid asexual T. gondii parasites quickly differentiate into pre-sexual merozoites in the small intestine. Within days, merozoites transition to gametes, which fuse to form diploid oocysts and are shed in the feces. These oocysts become highly infectious after they sporulate in the environment and can persist for years, making them key to T. gondii transmission. Despite their importance, T. gondii’s pre-sexual and sexual cat stages have been refractory to cell culture since their discovery 50 years ago.

Figure 1. The T. gondii life cycle and ApiAP2 transcription factors.

Intermediate hosts contract T. gondii by consuming oocyst- or tissue cyst-contaminated food or water. Fast-replicating tachyzoites cause disease in immunocompromised hosts, but quickly convert to slow-replicating bradyzoites that perpetuate subclinical lifelong infection. Feline predators consume bradyzoites in tissue cysts, which begins pre-sexual and sexual development. Cat-restricted stages are depicted in purple. Asexual stages (pink) occur in intermediate hosts and cats. Center: CRISPR-mediated knockdown (KD) of the ApiAP2 transcription factors AP2XI-2 and AP2XII-1 removes epigenetic proteins microrchidia (MORC) and histone deacetylase 3 (HDAC) from promoter regions and derepresses merozoite genes, converting tachyzoites to merozoites. Created with biorender.com.

Life stage transitions in T. gondii are carefully coordinated by host cues, epigenetics, and transcriptional reprogramming [1,2]. At the transcriptional level, ApiAP2 transcription factors are well-known controllers of life stage transitions in T. gondii and other apicomplexan parasites [2]. In T. gondii, AP2IX-9 was the first ApiAP2 shown to control T. gondii life stage conversion. In this case, AP2IX-9 “locked” parasites in the fast-replicating asexual tachyzoite stage, preventing their transition to the slow-replicating asexual bradyzoite stage [3]. Now, Antunes et al. show that simultaneous and conditional knockdown of two other pro-tachyzoite ApiAP2s, AP2XI-2 and AP2XII-1, converts asexual tachyzoites to cat-restricted pre-sexual merozoites [4] (Figure 1). With nearly uniform (98%) expression of the bona fide merozoite marker GRA11b in these parasites [5], Antunes et al. report what is by far the most successful in vitro method for early stage merozoite conversion reported to date.

Going beyond GRA11b, Antunes et al. also confirm that the transcriptional and proteomic profiles of their in vitro merozoites match those of in vivo merozoites with limited expression of asexual stage genes [5]. Their dual-omics profiling also identified GRA80, a dense granule protein, as a promising new merozoite marker that co-stains with GRA11b in both cat intestinal sections and cell culture. Ongoing studies [6] are already using GRA80 to document stage conversion, and there is no doubt that the molecular characterization of these new cell culture merozoites will help identify additional markers for pre-sexual and sexual forms of T. gondii.

Early studies of cat-stage T. gondii parasites revealed a unique method of cell division called endopolygeny that is characteristic of merozoites [1]. In Antunes et al.’s study, electron micrographs and immunofluorescence of the T. gondii inner membrane complex further confirm that in vitro merozoites use the same replication strategy as in vivo merozoites. The high efficiency of this system will make it useful for examining the precise mechanism of endopolygeny for T. gondii and other apicomplexan parasites.

A key insight of Antunes et al. is confirming a link between ApiAP2 transcription factors and parasite epigenetics in controlling T. gondii life stage transitions. Histone deacetylases (HDACs) are epigenetic enzymes that control eukaryotic gene expression by influencing chromatin accessibility. The Hakimi lab previously showed that inhibition of T. gondii HDAC3 changes expression of life stage-specific parasite genes [7]. More recently, they showed that HDAC3 is just one member of a protein complex that contains numerous ApiAP2s and a microrchidia (MORC) protein [8]. Disruption of that complex by targeting either HDAC3 or MORC skewed parasite gene expression to a mixed profile of bradyzoite and merozoite genes [8]. Antunes et al. build on that story by showing that targeted disruption of the ApiAP2 members of the protein complex – specifically AP2XI-2 and AP2XII-1 – derepresses merozoite gene expression without derepressing bradyzoite genes (Figure 1). Collectively, these studies suggest that AP2XI-2 and AP2XII-1 are powerful and precise transcriptional adaptors for HDAC3- and MORC-mediated repression of merozoite-specific genes.

Looking ahead, we can expect the T. gondii field to quickly adopt the CRISPR approach to merozoite production. In fact, another group working on ApiAP2 transcription factors already showed in 2023 that depletion of AP2XII-1 alone induced changes in transcription and cell division similar to those observed by Antunes et al. [9]. This efficient and now relatively straightforward gene editing technique will have many implications for the T. gondii field. Parasitologists have wondered for decades how merozoites differ from asexual stage parasites in their cell invasion and replication strategies, effector protein repertoires, gene regulation, and metabolism. With efficient merozoite production, those questions can begin to be answered.

Antunes et al. raise especially exciting questions for those studying T. gondii stage conversion. First, in vitro depletion of AP2XI-2 and AP2XII-1 in this study “locked” parasites in a merozoite state. Why is it that parasites do not spontaneously proceed to gamete and oocyst development like they do in cats? Perhaps feline-specific environmental factors act as additional checkpoints in the developmental cascade [10]. Or perhaps it is something related to the RH parasite strain used in this study. Having been passaged asexually for 100 years, RH parasites may have lost the genetic or epigenetic machinery required for full sexual development. Creation of a AP2XI-2/AP2XII-1 double knockdown in a Type II strain like Pru or ME49 may help answer that question. Another question is how the parasites in this study are able to proceed directly from tachyzoite to merozoite, skipping the presumably requisite life stage of bradyzoite that precedes sexual development in cats. Did Antunes et al. discover a shortcut, or do these two tachyzoite-to-merozoite ApiAP2s also control the bradyzoite-to-merozoite transition we see in nature? Finally, the field still seeks the cat-specific factor(s) that initiate and propagate sexual development. AP2XI-2 and AP2XII-1 represent new and promising targets in identifying what first signals to T. gondii that it has reached its definitive host.