The Toxoplasma gondii F-Box Protein L2 Functions as a Repressor of Stage Specific Gene Expression.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages require substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct perinucleolar sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNAseq data show that TgFBXL2 conditional depletion induces the expression of stage-specific genes including a large cohort of genes necessary for sexual commitment. Together, these data suggest that TgFBXL2 is a latent guardian of stage specific gene expression in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of development.

"Reading" a new chapter in protozoan parasite transcriptional regulation.

Protozoan parasites continue to cause a significant health and economic burden worldwide. As infectious organisms, they pose unique and difficult challenges due to a level of conservation of critical eukaryotic cellular pathways with their hosts. Gene regulation has been pinpointed as an essential pathway with enough divergence to warrant investigation into therapeutically targeting. Examination of human parasites such as Plasmodium falciparum, Toxoplasma gondii, and kinetoplastids have revealed that epigenetic mechanisms play a key role in their gene regulation. The enzymes involved in adding and removing epigenetic posttranslational modifications (PTMs) have historically been the focus of study. However, the reader proteins that recognize and bind PTMs, initiating recruitment of chromatin-modifying and transcription complexes, are now being realized for their critical role in regulation and their potential as drug targets. In this review, we highlight the current knowledge on epigenetic reader proteins in model parasitic protozoa, focusing on the histone acyl- and methyl-reading domains. With this knowledge base, we compare differences between medically relevant parasites, discuss conceivable functions of these understudied proteins, indicate gaps in knowledge, and provide current progress in drug development.

Evaluating the GCN5b bromodomain as a novel therapeutic target against the parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite of great importance in human and veterinary health. The frontline treatment of antifolates suffers a variety of drawbacks, including toxicity and allergic reactions, underscoring the need to identify novel drug targets for new therapeutics to be developed. We previously showed that the Toxoplasma lysine acetyltransferase (KAT) GCN5b is an essential chromatin remodeling enzyme in the parasite linked to the regulation of gene expression. We have previously established that the KAT domain is a liability that can be targeted in the parasite by compounds like garcinol; here, we investigate the potential of the bromodomain as a targetable element of GCN5b. Bromodomains bind acetylated lysine residues on histones, which helps stabilize the KAT complex at gene promoters. Using an inducible dominant-negative strategy, we found that the GCN5b bromodomain is critical for Toxoplasma viability. We also found that the GCN5-family bromodomain inhibitor, L-Moses, interferes with the ability of the GCN5b bromodomain to associate with acetylated histone residues using an in vitro binding assay. Moreover, L-Moses displays potent activity against Toxoplasma tachyzoites in vitro, which can be overcome if parasites are engineered to over-express GCN5b. Collectively, our data support the GCN5b bromodomain as an attractive target for the development of new therapeutics.

A latent ability to persist: differentiation in Toxoplasma gondii.

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching.

Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii.

Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasiteToxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required forToxoplasmatachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibitsToxoplasmatachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibitsPlasmodium falciparumasexual replication with a 50% inhibitory concentration (IC50) similar to that forToxoplasma Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication.

Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation.

Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a "core complex" that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.

Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development.

Lysine acetylation has emerged as a major post-translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.

Histone code: a common language and multiple dialects to meet the different developmental requirements of apicomplexan parasites.

Apicomplexan parasites have complex life cycles, often requiring transmission between two different hosts, facing periods of dormancy within the host or in the environment to maximize chances of transmission. To support survival in these different conditions, tightly regulated and correctly timed gene expression is critical. The modification of histones and nucleosome composition makes a significant contribution to this regulation, and as eukaryotes, the fundamental mechanisms underlying this process in apicomplexans are similar to those in model eukaryotic organisms. However, single-celled intracellular parasites face unique challenges, and regulation of gene expression at the epigenetic level provides tight control for responses that must often be rapid and robust. Here, we discuss the recent advances in understanding the dynamics of histone modifications across Apicomplexan life cycles and the molecular mechanisms that underlie epigenetic regulation of gene expression to promote parasite life cycle progression, dormancy, and transmission.

Seeing the unseen: illuminating Toxoplasma gondii's metabolic manipulation.

Intracellular infection by a pathogen induces significant rewiring of host cell signaling and biological processes. Understanding how an intracellular pathogen such as Toxoplasma gondii modulates host cell metabolism with single-cell resolution has been challenged by the variability of infection within cultures and difficulties in separating host and parasite metabolic processes. A new study from Gallego-Lopez and colleagues (G. M. Gallego-López, E. C. Guzman, D. E. Desa, L. J. Knoll, M. C. Skala, mBio e00727-24, 2024, https://doi.org/10.1128/mbio.00727-24) applies a quantitative imaging approach to evaluate the host cell metabolism during intracellular infection with Toxoplasma. This study provides important insights into host metabolic responses to Toxoplasma infection and offers a valuable tool to dissect the mechanisms underlying parasite infection and pathophysiology.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii.

While histone proteins are the founding members of lysine acetylation substrates, it is now clear that hundreds of other proteins can be acetylated in multiple compartments of the cell. Our knowledge of the scope of this modification throughout the kingdom of life is beginning to emerge, as proteome-wide lysine acetylation has been documented in prokaryotes, Arabidopsis thaliana, Drosophila melanogaster, and human cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we produced the first proteome-wide analysis of acetylation for a protozoan organism, the opportunistic apicomplexan parasite Toxoplasma gondii. The results show that lysine acetylation is abundant in the actively proliferating tachyzoite form of the parasite, which causes acute toxoplasmosis. Our approach successfully identified known acetylation marks on Toxoplasma histones and α-tubulin and detected over 400 novel acetylation sites on a wide variety of additional proteins, including those with roles in transcription, translation, metabolism, and stress responses. Importantly, an extensive set of parasite-specific proteins, including those found in organelles unique to Apicomplexa, is acetylated in the parasite. Our data provide a wealth of new information that improves our understanding of the evolution of this vital regulatory modification while potentially revealing novel therapeutic avenues. We conclude from this study that lysine acetylation was prevalent in the early stages of eukaryotic cell evolution and occurs on proteins involved in a remarkably diverse array of cellular functions, including those that are specific to parasites.

An apicomplexan bromodomain protein, TgBDP1, associates with diverse epigenetic factors to regulate essential transcriptional processes in Toxoplasma gondii.

The protozoan pathogen Toxoplasma gondii relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of Toxoplasma and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for Toxoplasma in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification are unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein, TgBDP1, which is conserved among Apicomplexa and within the Alveolata superphylum, is essential for Toxoplasma asexual proliferation. Using cleavage under targets and tagmentation, we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodeling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the Toxoplasma tachyzoite. IMPORTANCE Histone acetylation is critical for proper regulation of gene expression in the single-celled eukaryotic pathogen Toxoplasma gondii. Bromodomain proteins are "readers" of histone acetylation and may link the modified chromatin to transcription factors. Here, we show that the bromodomain protein TgBDP1 is essential for parasite survival and that loss of TgBDP1 results in global dysregulation of gene expression. TgBDP1 is recruited to the promoter region of a large proportion of parasite genes, forms a core complex with two other bromodomain proteins, and interacts with different transcriptional regulatory complexes. We conclude that TgBDP1 is a key factor for sensing specific histone modifications to influence multiple facets of transcriptional regulation in Toxoplasma gondii.

Toxoplasma gondii F-Box Protein L2 Silences Feline-Restricted Genes Necessary for Sexual Commitment.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages requires substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct nuclear sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNA seq data show that TgFBXL2 conditional depletion induces the expression of genes necessary for sexual commitment. We suggest that TgFBXL2 is a latent guardian of sexual stage development in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of sexual development.

No acetyl-CoA keeps Plasmodium at bay.

Acetyl-coenzyme A is an important metabolite and regulates diverse cellular processes, including metabolism and epigenetics. In this issue of Cell Chemical Biology, Summers et al. (2022) describe an essential parasite enzyme, acetyl-coenzyme A synthetase, as a target of two antimalarial small molecules active against liver and blood stages of the parasite.

Protein acetylation in the critical biological processes in protozoan parasites.

Protein lysine acetylation has emerged as a major regulatory post-translational modification in different organisms, present not only on histone proteins affecting chromatin structure and gene expression but also on nonhistone proteins involved in several cellular processes. The same scenario was observed in protozoan parasites after the description of their acetylomes, indicating that acetylation might regulate crucial biological processes in these parasites. The demonstration that glycolytic enzymes are regulated by acetylation in protozoans shows that this modification might regulate several other processes implicated in parasite survival and adaptation during the life cycle, opening the chance to explore the regulatory acetylation machinery of these parasites as drug targets for new treatment development.

A novel GCN5b lysine acetyltransferase complex associates with distinct transcription factors in the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that has a tremendous impact on human health and livestock. High seroprevalence among humans and other animals is facilitated by the conversion of rapidly proliferating tachyzoites into latent bradyzoites that are housed in tissue cysts, which allow transmission through predation. Epigenetic mechanisms contribute to the regulation of gene expression events that are crucial in both tachyzoites as well as their development into bradyzoites. Acetylation of histones is one of the critical histone modifications that is linked to active gene transcription. Unlike most early-branching eukaryotes, Toxoplasma possesses two GCN5 homologues, one of which, GCN5b, is essential for parasite viability. Surprisingly, GCN5b does not associate with most of the well-conserved proteins found in the GCN5 complexes of other eukaryotes. Of particular note is that GCN5b interacts with multiple putative transcription factors that have plant-like DNA-binding domains denoted as AP2. To understand the function of GCN5b and its role(s) in epigenetic gene regulation of stage switching, we performed co-immunoprecipitation of GCN5b under normal and bradyzoite induction conditions. We report the greatest resolution of the GCN5b complex to date under these various culture conditions. Moreover, reciprocal co-IPs were performed with distinct GCN5b-interacting AP2 factors (AP2IX-7 and AP2XII-4) to delineate the interactomes of each putative transcription factor. Our findings suggest that GCN5b is associated with at least two distinct complexes that are characterized by two different pairs of AP2 factors, and implicate up to four AP2 proteins to be involved with GCN5b-mediated gene regulation.

Bromodomains in Protozoan Parasites: Evolution, Function, and Opportunities for Drug Development.

Parasitic infections remain one of the most pressing global health concerns of our day, affecting billions of people and producing unsustainable economic burdens. The rise of drug-resistant parasites has created an urgent need to study their biology in hopes of uncovering new potential drug targets. It has been established that disrupting gene expression by interfering with lysine acetylation is detrimental to survival of apicomplexan (Toxoplasma gondii and Plasmodium spp.) and kinetoplastid (Leishmania spp. and Trypanosoma spp.) parasites. As "readers" of lysine acetylation, bromodomain proteins have emerged as key gene expression regulators and a promising new class of drug target. Here we review recent studies that demonstrate the essential roles played by bromodomain-containing proteins in parasite viability, invasion, and stage switching and present work showing the efficacy of bromodomain inhibitors as novel antiparasitic agents. In addition, we performed a phylogenetic analysis of bromodomain proteins in representative pathogens, some of which possess unique features that may be specific to parasite processes and useful in future drug development.

TgPRELID, a Mitochondrial Protein Linked to Multidrug Resistance in the Parasite Toxoplasma gondii.

New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite's replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the "reader" module of acetylated lysines. In independent studies, we sought to determine the targets of these two compounds using forward genetics, generating resistant mutants and identifying the determinants of resistance with comparative genome sequencing. Despite the dissimilarity of the two compounds, we recovered resistant mutants with nonsynonymous mutations in the same domain of the same gene, TGGT1_254250, which we found encodes a protein that localizes to the parasite mitochondrion (designated TgPRELID after the name of said domain). We found that mutants selected with one compound were cross resistant to the other compound, suggesting a common mechanism of resistance. To further support our hypothesis that TgPRELID mutations facilitate resistance to both I-BET151 and F3215-0002, CRISPR (clustered regularly interspaced short palindromic repeat)/CAS9-mediated mutation of TgPRELID directly led to increased F3215-0002 resistance. Finally, all resistance mutations clustered in the same subdomain of TgPRELID. These findings suggest that TgPRELID may encode a multidrug resistance factor or that I-BET151 and F3215-0002 have the same target(s) despite their distinct chemical structures. IMPORTANCE We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic infection, and understanding how TgPRELID mutations augment resistance to multiple, distinct compounds will reveal needed insights into the development of new therapies for toxoplasmosis and other related parasitic diseases.

Proteome-wide lysine acetylation in cortical astrocytes and alterations that occur during infection with brain parasite Toxoplasma gondii.

Lysine acetylation is a reversible post-translational modification (PTM) that has been detected on thousands of proteins in nearly all cellular compartments. The role of this widespread PTM has yet to be fully elucidated, but can impact protein localization, interactions, activity, and stability. Here we present the first proteome-wide survey of lysine acetylation in cortical astrocytes, a subtype of glia that is a component of the blood-brain barrier and a key regulator of neuronal function and plasticity. We identified 529 lysine acetylation sites across 304 proteins found in multiple cellular compartments that largely function in RNA processing/transcription, metabolism, chromatin biology, and translation. Two hundred and seventy-seven of the acetylated lysines we identified on 186 proteins have not been reported previously in any other cell type. We also mapped an acetylome of astrocytes infected with the brain parasite, Toxoplasma gondii. It has been shown that infection with T. gondii modulates host cell gene expression, including several lysine acetyltransferase (KAT) and deacetylase (KDAC) genes, suggesting that the host acetylome may also be altered during infection. In the T. gondii-infected astrocytes, we identified 34 proteins exhibiting a level of acetylation >2-fold and 24 with a level of acetylation <2-fold relative to uninfected astrocytes. Our study documents the first acetylome map for cortical astrocytes, uncovers novel lysine acetylation sites, and demonstrates that T. gondii infection produces an altered acetylome.

Protein intrinsic disorder in the acetylome of intracellular and extracellular Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, which includes a number of species of medical and veterinary importance. Inhibitors of lysine deacetylases (KDACs) exhibit potent antiparasitic activity, suggesting that interference with lysine acetylation pathways holds promise for future drug targeting. Using high resolution LC-MS/MS to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we recently produced an acetylome of the proliferative intracellular stage of Toxoplasma. In this study, we used similar approaches to greatly expand the Toxoplasma acetylome by identifying acetylated proteins in non-replicating extracellular tachyzoites. The functional breakdown of acetylated proteins in extracellular parasites is similar to intracellular parasites, with an enrichment of proteins involved in metabolism, translation, and chromatin biology. Altogether, we have now detected over 700 acetylation sites on a wide variety of parasite proteins of diverse function in multiple subcellular compartments. We found 96 proteins uniquely acetylated in intracellular parasites, 216 uniquely acetylated in extracellular parasites, and 177 proteins acetylated in both states. Our findings suggest that dramatic changes occur at the proteomic level as tachyzoites transition from the intracellular to the extracellular environment, similar to reports documenting significant changes in gene expression during this transition. The expanded dataset also allowed a thorough analysis of the degree of protein intrinsic disorder surrounding lysine residues targeted for this post-translational modification. These analyses indicate that acetylated lysines in proteins from extracellular and intracellular tachyzoites are largely located within similar local environments, and that lysine acetylation preferentially occurs in intrinsically disordered or flexible regions.