Bradyzoite subtypes rule the crossroads of Toxoplasma development

Domain: neuroscience
Published: 2026-01-24
Editorial outcome: minor_revision

Abstract
Toxoplasmosis is a major risk to chronically infected individuals, especially those who become immunocompromised. Although one-third of the globe is infected with Toxoplasma, no treatments prevent or eliminate cysts in part due to limited understanding of bradyzoite biology. The cyst is central to Toxoplasmosis, as transition from bradyzoites to tachyzoites drive pathology. In this study, we aim to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. Here, we discover ME49EW cysts from infected mice harbor multiple bradyzoite subtypes with distinct fates. Purified subtypes exhibit defined developmental pathways in animals and in primary astrocytes. Single-bradyzoite RNA-sequencing reveals five major subtypes within cysts. We further show that a crucial subtype in chronically infected mice is absent from a widely used in vitro model of bradyzoite development. Altogether, this work establishes new foundational principles of Toxoplasma cyst development and reactivation that operate during the intermediate lifecycle of Toxoplasma.

Introduction
Toxoplasma gondii is a globally prevalent protozoan parasite, infecting nearly one-third of the human population. During chronic infection, the tissue cyst form is essential for both parasite survival and disease pathogenesis. In immunocompromised individuals, reactivation of these cysts or recrudescence, is the only known cause of acute, and sometimes fatal, toxoplasmosis1,2. Cyst reactivation is typically controlled by IFN-γ mediated immune responses3. However, with rising immunosuppression in the U.S. population, 6.6% of adults are immunocompromised, and with over a million cancer patients undergo chemotherapy annually, the risk of cyst reactivation becomes a growing public health concern4. Given its high prevalence5,6, and potential for severe disease, understanding bradyzoite biology and tissue cyst dynamics is critical.

Broad host range and durable Toxoplasma tissue cysts underlie its success as an intracellular parasite. Transmission occurs via feline oocysts and ingestion of cyst-contaminated foods. Bradyzoites within tissue cysts are the pivotal stage of the Toxoplasma lifecycle. They serve as a reservoir of infection capable of differentiating directly into tachyzoites responsible for dissemination and cell lysis7–9 or switching to the merozoite stage in the feline definitive host that lead to sexual reproduction in the cat gut10–12. Bradyzoites are also capable of direct replication, which is thought to be required for maintaining cyst burden and chronic infection10,11,13–15. Investigating this slow growing, in vivo bradyzoites is difficult, but our ex vivo recrudescence system using in vivo-derived cysts13 allows characterization of growth dynamics and heterogeneity around reactivation that cannot be fully replicated in vitro.

In the Toxoplasma Type II ME49 strain, genes encoding SAG1-related surface proteins (SRS) includes 144 genes; 109 that match gene models and 35 incomplete pseudogenes16,17. SRS genes are present on all ME49 chromosomes with clusters of SRS paralogs found on seven chromosomes. SRS antigens discovered 40 years ago remain an enigmatic class of Toxoplasma factors18,19. SRS antigens influence host cell adhesion20, and it is postulated that immunodominant SRS antigens, such as SAG1, elicit immune responses against the tachyzoite stage so that bradyzoites, which lack SAG1 are spared21. Notwithstanding, the realization of purpose for a few SRS antigens, the function of most SRS antigens remains unknown. However, antibodies raised against developmentally regulated SRS antigens are valuable reagents in the study of Toxoplasma developmental pathways19,22. Similarly, SAG1 is a primary marker of the tachyzoite stage, while induction of SRS9 expression (i.e. SRS16B) indicates early bradyzoite development13. In this study we investigated the SRS22A protein that belongs to a cluster of 9 paralogs (SRS22A-I) and one pseudogene tandemly arrayed on the left end of chromosome VI. The SRS22 gene family are highly expressed in merozoites of the definitive lifecycle23,24 with the exception of SRS22A mRNA, which is additionally expressed in bradyzoites23.

In this study, we demonstrate SRS22A is a dominant bradyzoite surface protein expressed in ME49EW bradyzoites from mice but is not detected in conventional in vitro models of bradyzoite development such as alkaline-stress model which now includes our ex vivo bradyzoite recrudescence model13. We determined that ME49EW tissue cysts from infected mice harbor at least two bradyzoite subtypes distinguishable by SRS22A expression that have different growth and developmental trajectories. Using single bradyzoite mRNA-sequencing we confirm that in vivo bradyzoites differentially express SRS22A mRNA and provide evidence that there are additional bradyzoite subtypes likely present in the tissue cysts of chronically infected mice. We next determined whether SRS22A was expressed in bradyzoites that develop in the in vitro alkaline stress-model.

Results
Bradyzoites from chronically infected mice have unique surface antigen composition
It is generally understood that conventional alkaline-stress models of tachyzoite-to-bradyzoite differentiation do not faithfully reproduce the full breadth of native developmental processes. The reliance on in vitro methods has contributed to significant gaps in our understanding of native bradyzoite developmental biology in the Toxoplasma intermediate lifecycle. To begin addressing this problem, we sought to identify potential in vivo bradyzoite markers by comparing the mRNA expression of SRS genes in ME49EW bradyzoites from 30-day infected mice against in vitro ME49B7 tachyzoite and alkaline-stressed parasite mRNA expression (FPKM_fold-change, for full RNA-seq data see ref. 13). In order to compare in vitro marker expression with the markers observed in vivo, we examined the most commonly employed in vitro system in which alkaline stress is used to generate in vitro ME49B7 bradyzoites. Seventeen SRS antigen mRNAs were expressed at least 10-fold higher in ME49EW bradyzoites from mice (Fig. 1a table). The mRNA encoding SRS9 (also designated as SRS16B) was highly expressed in ME49EW bradyzoites as well as alkaline-stressed in vitro bradyzoites validating the use of anti-SRS9 antibodies as an early bradyzoite marker. The gene encoding the SAG-related protein SRS22A was the top induced SRS mRNA in ME49EW bradyzoites from in vivo cysts at >1000-fold higher levels than SRS22A mRNA levels in tachyzoites. The in vivo expression of SRS22A mRNA was also well above SRS22A mRNA levels from alkaline-stressed ME49B7 parasites (~17 fold higher, Fig. 1a). We selected a peptide from the N-terminal region of the SRS22A protein coding sequence (LRGNDGRSSRVIEKEAEVAK; Fig. S1A) based on informatic prediction of immunogenicity and produced peptide-specific antibodies in rabbits. The SRS22A antisera successfully stained in vivo tissue cysts from mice (30-days post-infection), whereas there was no reactivity of the pre-immune serum control (Fig. S1B). Furthermore, Western blot analysis detected a strong, single band in excysted bradyzoites obtained from 30-day infected mice with no signal detected in RH tachyzoites grown in vitro (Fig. 1b) indicating specificity for bradyzoites25.

Utilizing the SRS22A antiserum, we determined the kinetics and cyst distribution of SRS22A expression in tissue cysts from infected brain homogenates at 14-, 21-, 28-, and 35-days post-infection (Fig. 1c–f). As examined by others, there are likely complex patterns of cyst growth, rupture and reformation over the course of infection, however, average cyst size generally increases over this period11–13, which is consistent with our results here (Fig. 1c). The proportion of cysts expressing SRS22A is generally correlated with increasing cyst size (Fig. 1d). By the last timepoint (Day 35), >75% of tissue cysts showed some expression of SRS22A. Over the course of infection, we estimated that cysts exhibited three SRS22A bradyzoite configurations; near 100% positive or negative or mixtures of SRS22A+/SRS22A- bradyzoites (Fig. 1f). The proportion of ~100% positive SRS22A cysts increased from Day 14 to Day 21 post-infection, and this level of expression was maintained at ~20% of total cysts thereafter (Fig. 1e). By contrast, the proportion of cysts with mixtures of SRS22A- and SRS22A+ bradyzoites continued to increase over time reaching >50% of the population by Day 35. A process of cyst maturation has been postulated to explain the differences in cysts generated by in vitro alkaline-stress models (considered to be early cysts) and the putative mature cysts recovered from >30-day infected mice. By probing for SRS22A, (an indicator of in vivo cysts) further cyst heterogeneity is revealed and indicates there is no single “mature” cyst in the native intermediate lifecycle, but that cyst heterogeneity exists throughout the course of Toxoplasma infection in the brain.

SRS22A is exclusively expressed by in vivo generated cysts
SRS22A mRNA expression in ME49EW cysts from mice is substantially higher than in cysts generated under alkaline-stress treatment (Fig. 1A). To determine if SRS22A protein expression differentiates in vivo from in vitro bradyzoites, we used ME49B7 tachyzoites26 to infect HFF monolayers, and following a 4h invasion period, shifted half the cultures to pH 7.4 media and the other half to pH 8.2 bradyzoite-media. We co-stained infected HFF cells with two pairs of antibodies; the tachyzoite-specific SAG1 monoclonal antibody with the bradyzoite-specific anti-SRS9 or anti-SRS22A rabbit antisera. Parasite vacuole size and SRS antigen expression was assessed at 48h after the shift to pH 8.2 media (Fig. 2a). ME49B7 tachyzoites grown in pH 7.4-media actively replicated and expressed SAG1 (Fig. 2a). In pH 8.2-media, ME49B7 tachyzoites grew much slower as evidenced by the smaller range of vacuole sizes. Tachyzoite vacuoles (SAG1+ only) were 41% of the total parasite population whereas 59% of vacuoles co-stained for SAG1+ and SRS9+ (SAG1+/SRS9+ vacuoles) or expressed only SRS9+ antigen and considered to be bradyzoite vacuoles. No parasites in either pH 7.4- or pH 8.2-media expressed detectable SRS22A antigen.

In our ex vivo recrudescence model, in vivo ME49EW bradyzoites are used to infect primary astrocytes cultivated under low oxygen (5%) conditions13. As expected, SRS22A antisera stained excysted ME49EW bradyzoites from in vivo cysts as well as single bradyzoites that had invaded astrocytes (Fig. 2b, free and Day 1 bradyzoites). Most vacuoles in Day 1-infected astrocytes contain single (pre-replication) bradyzoites reflecting time needed for dormant bradyzoites to reawaken13. In advance of the first parasite doubling, we detected switches in SRS antigen composition indicating changes in developmental transcription (e.g. SRS22A) occur before replication commences in full. The two-representative single bradyzoite vacuoles shown here (Fig. 2b, D1 EW) both express SRS22A, while only one vacuole co-stained brightly for SAG1. In the ex vivo recrudescence model13 replicating bradyzoite vacuoles occur alongside tachyzoite vacuoles (SAG1+) as visualized by DBA positive cyst walls (Fig. 2b); D3 EW and D7 EW image panels). Importantly, SRS22A expression was not observed in these mixed populations (Fig. 2b) in either the fast-replicating tachyzoite period at Day 3 (D3 EW) or the post-growth shift tachyzoite/bradyzoite population at Day 7 (D7 EW). Similar to the alkaline-stress model SRS22A was not detected in any parasite vacuole beyond Day 1 in the infected astrocytes.

Bradyzoite subtypes initiate different developmental pathways
The discovery of SRS22A+ bradyzoites in tissue cysts of infected mice, but not in the alkaline stress-model of cyst development, raised questions about the developmental trajectory of both SRS22A- and SRS22A+ bradyzoite subtypes. The finding that cysts from chronically infected mice had mixed SRS22A (+ and -) bradyzoite populations (Fig. 1e) gave equal importance to investigating SRS22A- bradyzoites. Therefore, to determine the developmental paths of these bradyzoite subtypes, we sorted SRS22A+ and SRS22A- bradyzoites from brain tissue cysts by FACS (Fig. 3, Fig. S2). Identification and sorting of parasite populations revealed a distinct SRS22A+ expressing population representing approximately 60-80% of all Day 0 bradyzoites (Fig. S2). To determine any differential SRS22A-associated growth patterns, sorted populations were used to infect primary astrocyte cultures (Fig. 3a). Population growth and SRS antigen expression patterns (SAG1 versus SRS9) were determined during the maximal ex vivo growth stage at Day 5 post-infection. The infected cultures revealed clear differences in growth rates. SRS22A+ seeded cultures expanded 3.1-fold by Day 5, whereas parasite populations from SRS22A- seeded cultures only reached replacement levels (1.1-fold). Remarkably, the end-stage parasites (tachyzoites or bradyzoites) produced by these cultures were also different. On average over 80% of sorted SRS22A expressing bradyzoites converted to replicating tachyzoites (SAG1+ only) with very few bradyzoite vacuoles (2.3%, SRS9+ only). In contrast, bradyzoites that were originally SRS22A- led to the opposite developmental profile and replicated as bradyzoites expressing SRS9 and forming DBA positive cysts (Fig. 3a, b). Here, bradyzoites (30% of vacuoles, SRS9+ only) were the principal end-stage produced with few tachyzoite vacuoles present (3.7% vacuoles SAG1+ only). Tissue cyst wall assembly was only detected in the SRS9+/SAG1- vacuoles (Fig. 3b). We used a restrictive criterion for calling end-stages, and thus, in both SRS22A infections there were intermediate parasites (see examples Fig. S1C) co-expressing varying levels of SAG1 and SRS9 antigens (pairwise dull to bright staining; 16% in SRS22A+, >60% in SRS22A-) and made up the remaining proportion of parasites.

The preference of SRS22A- bradyzoites to initiate further bradyzoite replication suggests two major aspects of bradyzoite-cyst biology: firstly, that there is heterogeneity of bradyzoites within cysts and secondly that these subpopulations have preferential developmental trajectories. Our previous work revealed heterogeneity of bradyzoites during ex vivo recrudescence with a proportion of parasites (~20%) replicating as SRS9+ bradyzoites (brady-brady replication) in addition to the canonical brady-tachy pathway associated with cell lysis and pathology during reactivation13. To determine if these subpopulations of bradyzoites also represent a stable developmental phenotype, SRS9+ bradyzoites were sorted from recrudescing populations in astrocytes and evaluated for growth using SAG1 versus SRS9 antigen expression at Day 1-, 3- and 5-days post-infection (Day 7 parasites, Fig. 3C). As we demonstrated in Fig. 2, Day 7-bradyzoites spontaneously assemble tissue cyst walls (DBA+) but do not express SRS22A, and are therefore, SRS9+/SRS22A-. Pre-sort, SRS9+ parasites (undergoing brady-brady replication) represented ~8% of the population (Fig. 3C) and as expected sorted parasites retained the SRS9+ only pattern 24h after inoculation (Fig. 3C, Day 1). However, these parasites continued to express only SRS9+ over the course of the next five days and did not express SAG1. It was apparent that these bradyzoites retained their ability to replicate, evidenced by the increase in SRS9+ only vacuole size (Fig. 3C, column numbers). After 5 days of sorted parasites being in culture ~75% of vacuoles remained brady-brady replicating parasites successfully making cyst wall proteins (Fig. 3D). A proportion of parasites (~20%) did convert to SAG1+ only vacuoles, however, these parasites were not growing well (average vacuole size of <2). It is notable that tachyzoites in the pre-FACS Day 7-population grew better with an average vacuole size of 14.3 likely because the parentage of these parasites was the fast-growing tachyzoite13. Altogether these data demonstrate that in vivo SRS22A- as well as in vitro SRS9+/SRS22A- bradyzoites are primary initiators of direct bradyzoite expansion, while in vivo SRS22A+ bradyzoites are responsible for recrudescence into the fast-growing tachyzoites.

Infection potency requires SRS22A+ bradyzoites
To evaluate whether FACS-purified SRS22A+ and SRS22A- bradyzoite populations are developmentally competent, we infected CBA mice with each subtype and assessed parasite burden during acute (5-days post-infection) and chronic infection (28-days post-infection). At the acute time point, SRS22A+ infections produced significantly more SAG1+ tachyzoites and vacuoles in peritoneal exudate cells (PECs) (Fig. 4a). Parasite burden in the liver, spleen and lung also trended higher in SRS22A+ infections (Fig. 4b). At 28-days post-infection, mice infected with SRS22A+ bradyzoites developed significantly more cysts than those infected with SRS22A- bradyzoites (Fig. 4c), consistent with the robust dissemination capacity of the fast-growing tachyzoites13 derived from SRS22A+ bradyzoites.

Single cell RNA-seq indicates evidence of multiple bradyzoite types
The above data using only three antigens, demonstrate heterogeneity of bradyzoites that predicts the developmental trajectory of parasites. To further understand the underlying basis for this heterogeneity, we analyzed the transcriptome of single, excysted ME49EW bradyzoites from 40-day infected mice (Figs. 5, 6, S3, and S4). Individual bradyzoites from two brain tissue cyst harvests collected more than 6 months apart (Fig. S3A, r=0.965) were captured using the 10x Chromium Single Cell Gel Bead kit and 3’- RNA-seq libraries sequenced, yielding ~22 billion reads with >80% mapping to the Toxoplasma genome and 4,190 high-quality bradyzoites (Fig. S3B). UMAP projections (R v4.4.2; Seurat 5.1.0) of bradyzoites from each cyst harvest produced matching parasite clusters (Groups A-E) (Fig. S3C). The merged data (Fig. 5a, UMAP projection) revealed ~1200–3300 mRNAs per bradyzoite Group (Pval<0.05, Supplementary Data 1) with 374 genes expressed in all Groups (Fig. 5b, Venn diagram). Group C, which interfaces with Groups A, B, and D, had the fewest (<75) unique transcripts. Approximately 40% of Toxoplasma genes are cell-cycle regulated, with peak transcription in G1 or in S/M phases27. Prior studies on tachyzoite cell cycle uncovered that bradyzoite-specific gene expression may also be linked to S/M phases27 and our scRNA-sequencing supports this: Group A-E shown early twice the expected (17%) proportion of S/M-enriched transcripts (except Group D), while G1 levels match predictions (~23%) (Fig. 5c). Nevertheless, these transcriptomes did not phenocopy the synchronous cell-cycle contours seen in ex vivo ME49EW tachyzoites13, and principal component analysis indicates that neither cell-cycle stage nor canonical bradyzoite markers (BAG1, LDH2, ENO1) drive major variation (Fig. S3E), unlike alkaline-stress studies28,29. Because cell-cycle markers are indirect measures of cell proliferation, DNA content remains the most accurate metric. Our previous flow cytometric analysis showed ~2% of these bradyzoites possess a 1N+ DNA content13. Group E, which expresses the most S/M transcripts (745 mRNAs) and elevated growth-related mRNAs (Fig. S3D; Fig. S4C), likely represents this small replicative subset within otherwise dormant cyst populations13.

To determine the possible functional differences among the clusters, differentially regulated transcripts in Groups A-E were compared to the transcriptome of Day-7 BAG1+ ME49EW bradyzoites. These bradyzoites spontaneously form in primary astrocytes following recrudescence13 and represent a replicating, cyst forming and developmentally competent population of bradyzoites in which to compare our originating bradyzoite cyst clusters. A combined UMAP projection of the in vivo versus ex vivo bradyzoites demonstrated clear separation of the in vivo bradyzoite Groups (A-E) from the distinct cluster of D7-BAG1+ bradyzoites from astrocytes (Fig. S3F). Hundreds of up- and down-regulated mRNAs were detected in Group A-E bradyzoites (Fig. S3F) compared to D7-BAG1+ bradyzoites (log2 fold-change results, Supplementary Data 2) with many of the highest up-regulated mRNAs encoding recognized canonical bradyzoite protein markers (e.g. BAG1, LDH2, ENO1), selective rhoptry and GRA proteins, ApiAP2 factors, and SRS proteins (Fig. 5c; Supplementary Data 2). Considering the significance of these proteins in Toxoplasma biology, we also compared their expression among Groups A-E. AP2 and SRS mRNA profiles (Supplementary Data 1) differed by bradyzoite Group, and AP2IX-9 was the highest expressed ApiAP2 factor in Groups A-D (Fig. S4A). The mRNA encoding tachyzoite-specific antigen, SAG1, was the most significantly down-regulated transcript in Group A-E bradyzoites (Fig. 5d, and Fig. 6a). The presence of SAG1 on the surface of parasites is a distinguishing marker of bradyzoites that develop in cell culture models from the in vivo bradyzoites of infected mice13.

In the experiments above, we established the patterns of in vivo bradyzoite SRS22A antigen expression and determined the distinct developmental of SRS22A+ and SRS22A- bradyzoite subtypes (Fig. 3). Thus, the expression profile of SRS22A mRNA in bradyzoite Groups A-E was of considerable interest. Indeed, SRS22A transcript expression was found highly focused in Group B bradyzoites accounting for 81% of the bradyzoites (Fig. 6a). Consistent with the lack of SRS22A protein expression in cell culture-formed bradyzoites (Fig. 2), we did not detect SRS22A mRNA in D7-BAG1+ bradyzoites (Fig. S3G). Several other features of Group B bradyzoite transcription were notable. (1) The small heat shock protein, BAG1 was the first bradyzoite-specific marker characterized30–32 and has served as a universal marker for over 30 years. It was therefore unexpected that over half of Group B bradyzoites lacked detectable expression of BAG1 mRNA (Fig. 6a). By contrast, Group B bradyzoites as well as bradyzoite Groups A, C & D and to a lesser extent Group E bradyzoites expressed high levels of LDH2 mRNA (Fig. 6a; Fig. S4A) supporting LDH2, a lactate dehydrogenase, as a more reliable marker for in vivo bradyzoites. (2) The SRS22 family of surface proteins are primarily merozoite-specific23,24 and Group B bradyzoites expressed two SRS22 members, SRS22A as mentioned and also SRS22i mRNA (Fig. 6b). The analysis of the Group B bradyzoites lacking BAG1-identified two other SRS22 family members, SRS22E and SRS22G, although the expression of these SRS22 family members were lower than SRS22A and SRS22i (Supplementary Data 1). (3) Group B bradyzoites also had the highest levels of 24 dense granule protein (GRA) mRNAs including bradyzoite-specific GRA46 and GRA63 (Fig. 6a) as well as merozoite GRA proteins GRA11A and GRA11B and GRA80-82 (Fig. S4A, B). (4) Finally, Group B bradyzoites expressed significantly higher levels (e.g. 21-fold higher Group B vs D, Supplementary Data 1) of merozoite transcription factor, AP2VIIa-1 (Fig. 6c)24. Altogether, these features may indicate Group B bradyzoites are poised to initiate merozoite development in the cat. Further study will be required to understand whether Group B bradyzoites or another in vivo bradyzoite group are the primary drivers of bradyzoite-to-merozoite development.

In addition to the distinct signature of Group B SRS22A expression, the in vivo bradyzoite Groups A-E displayed other distinctive SRS mRNA profiles (Fig. 6b). Ten SRS protein mRNAs had peak levels in Group E bradyzoites (Fig. 6b, Supplementary Data 1), including four merozoite-specific SRS proteins, SRS16E, 23, 30C, 53F and one SRS mRNA encoding a tachyzoite-associated protein SRS20A. The SRS mRNA profiles of bradyzoite Groups C and D were partially shared reflecting the nearly 70% overlap of Group C transcripts with the transcriptome of Group D bradyzoites. In particular, the expression of the SRS49 gene family mRNAs (SRS49A-D) was a distinct feature of bradyzoite Groups C and D (Fig. 6a, b). Group D bradyzoites had the highest proportion of unique mRNA expression (Fig. 5b, 1074 mRNAs) and also one of the lowest levels of SRS22A expression indicating Group D is likely a major source of SRS22A- bradyzoites. Intriguingly, the highest levels of SRS9 mRNA expression were in Group C and D, which correlates with the SRS22A- bradyzoite phenotype (see Fig. 3). The results above demonstrated SRS22A- bradyzoites initiate bradyzoite-to-bradyzoite development and it was SRS22A- bradyzoite infections that were responsible for spontaneous cyst wall formation (Fig. 3 and 13). Consistent with these findings, Group C and D bradyzoites expressed the highest levels of SRS mRNAs that encode major cyst wall proteins, with 75% of bradyzoites expressing SRS44 (CST1) (Fig. 6a) and 62% of bradyzoites expressing SRS13 (Fig. S4A).

Group A bradyzoites express the 3rd highest number of unique transcripts (Fig. 5b, 172 mRNAs) including many hypothetical proteins and increased expression of selective rhoptry and RON mRNAs (Fig. 6a, Fig. S4C). This characteristic partially overlaps with Group E, which also shows increased BRP1 and bradyzoite-specific ROP transcripts (e.g. ROP28, 30, 42, 43; RON2L1, Fig. S4A)23,24,33 and the Group A-enhanced ROP kinase (TGME49_308093, Fig. S4)). In Group A, the strong upregulation of rhoptry mRNAs (e.g. >50-fold for some), is not due to S/M cell cycle enrichment34, unlike Group E, where rhoptry expression correlates with S/M transcripts (Fig. S4C, A vs E heatmap). Both Group A and E display elevated AP2XII-9 (Fig. 6a), a known activator of rhoptry and RON genes35,36. We do not fully understand the biological role for increased rhoptry gene transcription in Group A bradyzoites; however, our previous cell cycle studies of rhoptry proteins in tachyzoites37 demonstrated the transcription of this class of mRNAs was uniquely sensitive to intercellular egress and invasion, which might indicate Group A bradyzoites were preparing to egress prior to tissue cyst harvest.

Finally, trajectory analysis, although not definitive, supports the presence of alternative bradyzoite developmental programs as seen with SRS22A+ and SRS22A- parasites. The inferred trajectory begins with parasites in Group D, progresses toward the transitional Group C/D population, and then bifurcates toward either Group B (SRS22A+) or A (Fig. S5B). These scRNA-seq data support the SRS22A and SRS9 findings that cysts contain distinct subsets of bradyzoites that lead to predictable patterns of development. These five populations separate based, not on cell cycle, but signatures that include characteristic proteins involved in parasite invasion and developmental regulation suggesting some of these populations are precursors to merozoites, tachyzoites and renewal of the bradyzoite population.

Methods
Parasite maintenance and mouse infections
Our research complies with all relevant ethical regulations (Biological Use Authorization, BUA-R202 approved by the Institutional Biosafety Committee at Environmental Health and Safety Office at the University of California, Riverside). All animal experiments (Protocol #108) were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Riverside. Six-week-old female CBA/J (strain #: 000656), C57BL/6J (strain #: 000664) and Swiss Webster (SWR/J) (strain #: SW-F, Taconic Biosciences) mice were obtained from Jackson Laboratories (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and Taconic Biosciences (Rensselaer, NY, USA) and maintained in a pathogen-free vivarium on a 12 h light/12 h dark cycle at 20–22 °C room temperature with ad libitum access to water and food. Type II ME49EW parasites were propagated in vivo by alternative passage between resistant (Swiss Webster, SWR/J) and sensitive (CBA/J) mouse strains47. Mice were infected via intraperitoneal injection with 10 tissue cysts (in 200 μl) prepared from brain homogenates of mice infected for 30 days. It has previously shown that it takes about a week for tissue cysts to appear in mouse infections62. Consistent with this, we have previously shown that excysted bradyzoites differentiate into fast-growing tachyzoites and then switch to a slower growth phase and reform bradyzoites after day 613. Considering these findings, we did not examine tissue cysts earlier than 14 days post-infection. For some experiments we needed a stable tachyzoite source for comparison purposes or to duplicate the conventional alkaline-stress model (Figs. 1a, 2a). In these experiments, we used the genome reference strain Type II ME49B7, which has been adapted to in vitro cell culture. A continuous replicating tachyzoite form for in vitro experiments is not available for the ME49EW strain as it is maintained exclusively in vivo. We also used tachyzoites of the Type I RH strain to check the bradyzoite-specificity of the anti-SRS22A antiserum in Western analysis (Fig. 1b).

Bradyzoite purification and ex vivo cultures following recrudescence
Following 40-day post-infection, ME49EW bradyzoites were isolated from infected mouse brains (CBA mice) via Percoll gradient13. Neonatal (from C57BL/6J mouse pups) cortical astrocytes grown in T-75 cm2 vented flasks were used for ex vivo parasite cultures initiated by excysted bradyzoites. Primary astrocyte cultures were inoculated with excysted bradyzoites or day 3 and 5 parasite cultures at an MOI of 0.5 as previously described13. Infected cells were cultured in DMEM media supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin (Genclone), 1% GlutaMax supplement (Thermo Fisher Scientific, Waltham, MA, USA), 1% Na pyruvate and 2.5% HEPES buffer (Thermo Fisher Scientific Waltham, MA, USA) and placed in humidified hypoxia chambers containing 5 % oxygen and 5 % CO2 (nitrogen balanced).

Production of antiserum against SRS22A
Rabbit antiserum was generated in-house against the Toxoplasma protein SRS22A using a selected peptide sequence (Fig. S1A, LRGNDGRSSRVIEKEAEVAK). Rabbits were immunized with the peptide, and antisera were collected at 28 days (first bleed), 56 days (second bleed), and 72 days (third bleed) post-immunization (Thermo Fisher Scientific Waltham, MA, USA). The peptide (20 amino acids) selected for production of anti-SRS22A antisera was confirmed by Protein BLAST to be uniquely present in the SRS22A protein sequence among SRS family and other proteins encoded in the Toxoplasma genome.

FACS sorting of bradyzoite populations
Upon excystation of purified ME49EW cysts, at least one million bradyzoites were stained for flow cytometry analysis using specific antibodies against SRS9 or SRS22A. Briefly, parasites were incubated with rabbit anti-SRS9 (kindly provided by John Boothroyd, Stanford University) or rabbit anti-SRS22A (generated in-house). Primary antibody incubation was performed at a 1:50 dilution in PBS in 5 ml round-bottom FACS tubes for 30 min at room temperature. Following incubation, each sample was washed with 1 ml of sterile PBS and centrifuged at 450 g for 5 min. Secondary antibodies were diluted 1:1000 in PBS, with Alexa Fluor 647 goat anti-rabbit IgG (A-21244, Lot: 2527970, Invitrogen, Carlsbad, CA, USA) used for SRS9 and Alexa Fluor 488 goat anti-rabbit IgG (A-11008, Lot: 1678787, Invitrogen, Carlsbad, CA, USA) used for SRS22A. Parasites were then resuspended in 50 µl of diluted secondary antibodies and incubated for 30 min at room temperature in the dark. After incubation, stained parasites were washed with 1 ml of PBS, centrifuged, and resuspended in 300 µL of PBS for FACS analysis. Flow cytometry and cell sorting were performed using the MoFlo Astrios EQ Cell Sorter (Beckman Coulter, Brea, CA, USA) at the UC Riverside School of Medicine Flow Cytometry Core Facility to identify and sort distinct SRS9+ or SRS22A+ parasite populations. SRS9 and SRS22A expression were detected in the APC and FITC channels, respectively, and parasite populations were collected in 1 ml of infection media. Primary astrocyte cultures grown on coverslips were infected either with purified SRS22A+ or SRS22A− sorted parasites (500 parasites per well in 6-well plates, approximately MOI of 0.0004) or with SRS9+ parasites (2500 parasites per well in 24-well plates, approximately MOI of 0.01). Full gating strategy, controls and reproducibility data are shown in Fig. S2A-D. FACS purification of the SRS22A+ and SRS9+ populations was highly reproducible as evidenced by low variability obtained from separate experiments run on different dates (Fig. S2B-D).

Immunofluorescence staining
Population growth and SRS antigen expression patterns (SAG1 vs. SRS9) were assessed in primary astrocytes infected with sorted parasite populations on Day 5 post-infection. Cells were fixed in 4% paraformaldehyde (PFA), followed by three 1 min washes with PBS. After permeabilization in acetone for 10 min, cells were washed three more times with PBS for 1 min each. Next, cells were blocked with 5% donkey serum in PBS and incubated with antibodies specific for bradyzoites and tachyzoites, including rabbit anti-SRS9 (1:1000 dilution), mouse anti-SAG1 (1:1000 dilution, catalog #9070-2020, Lot: 162026, Bio-Rad, Hercules, CA, USA), and biotinylated DBA (1:500 dilution, B-1035-5, Lot: ZJ1117, Vector Laboratories, Newark, CA, USA) to identify the cyst wall. Primary antibody incubation was carried out overnight at 4°C. The following day, cells were washed three times with PBS for 5 min each and incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies used included donkey anti-rabbit Alexa Fluor 568 (1:1000 dilution, A10042, Lot: 1964370, Invitrogen, Carlsbad, CA, USA), goat anti-mouse Alexa Fluor 488 (1:1000 dilution, A-11029, Lot: 2821059, Invitrogen, Carlsbad, CA, USA), and Alexa Fluor 647-streptavidin conjugate (1:1000 dilution, S32357, Lot: 2179341, Sigma-Aldrich, St. Louis, MO, USA) to detect rabbit anti-SRS9, mouse anti-SAG1, and biotinylated DBA, respectively. After incubation, cells were washed three times with PBS for 10 minutes each and rinsed once with autoclaved water. DAPI VIBRANCE (Vectashield Vibrance, H-1800, Vector Laboratories, Newark, CA, USA) was then applied to each coverslip before mounting onto a glass slide. Parasite vacuoles were counted using a Leica DMI 6000 immunofluorescence microscope with a 40X objective, and vacuoles were enumerated.

Single-cell RNA-sequencing of in vivo bradyzoites
Single-cell RNA sequencing (scRNA-seq) was performed on excysted bradyzoites and parasite cultures obtained from ex vivo Day 7 astrocytes at a sequencing depth of one billion reads13. This was achieved using the 10X Genomics Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (10X Genomics, Inc., Pleasanton, CA, USA)13. Sequencing was conducted at the San Diego IGM Genomics Center using an Illumina NovaSeq 6000. As before, Cell Ranger version 5.0.1 was used for read alignment to a custom reference genome (ME49 Toxoplasma gondii reference genome) and for count matrix generation. The scRNA-seq data were embedded using Uniform Manifold Approximation and Project (UMAP) and parasite transcriptome intersection determined using the Seurat package (R version 4.4.2 and Seurat 5.1.0). scRNA-seq data were analyzed using the Seurat package (R version 4.4.2 and Seurat 5.1.0) with libraries ‘ggplot2’, ‘tidyverse’, ‘conflicted’, ‘gridExtra’, ‘dplyr’, ‘EnhancedVolcano’ and ‘ggrepel’. Dimensionality reduction was first performed using Principal Component Analysis (PCA), followed by construction of a Shared Nearest Neighbor (SNN) graph via the ‘FindNeighbors’ function. Cells were then clustered using the Louvain algorithm at a resolution of 0.2, yielding five transcriptionally distinct clusters. For visualization, cells were embedded in two dimensions using (UMAP).’ A non-parametric Wilcoxon rank test was applied to identify differentially expressed genes among Groups A-E. Volcano plots were generated from a comparison of each bradyzoite Group A-E (chronic phase infection) and the BAG1+ bradyzoites (early phase) that spontaneously arise in our ex vivo bradyzoite recrudescence model at Day-7 as explained in Results.

Trajectory analysis was performed using the dyno R package (dyneverse.org). We followed the recommendations of the Guidelines Shiny application to select trajectory-inference methods appropriate for our dataset. Based on these guidelines, we implemented four recommended methods including Slingshot, PAGA, PAGA-tree, and MST.

Bradyzoite differentiation assay under alkaline stress
Alkaline stress assay was performed in HFFs (ATCC SCRC-1041)63,64. A bradyzoite differentiation media was prepared using RPMI 1640 (GIBCO) medium supplemented with 5% FBS, 50 mM HEPES, 100 unit/ml penicillin, and 100 μg/ml streptomycin. The alkaline medium pH was adjusted to 8.2 using NaOH. Briefly, HFFs were infected with ME49B7 parasites for 4 h, and then media was removed and rinsed four times with parasite media (5% FBS, 1% Gln, 100 unit/ml penicillin, and 100 μg/ml streptomycin) to remove unattached parasites. The bradyzoite differentiation media was added onto the cells and cell culture flasks were placed at 37°C for 48 h. Cells were fixed in 4% PFA post-infection and then stained for SAG1, SRS9 and SRS22A using immunofluorescence staining as described above.

Preparation of PECs and brain homogenates at days 5- and 28-post-infection
On 5-days post-infection, CBA mice were anesthetized and injected intraperitoneally with 5 ml of cold PBS with 1% BSA. Peritoneal exudate cells were collected by withdrawing the injected solution and subsequently enumerated. For each mouse, 100,000 cells were deposited onto microscope slides using cytospin funnels and centrifugation at 300 g for 5 min. On 28-days post-infection, infected mouse brains were harvested in 3 ml cold PBS, and brain homogenates were prepared by sequentially passing the tissue through 18G, 20G, and 22G needles. A 30 ul aliquot of brain homogenate was deposited on a microscope slide, smeared using a coverslip and allowed to dry. Slides were then fixed in methanol and stored at −20°C until immunofluorescence staining was performed.

Detection of parasite burden in mouse tissues
Liver, spleen and lung were harvested from infected CBA mice and stored at −80°C prior to DNA purification. DNA was purified using a DNeasy mini kit (Qiagen) according to manufacturer’s instructions. The qPCR reaction used 600 ng of DNA from each organ, 0.375 μM B1 gene primers (Forward: 5′- TCCCCTCTGCTGGCGAAAAGT-3′; Reverse: 5′- AGCGTTCGTGGTCAACTATCG-3′), and 2x SensiFAST SYBR No-ROX qPCR mastermix (catalog #C755J00, Thomas Scientific, Swedesbro, NJ, USA) in a 20 μl total volume. The qPCR program was: 3 min at 95°C, 40 cycles of 10 s at 95°C for denaturation, 30 s at 60°C for annealing, followed by 5 s at 65°C and 5 s at 95°C for extension. The real-time data was collected on a BioRad CFX96 Touch Real-Time PCR Detection system, using Bio-Rad CFX software. A standard curve was generated with serial dilution of purified ME49 genomic DNA and used to calculate pg amount from the equation displayed on the linear trendline (R2=0.99). All results were expressed as the pg of parasite genomic DNA calculated from an experimentally generated standard curve and relative expression was presented on each graph as compared to naïve controls.

Western blot analysis of SRS22A
Cysts were purified from the brains of three CBA mice infected with ME49EW for 30 days. Bradyzoites were excysted from the pooled cysts by 90 seconds of pepsin-HCl treatment at 37°C. RH grown in HFFs were used as a negative control. These parasites were lysed by a 25G needle and filtered in a 3 µm filter. After washing the parasites, protein lysates were made by adding 100 μL NP-40 buffer with protease inhibitors for each 1×10⁶ pelleted parasites. After keeping on ice for 10 minutes, the lysate was centrifuged at 14,000 xg for 10 min at 4°C, and the supernatant collected. 1 μg of each protein lysate was separated by SDS-PAGE (15% resolving gel, 5% stacking gel) for 2 h and transferred onto a 0.2 µm nitrocellulose membrane for 35 minutes. Membranes were incubated in 5% milk in 0.1% PBS-T for 30 minutes at room temperature, then probed with rabbit-α-SRS22A antibody (1:500) or pre-immune serum (1:500) overnight at 4°C. After washing, membranes were probed with goat-α-rabbit HRP conjugate (Bio-Rad #1705046, 1:10,000, Hercules, CA, USA) for one hour at room temperature. The membranes were then reprobed with rabbit-α-PCNA antibody (1:2,500, a gift from Dr. Michael White’s laboratory65,66) overnight at 4°C, followed by goat-α-rabbit HRP conjugate for one h at room temperature. Importantly, signal was detected with the SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific #34094). Membranes were imaged on the Bio-Rad Gel Doc XR+ System at an exposure of 5 seconds.

Statistical analysis
Statistical analyses for tissue cyst diameter, cyst counts, SRSR22A staining intensity, vacuole counts, and parasite burden were conducted in GraphPad Prism (version 10.2.3) using one-way or two-way ANOVA or two-tailed t-tests, as appropriate. A P-value<0.05 was considered statistically significant. scRNA-seq data were analyzed using Seurat67 as described above.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper provides significant new insights into the biology of T. gondii, which is an important human and veterinary pathogen. In particular, the observation on bradyzoite subtypes provides a rigorous definition of the heterogeneity of the T. gondii cyst (as established by work by Sinai's laboratory (PMID:26350965 PMCID:PMC4600105)). The identification of SRS22A as a marker for bradyzoites that are committed to the rapid growth (and merozoite transition) in the cat intestine is an important finding for understanding this developmental pathway. Furthermore the identification of SRS9 positive SRS22A negative bradyzoites as a group committed to brady to brady growth is also an important insight into infection dynamics. The ability to FACS sort these populations is an important observation and a significant advance for dissection of what is going on in the tissue cyst. As has been elucidated by other investigators, the in vivo cyst is different/ more mature then the cysts formed in vitro using high pH or low CO2 differentiation conditions. The scRNA data supports the staining and in vitro data on FACS sorted bradyzoites and establishes the presence of 5 populations (although functionally this may only be 3, however this remains to be determined).\n\nA few questions and suggestions\n1. Did the investigators try to examine in vitro long term cultures in muscle myoblasts (PMID: 38213326) or similar cells. The data suggests that cysts formed in these culture systems may be more similar to in vivo cysts? This longer term in vitro culture data (day 21 etc.) might be interesting as a comparison for your scRNA data in vivo. However, given the difficulty of this in vitro myoblast culture system it is not essential for the current manuscript.\n2. To that end, it would be useful if it was more clearly stated what the data of in vivo cysts would look like at 2 to 4 days after formation of cysts; as the culture systems are being examined at very early time points after formation of the bradyzoite parasitophorous vacuole\n3. Work by Sinai should be added to the paper in the discussion of cyst heterogeneity (e.g.PMID:26350965, PMID: 40492719, PMID: 3673022)\n4. As to scRNA there is also work by Sugi on this (as it applies to host cells), e.g. PMID: 35372115. Is there anything in this data that corresponds to what was found in your scRNA For the in vitro cysts.\n5. There is no accession number for the scRNA data, as noted this needs to be deposited in GEO and an accession number provided. I also suggest this data be provided to ToxoDB (EuPathDB) for incorporation into that widely used web resource.\n6. References 25 and 26 are not fully cited (the titles are cut off).\n7. For BAG1/BAG5 you are missing PMID: 10027984 which describes the phenotype of a BAG1 knockout.", "reviewer_2": "Summary and Overall Assessment\nThis manuscript presents evidence that native Toxoplasma gondii ME49EW cysts harbor heterogeneous bradyzoite subtypes distinguished by SRS22A expression. SRS22A⁺ bradyzoites preferentially recrudesce to fast-growing tachyzoites, whereas SRS22A⁻ bradyzoites tend toward brady-brady replication and cyst-wall reinforcement. Single-cell RNA-seq (~4,190 excysted bradyzoites) resolves five clusters (A–E): Group B is enriched for SRS22A and frequently lacks BAG1, and Group E shows S/M-biased “replicative” signatures. Conventional in-vitro models fail to detect SRS22A protein. The question is important and the dataset is potentially impactful; however, several controls, standardizations, and analyses are required to rigorously support the central claims.\n\nMajor Strengths\n• Addresses a core gap in native bradyzoite biology using complementary approaches (IFA/FACS, ex vivo astrocyte infection, mouse infection, 10x scRNA-seq).\n• Proposes a mechanistic framework linking bradyzoite subtypes to divergent developmental outcomes.\n• Generates a resource that could recalibrate how we model chronic infection in vitro.\n\nSuggested Revisions\n1. Antibody validation and specificity for SRS22A: You generated a polyclonal antiserum against a single SRS22A peptide and show bradyzoite-specific bands/staining, but there’s no peptide competition (antigen pre-absorption) control, isotype control, or demonstration of cross-reactivity against closely related SRS22 paralogs (SRS22i, SRS22E/G) that you discuss extensively. Add peptide competition IFA and immunoblot; test binding to recombinant/overexpressed SRS22 paralogs, or perform CRISPR knockout/conditional knockdown rescue of SRS22A to demonstrate loss and specificity of signal.\n2.Reliance on two different ME49 lineages for in vivo vs in vitro. Key contrasts hinge on ME49EW (maintained in vivo) vs ME49B7 (adapted to culture) and RH (Type I) for some blots. Genetic and epigenetic divergence makes it hard to attribute differences (e.g., absence of SRS22A in vitro) to “model limitations” rather than strain history. Please (i) use the same lineage wherever possible (e.g., derive in vitro bradyzoites from ME49EW via limited short-term passage), or (ii) complement with additional Type II lab strains and show the phenotype generalizes. At minimum, acknowledge and temper causal claims.\n3. FACS gating and sorting controls. The core conclusion (distinct developmental fates of SRS22A+ vs SRS22A− bradyzoites) rests on antibody-based sorting. Provide full gating strategy, FMO/isotype controls, doublet discrimination, viability dye, compensation matrices, sort purity checks, and a re-sort or post-sort IFA to confirm identity and purity of each fraction. Include replicate sorts across independent mouse infections and show between-experiment variance.\n4. Causality vs correlation of SRS22A. Data support that SRS22A+ bradyzoites are associated with tachyzoite recrudescence, but do not prove SRS22A is functionally required. Consider genetic perturbation (SRS22A KO/conditional depletion or overexpression) to test whether altering SRS22A levels changes the brady-tachy vs brady-brady outcome. If not feasible now, the manuscript should clearly reframe the claim as associative and propose SRS22A as a marker, not a driver.\n5. Alkaline stress model conclusions. The statement that conventional in vitro models “do not form SRS22A+ bradyzoites” is based on 48-h pH 8.2 HFF culture. Test additional differentiation protocols (longer durations, neuronal/astrocytic cells, hypoxia, different CO₂, serum deprivation, host cytokine milieu) before making general claims. Alternatively, narrow the claim to the specific conditions tested.\n6. Interpretation about merozoite priming. The suggestion that Group B bradyzoites may be poised for feline merozoite development is speculative. Either (i) provide orthogonal evidence (e.g., AP2VIIa-1 protein, promoter accessibility, or functional data in feline enterocytes/organoids), or (ii) clearly label as hypothesis and tone down the conclusion.\n7. As a suggestion for Figure 7 and the discussion, we encourage you to integrate recent evidence that elements of the chronic-stage program can initiate independently of BFD1 (Robert et al., EMBO Mol. Med., 2025). Concretely, testing BFD1 dependence in your scRNA-seq by examining BSM and BCLA across clusters A–E would help identify BFD1-dependent versus BFD1-independent subpopulations and allow you to incorporate those assignments directly into the model and narrative. We also recommend situating your framework explicitly within a continuum—rather than a binary switch—by comparing your inferred trajectories to proposals of epigenetically primed, transcriptionally poised, context-responsive intermediate states (Shahinas et al., COM, 2025), and mapping clusters A–E onto that spectrum to highlight points of convergence and divergence. Finally, it would be valuable to assess whether your dataset contains a hybrid P6-like population co-expressing tachyzoite and bradyzoite markers (Xue et al., Boothroyd JC. eLife, 2020). Marker overlays, module scores, and cell-cycle–aware analyses could clarify whether Group B or E aligns with this hybrid profile and whether such cells act as progenitor-like intermediates within the bradyzoite lineage.", "reviewer_3": "This manuscript represents a significant advance in our understanding of Toxoplasma gondii bradyzoite biology. The authors provide compelling evidence that bradyzoite populations are heterogeneous and that distinct subgroups possess unique developmental potentials. Their findings explain several long-standing observations related to bradyzoite infection dynamics and reactivation.\n\nThe authors identified a novel surface marker, SRS22A, which is specifically expressed on bradyzoites derived from in vivo tissue cysts. They also demonstrated that SRS22A-positive and SRS22A-negative bradyzoites have distinct functions—the former are capable of robust conversion to fast-replicating tachyzoites in astrocytes, while the latter appear to participate primarily in bradyzoite-bradyzoite replication. Supporting this finding, they show using an in vivo infection model that SRS22A-positive bradyzoites disseminate more efficiently and form tissue cysts more readily than their SRS22A-negative counterparts. Finally, using single-cell RNA sequencing, they identified five distinct bradyzoite subpopulations (Groups A–E) within in vivo cysts. Each group exhibits unique transcriptional profiles suggestive of specific developmental trajectories. For example, Group B bradyzoites—expressing the highest levels of SRS22A—appear transcriptionally primed for conversion to tachyzoites or merozoites, whereas Groups C and D, which lack SRS22A expression, show elevated expression of cyst wall mRNAs consistent with continued bradyzoite replication.\n\nCollectively, these results convincingly demonstrate that bradyzoite development is not a linear process and that multiple developmental fates coexist within chronic-stage cysts. This work provides strong evidence for the functional heterogeneity of bradyzoite populations and offers a refined framework for understanding chronic toxoplasmosis.\n\nThe study is well-designed, the experiments are appropriately controlled, and the manuscript is clearly written. The authors also transparently address the limitations of their approach. Importantly, this work opens several exciting avenues for future research, such as identifying the triggers that drive each bradyzoite subgroup’s developmental trajectory.\n\nIn this reviewer’s opinion, the quality, novelty, and depth of this study meet the standards expected for Nature Communications.\n\nMinor comment:\nPage 8, line 2: The text should refer to Figure 2B rather than 2A."}, "author_response": "We thank all the reviewers for their time and comments on our manuscript. We\nanswered their questions point-by-point below in bold font, and modified the\nmanuscript accordingly. The modifications were highlighted in the manuscript in red\nin the Marked copy.\nReviewer’s Comments:\nReviewer #1 (Remarks to the Author)\nThis paper provides significant new insights into the biology of T. gondii, which is an\nimportant human and veterinary pathogen. In particular, the observation on bradyzoite\nsubtypes provides a rigorous definition of the heterogeneity of the T. gondii cyst (as\nestablished by work by Sinai's laboratory (PMID:26350965 PMCID:PMC4600105)). The\nidentification of SRS22A as a marker for bradyzoites that are committed to the rapid growth\n(and merozoite transition) in the cat intestine is an important finding for understanding this\ndevelopmental pathway. Furthermore the identification of SRS9 positive SRS22A negative\nbradyzoites as a group committed to brady to brady growth is also an important insight into\ninfection dynamics. The ability to FACS sort these populations is an important observation\nand a significant advance for dissection of what is going on in the tissue cyst. As has been\nelucidated by other investigators, the in vivo cyst is different/ more mature then the cysts\nformed in vitro using high pH or low CO2 differentiation conditions. The scRNA data\nsupports the staining and in vitro data on FACS sorted bradyzoites and establishes the\npresence of 5 populations (although functionally this may only be 3, however this remains\nto be determined).\nWe appreciate the reviewer’s assessment and recognition of the significance of the\nwork.\nA few questions and suggestions\n1. Did the investigators try to examine in vitro long term cultures in muscle myoblasts\n(PMID: 38213326) or similar cells. The data suggests that cysts formed in these culture\nsystems may be more similar to in vivo cysts? This longer term in vitro culture data (day 21\netc.) might be interesting as a comparison for your scRNA data in vivo. However, given the\ndifficulty of this in vitro myoblast culture system it is not essential for the current\nmanuscript.\nWe thank the reviewer for this suggestion and agree the myoblast culture system\noffers one of the best options for long term culture and in vitro propagation of cysts.\nSuch culture systems will likely be valuable to directly assess questions of cyst\nmaturity in vitro. Of note, the Luder lab examined parasite gene expression in four\ndifferent murine cell types including neurons, astrocytes, fibroblasts and skeletal\nmuscle cells1. The expression of SRS22A was very low in all four cell types agreeing\nwith our findings with the alkaline-stress model and ex vivo recrudescence models\nshown in Figure 2. We have now included this citation in the paper on page 15 (in red).\nA more complete evaluation of the available in vitro bradyzoite developmental models\nwill be an important future addition to the field.\n2. To that end, it would be useful if it was more clearly stated what the data of in vivo cysts\nwould look like at 2 to 4 days after formation of cysts; as the culture systems are being\nexamined at very early time points after formation of the bradyzoite parasitophorous\nvacuole.\nThe question of when functional bradyzoites are present in mice was historically\nexamined in an early 1997 paper2 using in vivo bradyzoites to orally infect naive mice.\nIt takes about a week for functional tissue cysts to appear in murine infections. In our\n2023 mBio paper we show that bradyzoites first differentiate into fast-growing\ntachyzoites and only reform bradyzoites after a shift to slower growth at Day 6-7,\nwhich explains why it takes a week in animals. The earliest cysts we examined (14dpi,\nFigure 1) will therefore be only 7 days after their formation. In addition, detailed\nkinetics of cyst dynamics by the Sinai group analyzed growth in cysts from 21dpi to\n56dpi with overall average size increasing over time. Our analysis of cysts even earlier\nthan this first time point with an average cyst size <20um (smaller than some of our in\nvitro cysts, Figure 3B) suggests we are indeed analyzing relatively recently formed\ncysts. We have clarified and expanded on these points in the Figure 1 experimental\nmethods (see page 19 Methods, in red).\n3. Work by Sinai should be added to the paper in the discussion of cyst heterogeneity\n(e.g.PMID:26350965, PMID: 40492719, PMID: 3673022)\nAs requested, we have included these citations on pages 5 (Results), 15 (Discussion).\n4. As to scRNA there is also work by Sugi on this (as it applies to host cells), e.g. PMID:\n35372115. Is there anything in this data that corresponds to what was found in your scRNA\nFor the in vitro cysts.\nWe thank the reviewer for bringing this to our attention. The paper by Sugi et al\nrevealed several clusters of host cell populations following infection and in vitro\ndifferentiation to bradyzoites supporting the concept of parasite heterogeneity at the\nbradyzoite level. In that work, the clustering reflects heterogeneity in the host cell\ntranscriptomes of bradyzoite-infected HFFs rather than in the parasites themselves. In\ncontrast, our analysis focuses on the transcriptional diversity among bradyzoites\nwithin cysts, distinguishing our findings from those reported by Sugi et al. We\nincorporated this point with a statement in the Discussion (page 16) as below:\n“…Another study found that BAG1⁺ bradyzoite-infected HFFs form two distinct\ntranscriptional subsets, suggesting that variability in host responses may reflect\nheterogeneity among the bradyzoites3.”\n5. There is no accession number for the scRNA data, as noted this needs to be deposited in\nGEO and an accession number provided. I also suggest this data be provided to ToxoDB\n(EuPathDB) for incorporation into that widely used web resource.\nWe deposited our data to GEO (Accession number: GSE311669), and will release\npublishing upon acceptance. In addition, we would be delighted to deposit our data\nwith ToxoDb upon invitation as soon as it is accepted so that it is easily accessible by\nToxoplasma research community.\n6. References 25 and 26 are not fully cited (the titles are cut off).\nWe corrected those references as suggested.\n7. For BAG1/BAG5 you are missing PMID: 10027984 which describes the phenotype of a\nBAG1 knockout.\nWe thank the reviewer for pointing out the missing citation on BAG1 knockout strains.\nWe now cite all BAG1 knockout references in the paper.\nReviewer #2 (Remarks to the Author)\nSummary and Overall Assessment\nThis manuscript presents evidence that native Toxoplasma gondii ME49EW cysts harbor\nheterogeneous bradyzoite subtypes distinguished by SRS22A expression.\nSRS22A⁺ bradyzoites preferentially recrudesce to fast-growing tachyzoites,\nwhereas\nSRS22A⁻ bradyzoites tend toward brady-brady replication and cyst-wall reinforcement.\nSingle-cell RNA-seq (~4,190 excysted bradyzoites) resolves five clusters (A–E): Group B is\nenriched for SRS22A and frequently lacks BAG1, and Group E shows S/M-biased\n“replicative” signatures. Conventional in-vitro models fail to detect SRS22A protein. The\nquestion is important and the dataset is potentially impactful; however, several controls,\nstandardizations, and analyses are required to rigorously support the central claims.\nMajor Strengths\n• Addresses a core gap in native bradyzoite biology using complementary approaches\n(IFA/FACS, ex vivo astrocyte infection, mouse infection, 10x scRNA-seq).\n• Proposes a mechanistic framework linking bradyzoite subtypes to divergent\ndevelopmental outcomes.\n• Generates a resource that could recalibrate how we model chronic infection in vitro.\nWe thank the reviewer for recognizing the data as potentially impactful and important.\nSuggested Revisions\n1. Antibody validation and specificity for SRS22A: You generated a polyclonal antiserum\nagainst a single SRS22A peptide and show bradyzoite-specific bands/staining, but there’s\nno peptide competition (antigen pre-absorption) control, isotype control, or demonstration\nof cross-reactivity against closely related SRS22 paralogs (SRS22i, SRS22E/G) that you\ndiscuss extensively. Add peptide competition IFA and immunoblot; test binding to\nrecombinant/overexpressed SRS22 paralogs, or perform CRISPR knockout/conditional\nknockdown rescue of SRS22A to demonstrate loss and specificity of signal.\nThe SRS antigens are a related gene family, however, they are also a divergent set of\nproteins. Overall, SRS protein sequences share only ~40% identity that is confined to\nspecific segments of protein structure, while other regions of SRS protein sequence\nshow little to no conservation. This feature has permitted successful antibody\nproduction in long term use for specific SRS proteins (e.g. commonly used SAG1 and\nSRS9 antibodies). To raise antibodies against SRS22A, we selected a peptide (20 amino\nacids) representing SRS22A protein sequence that is unique among SRS proteins as well\nas all other proteins encoded in the Toxoplasma genome. For example, the selected\nSR22A peptide sequence shares one amino acid with SRS22G protein sequence (see\nexample alignment of SRS22A vs SRS22G, 22i and 22E below, peptide sequence highlighted).\nFor SRS22i only four noncontiguous amino acids are shared with\nSRS22A. Consequently, there is very little chance the anti-SRS22A antisera raised\nagainst this peptide will cross react with other SRS antigens, which is borne out by the\nunique expression patterns of SRS22A in bradyzoites and tachyzoite\ninfections. Moreover, SRS22A mRNA is expressed at levels 20-75 fold higher in our\nbradyzoite subtypes than either SRS22i or SRS22G mRNAs decreasing the near zero\nchance of cross reactivity to these antigens to even less.\n2.Reliance on two different ME49 lineages for in vivo vs in vitro. Key contrasts hinge on\nME49EW (maintained in vivo) vs ME49B7 (adapted to culture) and RH (Type I) for some\nblots. Genetic and epigenetic divergence makes it hard to attribute differences (e.g.,\nabsence of SRS22A in vitro) to “model limitations” rather than strain history. Please (i)\nuse the same lineage wherever possible (e.g., derive in vitro bradyzoites from ME49EW via\nlimited short-term passage), or (ii) complement with additional Type II lab strains and show\nthe phenotype generalizes. At minimum, acknowledge and temper causal claims.\nThe ME49EW strain was chosen because it is a Type II strain that has not been adapted\nto cell culture as this causes major permanent developmental changes. ME49B7 is\nused here as a stable HFF-adapted strain to measure SRS22A expression in the most\ncommonly used method for in vitro bradyzoite induction (alkaline-stress). This was\ncompared to spontaneous induction of bradyzoites in the ex vivo recrudescence\nmodel we developed for the ME49EW strain. There was no SRS22A expression in\neither in vitro model indicating this is not due to strain differences. The use of RH\ntachyzoites is a negative control in Western Blots and was chosen because it is unable\nto differentiate. We have clarified in the paper that SRS22A was not expressed in\neither in vitro model we tested (Abstract, and page 15, in red). Future work will be\nneeded to fully evaluate other in vitro models as to the ability of these models to form\nthe different bradyzoite subtypes, which is beyond the scope of this paper.\n3. FACS gating and sorting controls. The core conclusion (distinct developmental fates of\nSRS22A+ vs SRS22A− bradyzoites) rests on antibody-based sorting. Provide full gating\nstrategy, FMO/isotype controls, doublet discrimination, viability dye, compensation\nmatrices, sort purity checks, and a re-sort or post-sort IFA to confirm identity and purity of\neach fraction. Include replicate sorts across independent mouse infections and show\nbetween-experiment variance.\nWe apologize that we did not provide all control data for the sorting analysis and thank\nthe reviewer for giving us the opportunity to include the following data, all of which are\nnow included in the Supplemental files, Supplemental Fig. S2A-D.\n1) Whole gating strategy with doublet discrimination.\n2) Pre- and post-sort viability and purity data of bradyzoite populations.\n3) Reproducibility of FACS sorting in experiments run on 4-5 different days.\n4) With regards to FMO control and compensation matrices, since we only have\none antibody, we only included an unstained control FACS plot.\nThis additional information is included in Methods, Section ‘FACS sorting of bradyzoite\npopulations’\n“Full gating strategy, controls and reproducibility data were shown in Fig. S2A-D. FACS\npurification of the SRS22A+ and SRS9+ populations was highly reproducible as evidenced\nby low variability obtained from separate experiments run on different dates (Fig. S2B-D).”\nWe included below the new Fig. S2 for ease of access to the reviewer:\n4. Causality vs correlation of SRS22A. Data support that SRS22A+ bradyzoites are\nassociated with tachyzoite recrudescence, but do not prove SRS22A is functionally\nrequired. Consider genetic perturbation (SRS22A KO/conditional depletion or\noverexpression) to test whether altering SRS22A levels changes the brady-tachy vs brady-\nbrady outcome. If not feasible now, the manuscript should clearly reframe the claim as\nassociative and propose SRS22A as a marker, not a driver.\nAt no point in the paper did we propose that SRS22A was responsible for driving\nbradyzoite development, indeed our goal was, as stated on page 5, “to identify\npotential in vivo bradyzoite markers”. We apologize if mechanism was implied. The\nfunction of SRS antigens has been a long and torturous goal that has largely been\nfruitless. SRS22A is used in this study as marker of bradyzoite development. Other\nfindings in this study do suggest avenues for future investigation in particular the\ndifferential regulation of ApiAP2 transcription factors.\n5. Alkaline stress model conclusions. The statement that conventional in vitro models “do\nnot form SRS22A+ bradyzoites” is based on 48-h pH 8.2 HFF culture. Test additional\ndifferentiation protocols (longer durations, neuronal/astrocytic cells, hypoxia, different\nCO₂, serum deprivation, host cytokine milieu) before making general claims. Alternatively,\nnarrow the claim to the specific conditions tested.\nAn exhaustive comparison of in vitro models is not the focus of this research and as\ndiscussed above we have modified the text to clarify this and the claims that we are\nmaking (see above and text in red on pages 5, 6, 15).\n6. Interpretation about merozoite priming. The suggestion that Group B bradyzoites may be\npoised for feline merozoite development is speculative. Either (i) provide orthogonal\nevidence (e.g., AP2VIIa-1 protein, promoter accessibility, or functional data in feline\nenterocytes/organoids), or (ii) clearly label as hypothesis and tone down the conclusion.\nAs part of the original discussion we wrote the sentence below that demonstrates we\nare in complete agreement with the reviewer that we do not currently know what\ndrives tachyzoite to merozoite development (page 12).\n“Altogether, these features may indicate Group B bradyzoites are poised to initiate\nmerozoite development in the cat. Further study will be required to understand\nwhether Group B bradyzoites or another in vivo bradyzoite group are the primary\ndrivers of bradyzoite-to-merozoite development.”\n7. As a suggestion for Figure 7 and the discussion, we encourage you to integrate recent\nevidence that elements of the chronic-stage program can initiate independently of BFD1\n(Robert et al., EMBO Mol. Med., 2025). Concretely, testing BFD1 dependence in your\nscRNA-seq by examining BSM and BCLA across clusters A–E would help identify BFD1-\ndependent versus BFD1-independent subpopulations and allow you to incorporate those\nassignments directly into the model and narrative. We also recommend situating your\nframework explicitly within a continuum—rather than a binary switch—by comparing your\ninferred trajectories to proposals of epigenetically primed, transcriptionally poised,\ncontext-responsive intermediate states (Shahinas et al., COM, 2025), and mapping\nclusters A–E onto that spectrum to highlight points of convergence and divergence. Finally,\nit would be valuable to assess whether your dataset contains a hybrid P6-like population\nco-expressing tachyzoite and bradyzoite markers (Xue et al., Boothroyd JC. eLife, 2020).\nMarker overlays, module scores, and cell-cycle–aware analyses could clarify whether\nGroup B or E aligns with this hybrid profile and whether such cells act as progenitor-like\nintermediates within the bradyzoite lineage.\nTo aid interpretation and incorporation of previously proposed bradyzoite\ndifferentiation pathways we examined BFD1, BSM and BCLA. BFD1 is lowly expressed\nacross all clusters, and thus may not be applicable to identify BFD1-\ndependent/independent populations. In contrast BSM is highly expressed across all\nclusters again minimizing its use for identifying parasite trajectories. BCLA,\nassociated with cysts, is the most differentially expressed being somewhat excluded\nfrom Group B. The UMAP projections for all these genes are provided here for the\nreviewer and in a new Supplemental Figure, Fig. S5A. We also discuss this analysis on\npage 18 (see text in red). Below are the violin plots and UMAPs associated with BFD1.\nSimilarly, genes mentioned in the P6 cluster (which defined a cluster of parasites\nseparated from the alkaline-induced population) in the paper Xue et al., Boothroyd JC.\neLife, 2020 are mostly lowly expressed or not defined to a single cluster. We included\nthe corresponding UMAPs below. While some genes appear to map onto Group B\nothers are minimally expressed or not defined to a single cluster. Therefore, we did\nnot include these additional analyses but provide all data via its deposit in GEO\n(Accession number: GSE311669).\nTo assist with interpretation and analysis of the scRNAseq data set, a trajectory\nanalysis is now provided (Fig. S5B). This supports one of our main conclusions that\nbradyzoite heterogeneity is a developmental process that defines their function by\nidentifying at least one branching point. This analysis was performed by Dr. Brandon\nLe, who is the Bioinformatics Core Coordinator at UC Riverside Institute for Integrative\nGenome Biology (IIGB). Due to his significant contribution, we now include him as a co-\nauthor in our manuscript.\nInterpretation of this additional analysis is on page 14 and Fig. S5B:\n“Finally, trajectory analysis, although not definitive, supports the presence of alternative\nbradyzoite developmental programs as seen with SRS22A+ and SRS22A- parasites. The\ninferred trajectory begins with parasites in Group D, progresses toward the transitional\nGroup C/D population, and then bifurcates towards either Group B (SRS22A+) or Group A\n(Fig. S5B).”\nFinally, we also mapped genes involved in the epigenetic regulation of stage transition\nas requested by the reviewer. However, none of these genes passed the p-value cutoff\nor displayed distinguishing patterns across our clusters, and therefore we could not\ndraw any conclusive inferences from this analysis. As a result, this analysis did not\nclarify the potential roles of the MORC/HDAC3 repressor complex or the AP2 factors.\nIn the paper by Shahinas et al4, we emphasized the importance of MORC/HDAC3\ncomplex in chromatin remodeling and stage transition. We similarly examined genes\nimplicated in merozoite stage transition, including AP2XII-1 and AP2X1-2, whose\ncombined deletion is known to initiate sexual stage transition in vitro. Yet, none of\nthese genes met our statistical thresholds. For completeness, we provide the UMAPs\nfor these genes below.\nTo respond to this question, we now included those citations and a statement on page\n17:\n“… Turning off BAG1 expression may signal a priming event that pre-stages SRS22A+\nGroup B bradyzoites to recrudesce into the tachyzoite or merozoite stage depending on the\nhost environment. Consistent with this, epigenetic priming events were proposed to be\nresponsible for stage transition. For example, one hypothesis involves regulation of\nchromatin remodeling by the MORC/HDAC3 repressor complex4, 5.”\nReviewer #3 (Remarks to the Author):\nThis manuscript represents a significant advance in our understanding of Toxoplasma\ngondii bradyzoite biology. The authors provide compelling evidence that bradyzoite\npopulations are heterogeneous and that distinct subgroups possess unique\ndevelopmental potentials. Their findings explain several long-standing observations\nrelated to bradyzoite infection dynamics and reactivation.\nThe authors identified a novel surface marker, SRS22A, which is specifically expressed on\nbradyzoites derived from in vivo tissue cysts. They also demonstrated that SRS22A-positive\nand SRS22A-negative bradyzoites have distinct functions—the former are capable of\nrobust conversion to fast-replicating tachyzoites in astrocytes, while the latter appear to\nparticipate primarily in bradyzoite-bradyzoite replication. Supporting this finding, they\nshow using an in vivo infection model that SRS22A-positive bradyzoites disseminate more\nefficiently and form tissue cysts more readily than their SRS22A-negative counterparts.\nFinally, using single-cell RNA sequencing, they identified five distinct bradyzoite\nsubpopulations (Groups A–E) within in vivo cysts. Each group exhibits unique\ntranscriptional profiles suggestive of specific developmental trajectories. For example,\nGroup B bradyzoites—expressing the highest levels of SRS22A—appear transcriptionally\nprimed for conversion to tachyzoites or merozoites, whereas Groups C and D, which lack\nSRS22A expression, show elevated expression of cyst wall mRNAs consistent with\ncontinued bradyzoite replication.\nCollectively, these results convincingly demonstrate that bradyzoite development is not a\nlinear process and that multiple developmental fates coexist within chronic-stage cysts.\nThis work provides strong evidence for the functional heterogeneity of bradyzoite\npopulations and offers a refined framework for understanding chronic toxoplasmosis.\nThe study is well-designed, the experiments are appropriately controlled, and the\nmanuscript is clearly written. The authors also transparently address the limitations of\ntheir approach. Importantly, this work opens several exciting avenues for future research,\nsuch as identifying the triggers that drive each bradyzoite subgroup’s developmental\ntrajectory.\nIn this reviewer’s opinion, the quality, novelty, and depth of this study meet the standards\nexpected for Nature Communications.\nWe thank the reviewer for their positive feedback and thoughtful analysis of our\nmanuscript, as well as for recognizing its significance.\nMinor comment:\nPage 8, line 2: The text should refer to Figure 2B rather than 2A.\nWe thank the reviewer for catching this mistake, we now corrected this as suggested."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have thoroughly and effectively addressed all of my prior comments. The revised manuscript is substantially strengthened by clearer methodological descriptions, improved figures, and the addition of key control data and analyses, including full FACS gating/purity documentation and trajectory analysis. The claims regarding SRS22A have been appropriately refined, antibody specificity is convincingly justified, and interpretations of in-vitro models and developmental trajectories are now well balanced. Speculative elements have been clearly framed as hypotheses. The manuscript is now rigorous and coherent and, in my opinion, represents an interesting and valuable study that provides the community with an important new resource for understanding chronic stage(s) of Toxoplasma gondii."}, "author_response": ""}]

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