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A positive feedback loop controls Toxoplasma chronic differentiation

An Author Correction to this article was published on 15 August 2023

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Abstract

Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. In this study we examine five factors that are transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc-finger family, which we name bradyzoite formation deficient 2 (BFD2). Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript under stress, and deletion of BFD2 reduces BFD1 protein levels but not messenger RNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces the chronic-stage gene-expression programme. Thus, our findings help explain how parasites both initiate and commit to chronic differentiation. This work provides new mechanistic insight into the regulation of T. gondii persistence, and can be exploited in the design of strategies to prevent and treat these key reservoirs of human infection.

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Fig. 1: Screening for downstream effectors of BFD1.
Fig. 2: BFD2 is a CCCH-type zinc-finger protein required for differentiation.
Fig. 3: Parasites lacking BFD2 fail to generate brain cysts in mice.
Fig. 4: BFD1 and BFD2 comprise a positive feedback loop.
Fig. 5: BFD2 overexpression induces chronic differentiation.
Fig. 6: BFD2 binds a cohort of transcripts during differentiation that includes BFD1.

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Data availability

For BFD1, BFD2, TGME49_253790 and TGME49_224630, the subsets of genes identified as significantly affected by depletion are listed in Supplementary Table 1. The results of quantitative proteome profiling in Δbfd2 parasites are provided in Supplementary Table 2. Primers used for RT–qPCR analysis of BFD1, BFD2, GCN5B and ASP5 are available in Supplementary Table 3. The Ct values and analyses of the RT–qPCR experiments are provided in Supplementary Tables 46. Raw reads and analysis of RNA IP and sequencing for BFD2 are provided in Supplementary Table 7. Minimally processed results from all bulk RNA-seq performed in this study are provided in Supplementary Table 8. The oligonucleotides used in this study for molecular cloning are in Supplementary Table 9. Unprocessed data from the transcriptional and proteome profiling described herein are available through Gene Expression Omnibus (GEO) and the Proteomics Identification Database (PRIDE), respectively, under the following accession numbers: GSE223819 (Fig. 1e–g and Extended Data Fig. 2), GSE223869 (Fig. 2i–k), PXD039648 (Extended Data Fig. 3c), GSE223621 (Extended Data Fig. 3d,e), GSE223877 (Fig. 4d) and GSE223620 (Fig. 6b–d and Extended Data Fig. 7). Source data are provided with this paper.

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Acknowledgements

We thank D. Soldati for the antibody to GAP45; D. Sibley for anti-SAG1; F. Araujo for the antibody to Toxoplasma; the Whitehead Institute Genome Technology Core, particularly T. Volkert, J. Love and S. Gupta for their expertise with library preparation and next-generation sequencing; and the Whitehead Bioinformatics and Research Computing Core for consultation and software support. This work relied on VEupathDB and we thank all contributors to this resource. This project was supported by grants from the NIH (grant no. R01AI158501) and the Smith Family Foundation (Odyssey Award) to S.L.

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Authors

Contributions

This project was conceptualized by M.H.L., B.S.W. and S.L. All experiments were performed by M.H.L., with contributions from A.W.C. for quantitative proteome profiling, C.J.G. for training and support with rodent infections, L.A.S. and J.N.E. for histology, and S. Chandrasekaran for infection of primary neurons. Resources were provided by S.L., A.A.K. and C.A.H. Data analysis was performed by M.H.L., S. Chakladar and S.L. The manuscript was prepared by M.H.L. and S.L., with input from all contributing authors.

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Correspondence to Sebastian Lourido.

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Extended data

Extended Data Fig. 1 Genotyping of conditional depletion strains.

a, Clones were screened to verify integration of mNG–mAID downstream of the targeted coding sequence (CDS) and the reciprocal loss of the untagged allele. The diagram (top) shows the binding sites for primers listed in the table (bottom), with regions used to direct construct integration (that is, H1 and H2; Fig. 1b) in dark grey. In each case a common gene-specific forward primer (P11–P16) was paired with reverse primers to either mNG (P17) or the respective endogenous 3′ UTR (P18–P23) downstream of the integration site. The expected PCR product size is given for each template and primer combination. Refer to Supplementary Table 9 for a complete list of the primer sequences. b, PCRs were performed on ME49/TIR1 gDNA (parental) as a control in addition to the respective tagged strain. Lanes are labelled with the reverse primer and gDNA template used in each reaction. For positive clones, bands were extracted and subjected to Sanger sequencing to verify in-frame integration of the tag.

Source data

Extended Data Fig. 2 Effects of AP2IX-9 and AP2IB-1 knockdown on the chronic-stage transcriptome.

Data reflect changes in knockdown strains (relative to the parental) after 96 h in alkaline-stress medium containing IAA. Differential expression analysis was performed as in Fig. 1e, based on n = 2 biological replicates. No genes were significantly affected (adjusted P < 0.05, calculated by DESeq2) by depletion of either factor.

Extended Data Fig. 3 Genotyping and characterization of BFD2-deficient parasites.

a, Δbfd2 clones were screened by PCR for replacement of the endogenous coding sequence with tdTomato using a common forward primer in the BFD2 5′ UTR (P31) and reverse primers binding to either BFD2 (P32) or the fluorescent reporter (P33). The diagram shows priming in the parental strain (top) and at the modified allele in Δbfd2 (bottom), with the expected product sizes indicated. The reverse primers and gDNA template used in each reaction are listed above the respective lane. b, Plaque assays after 16 d of undisturbed growth. c, Comparison of protein abundance in unstressed parental (Δbfd1::BFD1-TY) and Δbfd2 parasites. Quantitative proteomics identified a total of 29,806 unique peptides corresponding to 4,303 individual proteins. Significantly affected proteins (magenta) were defined as those meeting three criteria: (1) a minimum of two unique peptides, (2) absolute fold change > 2 and (3) P < 0.05. Differences are limited to canonical bradyzoite markers (CST1, LDH2 and SRS35A) or other developmentally regulated genes (TgSPT2)76. d,e, Effects of BFD1 or BFD2 deletion on the parasite transcriptome during infection of mouse primary neurons. Data are based on n = 3 independent infections with colour assigned based on log2(fold change) during conditional BFD1 expression23. Significantly affected genes (adjusted P < 0.05, Wald test in DESeq2) are indicated by larger point size. d, Differential expression analysis was performed for parasites lacking either factor, as compared with the parental strain. The number of genes meeting the cutoff for statistical significance is indicated. e, Comparison of the effects of BFD1 versus BFD2 deletion reveals a comparatively larger impact for the former. Pearson’s correlation was performed on all significant points with a trend line fit by linear regression.

Source data

Extended Data Fig. 4 Re-analysis of BFD2-deficient parasites in previous ME49 screens.

a, Overview of the CRISPR-based screen that identified BFD1 (ref. 23). A CRISPR-compatible ME49 strain was modified to express mNG under the bradyzoite-specific BAG1 promoter (pBAG1), enabling isolation of chronic stages by fluorescence-activated cell sorting (FACS). The reporter strain was transfected with gRNA libraries targeting approximately 200 predicted nucleic acid-binding proteins with five gRNAs per gene. After initial passages allowing for guide integration and gene disruption, the transfectants were split between alkaline-stress and unstressed (standard media) conditions. Samples were collected from each population over a 10-d period, with bradyzoites (mNG+-stressed parasites) isolated by FACS. Integrated gRNAs from all samples were enumerated by next-generation sequencing and the abundance of each guide was assessed relative to the input library. The log2(fold change) for guides targeting each gene is referred to as its fitness or differentiation score, based on representation in unstressed or bradyzoite samples, respectively. b,c, Analysis of BFD2-targeting gRNAs from the screen described in a. b, Four of the five guides targeting BFD2 were lost from the transfectant pool under standard conditions over the course of serial passaging. Subsequent sequence-level analysis revealed that the single guide that remains abundant (black) is likely to be non-cutting due to a mismatch in the protospacer and its intended genomic target. c, Among the alkaline-stressed cultures at both time points examined—with the exception of the non-cutting guide (black)—gRNAs targeting BFD2 are under-represented in bradyzoite samples (mNG+) relative to the unsorted alkaline-stressed population (bulk).

Extended Data Fig. 5 BFD2 deletion in a conditional BFD1 strain.

a, Schematic of ligand-inducible BFD1. The DD-BFD1-TY strain was constructed previously23 by integration at the HXGPRT locus in the Δbfd1 genetic background. A heterologous promoter (pTUB1) drives expression of the transgene but DD-BFD1-TY protein is only stabilized following treatment with Shield-1. b, Validation of BFD2 knockout in DD-BFD1-TY. Selected clones were screened using the same strategy described in Extended Data Fig. 3, verifying both loss of endogenous BFD2 and replacement with the fluorescence cassette. The gel (bottom) shows PCRs performed on gDNA from both the parental (DD-BFD1-TY) and DD-BFD1-TYΔbfd2 strains. The reverse primers used are listed over the respective well, with the expected product sizes indicated in the diagram (top).

Source data

Extended Data Fig. 6 Genotyping BFD2-complemented and conditional overexpression strains.

a, Complementation with wild-type (HABFD2) or non-RNA-binding (HABFD2ΔZF) BFD2 was verified by PCR using the same primers as in Extended Data Fig. 3, screening for reintroduction of the coding sequence and reciprocal loss of tdTomato at the endogenous BFD2 locus. The diagram shows PCR priming in Δbfd2 (top) versus the complemented loci (bottom), with the expected product sizes indicated. The reverse primers and gDNA template used in each reaction are listed above the respective lane. b, For conditional BFD2 expression, pTUB1-DD-HA was integrated at the endogenous BFD2 locus (P51 and P32), resulting in coincident loss of the untagged allele (P50 and P32). As in a, the diagram shows the relative positions of primer binding with the expected product sizes and lanes are labelled above with the gDNA template and forward primer used.

Source data

Extended Data Fig. 7 Neither mRNA abundance nor differential expression are predictive of interaction with BFD2.

a,b, Comparison of log-transformed enrichment ratios (TPMIP/TPMinput) for all transcripts detected in stressed HABFD2 parasites and either abundance in the unenriched input (a) or change in expression after 48 h of stress, based on a previously published dataset (b)23. The 375 most highly enriched genes identified by Gaussian mixture modelling are highlighted in green.

Extended Data Fig. 8 Strain construction using the HiT vector strategy.

a, Schematic of C-terminal tagging HiT vectors (top), as described previously38. Targeted integration of BsaI-linearized constructs (bottom) is facilitated by a gRNA specific to the 3′ end of the coding sequence and 40-bp homology regions (H1 and H2), both encoded in the gene-specific cutting unit. Transcription of the gRNA is driven by a type III promoter (pU6). A heterologous 3′ untranslated region (3′CDPK3) allows expression of the gene product. DHFR denotes a pyrimethamine-resistance cassette to enable mutant selection. b, N-terminal HiT vector configuration for the generation of conditional overexpression strains. Construct integration endogenously tags the targeted gene with the Shield-1-stabilized DD domain and replaces the native promoter with that of α-tubulin (pTUB1). Cutting units were designed similarly to those in a, with a gRNA that targets the 5′ end of the coding sequence encoded in the reverse orientation. For the inducible BFD2 strain in particular, HA was also designed into the cutting unit to enable detection of the protein by the same epitope used for the examination of endogenously regulated BFD2.

Supplementary information

Reporting Summary

Supplementary Tables 1–9

Table 1. Subsets of genes significantly affected by conditional depletion of BFD2 (Table 1a), BFD1 (Table 1b), TGME49_253790 (Table 1c) and TGME49_224630 (Table 1d) after 96 h under alkaline stress. Related to Fig. 1. The behaviour of listed genes in previous transcriptional profiling (columns labelled as ‘.2020’) is shown. Table 2. Results of TMT quantitative proteome profiling in Δbfd2 parasites and the parental strain. Related to Extended Data Fig. 3c. Parasites were cultured in HFFs under standard culture conditions for 48 h before harvest. Table 3. RT–qPCR primers used for the analysis of BFD1, BFD2, GCN5B and ASP5 transcripts. Related to Figs. 4 and 5. Table 4. Analysis of BFD1 and BFD2 mRNA abundance in Δbfd1::BFD1-Ty, Δbfd1, Δbfd1::BFD1ΔMYB-Ty and Δbfd2 parasites after 48 h under alkaline-stress or unstressed conditions. Related to Fig. 4a. Mean Ct values are the average of three technical replicates. All reactions were performed in parallel to a ‘No reverse transcriptase’ (No-RT) control. Table 5. Analysis of DD-BFD1-Ty mRNA abundance in DD-BFD1-Ty and DD-BFD1-TyΔbfd2 parasites following 48 h of treatment with Shield-1 or vehicle. Related to Fig. 4e. Mean Ct values are the average of two technical replicates. All reactions were performed in parallel to a ‘No-RT’ control. Table 6. Analysis of BFD1, GCN5B and ASP5 mRNA abundance in input and enriched pulldown samples. Related to Fig. 6a. Data were analysed using the Percent Input method. Mean Ct values are the average of two technical replicates. All reactions were performed in parallel to a ‘No-RT’ control. Analysis of BFD1, GCN5B and ASP5 mRNA abundance in input and enriched pulldown samples. Related to Fig. 6a. Data were analysed using the Percent Input method. Mean Ct values are the average of two technical replicates. All reactions were performed in parallel to a ‘No-RT’ control. Table 7. Results of RNA IP and sequencing analysis performed on HABFD2 parasites after 72 h of alkaline stress. Related to Fig. 6. Raw read counts for all identified transcripts (Table 7a), the trimmed dataset excluding poorly detected messages (that is, fewer than five reads assigned in either IP or input samples, or read coverage less than ten) with enrichment analysis (Table 7b) and the final list of transcripts identified as highly enriched by HA–BFD2 pulldown (Table 7c) are provided. The behaviour of listed genes in previous transcriptional profiling (columns labelled as *.2020) is shown. Table 8. Compiled results of differential expression analyses performed in this work. Related to Figs. 1,2,4 and Extended Data Fig. 3d. The behaviour of listed genes in previous transcriptional and chromatin profiling (columns labelled as ‘.2020’) is shown. Table 9. Primers used for molecular cloning and genotyping.

Source data

Source Data Fig. 1

Statistical source data; corresponds to Fig. 1d.

Source Data Fig. 2

Statistical source data; corresponds to Fig. 2c,e–h.

Source Data Fig. 3

Statistical source data; corresponds to Fig. 3b,c,e.

Source Data Fig. 4

Statistical source data; corresponds to Fig. 4b,f,g.

Source Data Fig. 5

Statistical source data; corresponds to Fig. 5d,e,g.

Source Data Extended Data Fig.1

Uncropped blot; corresponds to Extended Data Fig. 1b.

Source Data Extended Data Fig. 3

Uncropped blot; corresponds to Extended Data Fig. 3a.

Source Data Extended Data Fig. 5

Uncropped blot; corresponds to Extended Data Fig. 5b.

Source Data Extended Data Fig. 6

Uncropped blot; corresponds to Extended Data Fig. 6a.

Source Data Extended Data Fig. 6

Uncropped blot; corresponds to Extended Data Fig. 6b.

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Licon, M.H., Giuliano, C.J., Chan, A.W. et al. A positive feedback loop controls Toxoplasma chronic differentiation. Nat Microbiol 8, 889–904 (2023). https://doi.org/10.1038/s41564-023-01358-2

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