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Transcriptional control of the Cryptosporidium life cycle

Abstract

The parasite Cryptosporidium is a leading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

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Fig. 1: The transcriptional program of the asexual cycle.
Fig. 2: The transcriptome of the full C. parvum life cycle.
Fig. 3: Female and male gametes use sex-specific gene sets.
Fig. 4: The transcription factor Myb-M commits parasites to a male fate.

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Data availability

The reference genomes for C. parvum Iowa II and C. parvum IOWA-ATCC were obtained from CryptoDB (https://cryptodb.org/cryptodb/app/downloads). The C. parvum IOWA-ATCC non-coding RNAs were acquired from GenBank accessions CP044415CP044422. Sorted asexual and female RNA-seq samples were downloaded from the GEO under accession number GSE129267. RNA-seq data generated in this study are available at the GEO under accession number GSE232438. The C. parvum single-cell atlas is available online (https://CryptoDB.org/).

Code availability

All code is found in Supplementary Data 1–6 and at GitHub (https://github.com/katelyn-walzer/Cryptosporidium_single_cell_atlas).

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health to B.S. (R01AI127798, R01AI112427, R01AI148249 and U01AI163671) and a postdoctoral fellowship to K.A.W. (F32 AI154666). We thank B. McLeod, E. Kugler, Z. Hutchins, E. Smith and G. Buenconsejo for help with animal care and strain passage; C. Huston and J. Gaertig for providing antibodies; the staff at the Penn Cytomics and Cell Sorting Resource Laboratory for assistance with cell sorting; the members of the Penn Vet Imaging Core for microscopy support; D. Cutillo for assistance with sequencing library preparation; staff at VEuPathDB for genomic resources; and M. Lebrun for reading the manuscript.

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Authors and Affiliations

Authors

Contributions

K.A.W., J.T., D.P.B. and B.S. conceived the study. K.A.W. and J.T. generated transgenic parasite strains. J.T. and J.A.G. assisted in cell sorting. E.C.W. and E.K. contributed to single-cell sequencing. K.A.W. performed bioinformatic analyses with support from E.C.W. and D.P.B.; A.M.D., S.D.C. and B.S. contributed to stage-specific gene annotation. K.A.W., J.T., J.H.B. and A.M.D. performed genotypic and phenotypic characterization of parasites. K.A.W. and J.H.B. conducted animal experiments. K.A.W. and B.S. secured funding and wrote the paper.

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Correspondence to Boris Striepen.

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Extended data figures and tables

Extended Data Fig. 1 Generation of transgenic parasite strains.

Integration maps show the homology repair, the native locus, and the modified locus, with the CRISPR/Cas9 induced break for each transfection marked by an arrowhead. Diagnostic PCR products are represented as inverted arrows on the integration maps and are shown on the corresponding gel, demonstrating successful insertion of the homology repair. Transgenic strains include a, tdNeon RPL22 HA, b, cgd7_5500 (GGC1) HA, c, cgd6_2250 (Myb-M) HA, d, cgd6_2250 (Myb-M) knockouts (no parasites recovered), e, cgd6_2250 (Myb-M) overexpression in thymidine kinase locus, f, cgd6_2250 (Myb-M) overexpression in cgd6_2670 (AP2-M) locus, g, cgd6_2250Δ (Myb-MΔ) overexpression in thymidine kinase locus, and h, cgd6_2250 (Myb-M) LoxP HA. All guide RNAs and primers noted are listed in Supplementary Table 13. Viable transgenic strains were each generated once, with the exception of cgd6_2250 (Myb-M) HA which was generated three separate times.

Extended Data Fig. 2 Enrichment of C. parvum infected cells and quality control for scRNA-seq.

a, b, Cell populations from a, culture or b, the ileum of an IFN-γ−/− mouse were sorted to enrich for viable cells infected with fluorescent green C. parvum. Cells sorted from the mouse ileum were also enriched for intestinal epithelial cells based on EPCAM positivity. Sorting gates are outlined in black. The plots were generated with FlowJo 10.8.1. c-e, Violin plots show the distribution of c, genes detected, d, UMIs, and e, percentage of transcripts mapping to rRNA genes across samples after filtering. Filtering cutoffs included a minimum of 100 detected genes and less than 60 percent of transcripts mapping to rRNA. The maximum cutoffs were set per sample according to the pre-processed distribution of detected genes and UMIs. For the in vivo sample, the maximum number of detected genes was 1800 and the maximum number of UMIs was 7500. For timepoints 24, 36, and 42 h, the maximum number of detected genes was 1200 and the maximum number of UMIs was 4000. For timepoints 46 and 54 h, the maximum number of detected genes was 500 and 400 and the maximum number of UMIs was 1000.

Extended Data Fig. 3 Temporal gene expression across the asexual cycle.

a, b, Scaled expression of organellar genes14 across pseudotime. Individual genes are shown in grey, the mean in black, and the 95 percent confidence interval in red (a). The means from each organelle (black in a) are plotted in colour (b). c, A heatmap shows the scaled expression of the organellar genes across asexual clusters. The colour for each gene group corresponds to that used in (b). These data represent the parasite transcriptomes captured at 24 and 36 h (see Supplementary Table 2 for gene lists).

Extended Data Fig. 4 Expression of cluster markers across the C. parvum life cycle.

A heatmap shows the scaled expression of 2880 genes that were identified as markers for one or more clusters defining the C. parvum transcriptome (see Supplementary Table 5 for gene lists).

Extended Data Fig. 5 The transcriptome of the full C. parvum life cycle including lncRNAs.

a, The UMAP shows 8962 individual C. parvum transcriptomes and is coloured for clustering across asexual (green), male (blue), and female (pink) parasites. b, Each UMAP shows the top lncRNA gene for each cluster with its normalized expression in blue.

Extended Data Fig. 6 Identification of meiosis-specific genes.

Previously published meiosis and DNA repair genes8 were examined for their female-specific expression, as expression during other stages would indicate that they are not specific for meiosis. Eight genes were identified as female-specific from the previous list and were designated as meiosis genes. Further examination of female-specific genes (Supplementary Table 9) revealed three additional meiosis genes not included in the previously published list. UMAPs show their normalized expression levels in blue.

Extended Data Fig. 7 Male gametes lack gliding and invasion machinery but exclusively express genes of the GGC exported protein family.

a-c, Signature scores were obtained for glideosome8(a), rhoptry14 (b), and microneme14 (c) genes and were painted onto the UMAP in green. Note that following successful fertilization and meiosis, in vivo females form sporozoites which are indistinguishable from merozoites with respect to their motility machinery. Late in vivo females therefore exhibit expression of glideosome genes. d, UMAPs show the normalized expression levels (in blue) of four paralogous GGC proteins. e, GGC1 (cgd7_5500) was epitope tagged and visualized in immunofluorescence assays after 54 h of growth in culture. Tagged GGC1 was stained with an antibody to HA, shown in green, while the male nucleus was stained with DAPI in blue. Males were stained with alpha tubulin, shown in magenta, and females were stained with DMC1, also shown in magenta, in two separate assays. GGC1 localizes to the periphery of the 8 N male gamont, surrounding the nuclei, and then localizes to the apical end of mature male gametes. It is not expressed in females (scale bar = 2 μm, data representative of two experiments).

Extended Data Fig. 8 Expression of AP2 transcription factors across the C. parvum life cycle.

A heatmap shows the normalized expression of 16 AP2 transcription factors across the C. parvum transcriptome. AP2s are largely stage- and sex-specific. Male and female expressed AP2s are shown in bold.

Extended Data Fig. 9 Myb-M is the earliest male transcription factor and the locus is refractory to disruption.

a, UMAPs show the normalized expression levels (in blue) of male- and female-specific transcription factors. Myb-M is expressed the earliest of all factors with female AP2s expressed particularly late in female developmental progression. b, c, Myb-M was targeted for deletion at the predicted N-terminal DNA binding domains (b) or after for C-terminal truncation (832 base pairs into the coding sequence, c) by CRISPR/Cas9 mediated marker insertions. Myb-M-HA was used as a control in parallel. Sporozoites transfected with a Cas9+gRNA plasmid and homology repair template (see Extended Data Fig. 1 for design) were given to mice via oral gavage. Oocyst shedding was monitored by faecal luminescence measurements. Data is represented as the mean of three technical replicates. Note that while epitope tagged mutants are readily recovered, no viable transgenics were isolated using the deletion and truncation constructs, suggesting that the gene is likely essential.

Extended Data Fig. 10 Ectopic expression of Myb-M drives parasites to a male fate.

a, Diagram showing conditional Myb-M overexpression AP2-M reporter strain. b, c, Infected cultures were treated with vehicle or Shield-1 at 12 h, fixed at 18 h, and AP2-M was detected with an antibody to Myc (scale bar = 2 μm) and quantified in 8 N parasites (c). Error bars represent the standard deviation of the mean from three biological replicates (**P = 0.0018, two-tailed unpaired t-test with Welch’s correction). d, Infected cultures were treated with vehicle or Shield-1 at 24 h, fixed at 28 h, and AP2-M was quantified as in c (**P = 0.0044, two-tailed unpaired t-test with Welch’s correction). e, Schematic comparing the conditional Myb-M-HA and Myb-MΔ-HA overexpression constructs. The conserved DNA-binding domains predicted by AlphaFold are marked in green, with the amino acid sequence annotated above. 315 nucleotides were deleted from conditional Myb-MΔ-HA, and the resulting open reading frame is shown indicating the amino acid sequence of the deletion. f, Inducible Myb-M-HA and Myb-MΔ-HA parasites were treated with Shield-1 and scored for HA staining. Error bars represent the standard deviation of the mean from three biological replicates. Significance was evaluated by a two-tailed Welch’s t-test. g, h, HCT-8 cultures infected with the inducible Myb-M-HA strain were treated with vehicle or Shield−1 at either 12 h (g) or 36 h (h). RNA was harvested at 18 h (g) or 48 h (h) and representative constitutive (grey), asexual (green), male (blue), or female (pink) transcripts were measured by qPCR. The log2 fold change is relative to the 18S rRNA control and vehicle-treated samples. Data is representative of two independent biological repeats and is plotted as the mean.

Supplementary information

Supplementary Figure 1

Uncropped source gel from Fig. 4k showing genomic excision of Myb-M after rapamycin treatment. DNA was extracted both from purified oocysts and cultures 48 h after infection. Rapamycin induces excision (note some excision before treatment). The cropped image shown in Fig. 4k is outlined in red lines.

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Supplementary Data 1–6.

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Walzer, K.A., Tandel, J., Byerly, J.H. et al. Transcriptional control of the Cryptosporidium life cycle. Nature 630, 174–180 (2024). https://doi.org/10.1038/s41586-024-07466-1

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