Abstract
Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These ‘abortive hepatocytes’, which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.
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Data availability
Code availability
The code used to process the raw data to a Scanpy/Seurat structure used for analysis is available at https://github.com/AmichayAfriat/SpatioTemporal_malaria_liver_stage_atlas/. Codes for further analysis and data visualization are available at https://doi.org/10.5281/zenodo.7081863.
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Acknowledgements
S.I. is supported by the Wolfson Family Charitable Trust, the Edmond de Rothschild Foundations, the Fannie Sherr Fund, the Dr. Beth Rom-Rymer Stem Cell Research Fund, the Helen and Martin Kimmel Institute for Stem Cell Research, the Minerva Stiftung grant, the Israel Science Foundation grant no. 1486/16, the Broad Institute‐Israel Science Foundation grant no. 2615/18, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme grant no. 768956, the Chan–Zuckerberg Initiative grant no. CZF2019‐002434, the Bert L. and N. Kuggie Vallee Foundation and the Howard Hughes Medical Institute (HHMI) international research scholar award. L.B. is supported by the European Molecular Biology Organization under EMBO Long-Term Fellowship ALTF 724–2019. This work was also financed by la Caixa Foundation (HR17/52150010) to M.M.M. and Fundação para a Ciência e Tecnologia (LISBOA-01-0145-FEDER-030751 and PTDC/BIA-MOL/30112/2017) to M.M.M. and V.Z.-L.
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S.I. and M.M.M. conceived the study. V.Z.-L., S.M., Â.F.C. and A.L. dissected the mosquitoes, obtained sporozoites and infected the mice. A.A., V.Z.-L., K.B.H., S.M., Â.F.C. and A.L. fixed and embedded liver tissue for imaging, performed liver perfusion, and FACS-sorted single-cell hepatocytes. A.A. and K.B.H. prepared and sequenced the MARS-seq libraries and designed, imaged and quantified smFISH protocols. A.A., L.B. and S.I. wrote the code and performed the data analysis. I.A. contributed to project design. S.I., A.A. and M.M.M. wrote the paper. All of the authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 scRNAseq sampling.
a-f, Representative FACS gating for scRNAseq. (a) FACS gating based on general size to find hepatocytes. (b-c) Gating to discern single-cells from clumps of cells. (d) Negative selection to exclude non-parenchymal cells. e, Gating to include only live hepatocytes. (f) gating for infected and uninfected hepatocytes based on GFP content. g-k, Quality control steps in the scRNAseq process. (g) UMIs (unique molecular identifier) are summed per gene per cell and denoted as either mouse or Plasmodium gene. (h) Quality control for infected tag assigned to cells. Fraction of PBA gene UMIs out of all reads was calculated per cell. Based on the log10 of that value (Y axis) a threshold was established (red line, Methods) to identify erroneous tagging of cells. Infected/Uninfected cells below/above the threshold are discarded. Bounds of boxes span IQR, horizontal black lines denote medians, whiskers span 1.5× IQR. (i) Identifying cells with high mitochondrial gene fraction. Per mouse, the 95th percentile of cells, based on their Mouse mitochondrial gene content (Y axis), was discarded from the dataset. (j) filtering all cells based on their high/low total transcriptome yield. X axis – total number of UMIs; Y axis – Total number of genes with at least 1 UMI assigned; cells colored by infection tag (green – Infected, blue – uninfected). Cells were discarded if they fell below (teal lines) or above (red lines) certain thresholds for each category (dashed line – number of UMIs; dotted line – number of genes). (k), Filtering plates outliers based on Z-score for the ratio between median UMIs per cell in plate compared to all cells from mouse. Per mouse, every cell total UMI count was summed, then the median value of log10 that sum for all cells assigned to plate was divided by the median value of log10 that sum for all cell assigned to mouse (Y axis). Based on that value a Z-score (white text on bars) was calculated per plate. Plates with Z-score lower than −1.5 (red outline) were excluded from further analysis.
Extended Data Fig. 2 Unsupervised clustering of the Plasmodium liver stage atlas.
a, UMAP of hepatocytes clustered based on both host and parasite transcriptome, colored by hours post-infection (hpi) and inoculation method. NI – non infected mouse, MB – mosquito bitten mouse. b, UMAP colored by Infected/Uninfected tag assigned to cells based on sorting. Infected cells are in red. c, UMAP colored by Louvain cluster (Methods). d, Heat map for marker genes per cluster. purple – low expression; yellow – high expression. e, Dotplot of specific markers for dividing (Mki67, Tubb5), immune (Ptprc, and specifically macrophages - Marco) and endothelial (Ptprb) cells, as well as interferon activation (Isg15, Ifit1, Irf7). f, The contribution of different sampled time points to the composition of individual clusters and the complete atlas. g, The contribution of different mice to the composition of individual clusters and the complete atlas.
Extended Data Fig. 3 Additional scRNAseq analysis.
a-g, ScRNAseq of uninfected cells acquired at 2hpi show increase in genes associated with tissue dissociation. a, Volcano plot of differentially-expressed hepatocyte genes between uninfected cells acquired at 2hpi and at every other time-point. Bold genes are highlighted in (d-f). b, UMAP plot colored by log10 summed relative expression of dissociation marker genes (van den Brink et al. 2017)17. c, Violin plot of stress indication in uninfected cells binned by time demonstrates notable increase in dissociation stress at 2hpi, both for injection inoculated and mosquito-bitten mice. Bounds of boxes span IQR, horizontal black lines denote the median, whiskers show data within 1.5*IQR. In order from left-to-right, n cells = 3,205/1,445/1,917/2,308/2,838/1,560/3,116/1,632/2,832 over 3/1/4/5/4/3/5/2/3 biologically independent mice. d-e, UMAP plots colored by normalized expression of representative up-regulated genes in the 2hpi cluster. UMAP projections reconstructed based on the combined mouse and Plasmodium transcriptomes. f, smFISH images of Cxcl10 and Mt1 at 2hpi and 24hpi uninfected hepatocyte show no discernible change in expression. Cxcl10 mRNA in red (positive control signal in non-parenchymal cell indicated by white arrow on left panel), Mt1 mRNA in green, Dapi in blue. Images are representatives of 40 imaging fields (15 for 2hpi and 25 for 24hpi) from 4 mice (2 per time point). g, Volcano plot of differentially-expressed hepatocyte genes between mock infected mouse acquired at 2hpi and hepatocytes from uninfected mice. h-i, Host hepatocyte transcriptome changes are also observed in mosquito-bitten mice. h, scatter plot of the log10 mean expression of genes in non-infected hepatocytes (Y axis) vs. infected hepatocytes (X axis) in the mosquito-bitten mice at 36hpi (n = 3 mice). Genes significantly increased or decreased in Fig. 2a are plotted in red or blue respectively. The names of selected genes from Fig. 2a are higlighted. i, Representative smFISH image of genes at 36hpi in mosquito-bitten mouse show similar changes to those observed in injected mice (Fig. 2d). Phalloidin (Phal) in white, Dapi blue; Ftl1 mRNA in red, Apob mRNA in green. Dashed white line - infected hepatocyte; Dotted yellow line - parasite. Image is representative of five imaging fields over two mice.
Extended Data Fig. 4 Differential gene expression in different spatio-temporal bins.
a, MA plots of the median expression ratio between infected and uninfected hepatocytes, split into 10 spatio-temporal bins. Y axis indicates log2 of the median ratio per gene. X axis indicates log10 of the gene’s max average expression. Genes significantly increased or decreased in infected hepatocytes are plotted in red or blue respectively (FDR q-value < 0.01). The names of selected genes from Fig. 2a marked with black circle are highlighted. b, Gene set enrichment analysis (GSEA) for highly expressed genes ranked based on ratio changes indicated in (a). (H) denote Hallmark gene sets, (K) denote KEGG gene sets. PC - pericentral, PP - periportal.
Extended Data Fig. 5 Pericentral infected hepatocytes become more abundant at late time points.
a, Violin plots of computationally-inferred zonation scores computed on the scRNAseq data. bounds of boxes span IQR, horizontal black lines denote the median, whiskers span 1.5*IQR (Interquartile range), dashed line demonstrates the median change between groups. Significance determined by two-sided Wilcoxon rank-sum test, with p-value adjusted using Benjamini–Hochberg procedure. b, smFISH of Albumin (Alb, white) and parasite Ch12 18S rRNA (18Sp, green). Yellow squares represents the 40x40 pixels window around the infected cells, number indicates median pixel intensity. Red dots represent the centers of 1248 40x40 pixel windows (“neighborhoods”) randomly placed onto the tissue area. “CV” denotes central-vein locations. Red blow up of 100x100 pixels (11x11 micron) around a pericentral infected cell. Letters indicate the infected cells plotted on c, Histogram of median pixel value of random neighborhoods in the image shown in (b), red X represent values of infected neighborhoods labeled correspondingly to the yellow labels in (b). Note that infected cell ‘C’ resides in a more periportal zone (as evident by the higher Alb levels in its neighborhood). d, smFISH quantification demonstrates lower proportions of infected periportal hepatocytes at 40hpi. Shown is the zonation score based on the smFISH images, namely the probability to observe a neighborhood zonation lower than the value observed in a random cell within the image field. Data points per category represent cells imaged from multiple non-consecutive tissue sections, sampled from 2 biologically independent mice per time point (Methods). e, scRNAseq analysis demonstrates that periportal parasites have lower transcript counts. Cells per time-point (Pericentral/periportal): 2hpi = 69/122, 12hpi = 179/715, 24hpi = 760/692, 30hpi = 182/307, 36hpi = 364/719 taken from 4/5/4/3/5 mice respectively. For (d) and (e), bounds of boxes span IQR, horizontal red lines denote the median, whiskers span 1.5*IQR (Interquartile range), black-bold circles indicate datapoints outside of said range. Significance determined by two-sided Wilcoxon rank-sum test, with p-value adjusted using Benjamini–Hochberg procedure.
Extended Data Fig. 6 Abortive hepatocytes show unique transcriptomic markers.
a-d, smFISH validations of host genes up regulated in abortive hepatocytes at 40hpi. Top row – Abortive; Bottom row – Productive. Per pair of images: Left panel - Phalloidin (Phal) in red, parasite Ch12 18S rRNA (18Sp, PBANKA_1245821) in green, Dapi blue; Right panel – Cxcl10/Cdkn1a/Sqstm1/Hes1 mRNA in white. Dashed yellow line: infected hepatocyte. Images are representatives of 35/30/30/49 (Cxcl10/Cdkn1a/Sqstm1/Hes1) imaging fields from 2 mice.
Extended Data Fig. 7 Properties of abortive hepatocytes.
a, Abortive hepatocytes are periportally zonated in-situ at 24hpi and 36hpi. Shown is the zonation score based on the smFISH images, namely the probability to observe a neighborhood zonation lower than the value observed in a random cell within the image field, bounds of boxes span IQR, horizontal black lines denote the median, whiskers span 1.5*IQR (Interquartile range). Significance was calculated using fisher’s method on one-sided Wilcoxon rank-sum test per mouse repeat (n = 2/3/2 for 24hpi/36hpi/40hpi respectevly). b-c, Pericentral bias of infection increase concordantly with the frequency of abortive cells. b, Average frequency of abortive infection at 36hpi in increasing inoculums. Error-bars denotes the standard deviation out of n = 3 biologically independent mice for injection model and n = 2 for mosquito bite model. c, zonation score in-situ similar to (a). d, MA plots of the mean PBA expression ratio between abortive and productive hepatocytes at 36hpi. Y axis indicates log2 of the mean ratio per gene. X axis indicates log10 of the gene’s max expression. PBA genes significantly increased or decreased in infected hepatocytes are plotted in red or blue respectively (FDR q-value < 0.01, ratio > 1.5). e, Abortive hepatocytes exhibit similar transcriptional changes in mosquito-bite model. Left panel - Phalloidin (Phal) in white, parasite Ch12 18S rRNA (18Sp, PBANKA_1245821) in green, Dapi blue; Right panel – Mdm2 mRNA in red, Myc mRNA in green. Dashed yellow line: infected hepatocyte. f, Abortive cells exhibit similar transcriptional changes even at 52hpi. Left panel - Phalloidin (Phal) in white, parasite Ch12 18S rRNA (18Sp, PBANKA_1245821) in green, Dapi blue; Right panel – Mdm2 mRNA in red, Myc mRNA in green. Dashed yellow line: infected hepatocyte. g, Abortive parasites exhibit loss of mRNA localization for some Plasmodium genes.
Extended Data Fig. 8 Unsupervised clustering of the infected hepatocytes based on Plasmodium transcriptome.
a, UMAP colored by hours post-infection (hpi) and inoculation method. b, UMAP colored by Abortive/Productive tag assigned to cells based on clustering analysis in Fig. 3a. c, UMAP colored by Louvain cluster (Methods). d, Heatmap for marker genes per cluster. purple – low expression; yellow – high expression. e, The contribution of different sampled time points to the composition of individual clusters and the complete infected hepatocytes atlas. g, Total Plasmodium (PBA) reads increase with time in productive clusters. Y axis - log10 of the sum of PBA reads. Pairwise significance between clusters was determined by two-sided Wilcoxon rank-sum test (ns:p > 0.05; ****:p < = 0.0001; ELS-MELS:p < 2.22e-16, MELS-MLLS:p < 2.22e-16, MLLS-LLS:p = 0.15, LLS-Abortive:p = 3.9e-7; n cells per cluster from left-to-right: 1,657/1,434/507/319/198). bounds of boxes span IQR, horizontal black lines denote the median, whiskers span 1.5*IQR (Interquartile range), black-bold circles indicate datapoints outside of said range.
Extended Data Fig. 9 Pseudotime and spatial analysis of the infected hepatocytes based on Plasmodium transcriptome.
a-d, Monocle UMAP visualization of the infected hepatocytes superimposed with their pseudotime trajectories. Initial node marked with white dot. a, Cells colored by pseudotime value. b, cells colored by sampled time point (hours post infection, hpi). c, Cells colored by their Abortive/Productive tag assigned based on clustering analysis in Fig. 3a. d, Cells colored by the 3 main branching trajectories. e, main UMAP figure colored by the 3 main branching trajectories. Note that branch B3 is in complete overlap with the “Abortive” cell cluster as shown in Extended Data Fig. 8c, while branch B2 is a subset of cluter “Mid-early LS”. f-g, B2 branch is a batch effect of 2 plates with elevated number of reads. f, Heatmap of marker genes per branch. purple – low expression; yellow – high expression. B2 markers are mainly ribosomal genes. g, log10 of total Plasmodium reads is significantly higher in branch B2 compared to B1 (two-sided Wilcoxon rank-sum test). h, Pseudotime distributions in cells binned by cluster. Pairwise significance between consecutive groups was determined by two-sided Wilcoxon rank-sum test (**:p < = 0.01; ****:p < = 0.0001; adjusted p-values for all comparisions p < 2.22e-16; n cells per cluster from left-to-right: 1,657/1,434/507/319/198). bounds of boxes span IQR, horizontal black lines denote the median, whiskers span 1.5*IQR, black-bold circles indicate datapoints outside of said range. i, MA plot (log ratio – M; to max average - A) showing zonally enriched genes in late pseudotime. Y axis indicates log2 of the mean ratio per gene. X axis indicates log10 of the gene’s max average expression. Genes significantly increased pericentral or periportal hepatocytes are plotted in red or blue respectively (FDR q-value < 0.2, Methods). The names of selected genes marked with black circle are highlighted.
Extended Data Fig. 10 Pseudotemporal expression in different Plasmodium gene sets.
a, Marker genes for salivated sporozoites (taken from Bogale et al23). b, Marker genes for Activating sporozoites (taken from Real et al24). c, Marker genes for sporozoites in the Malaria Cell Atlas (taken from Howick et al13). d, Marker genes for exoerythrocytic forms (EEF) in the Malaria Cell Atlas (taken from Howick et al13). e-f, RNA/DNA polymerases. g, Genes related to Plasmodium KEGG pathway “Glycerolipid metabolism”. h, Genes related to Plasmodium KEGG pathway “Glycerophospholipid metabolism”. i, Genes related to Plasmodium KEGG pathway “Pyruvate metabolism”. j, Genes related to Plasmodium KEGG pathway “Glycolysis / Gluconeogenesis”. k, Genes related to Plasmodium KEGG pathway “Biotin metabolism”. l, Genes related to various Plasmodium KEGG pathways relating to fatty acids (e.g. biosynthesis, elongation, degradation). Pseudotemporal expression Color indicates gene-wise normalization over pseudotime with yellow = 1 = max expression. m, Pie chart showing the distributions of the times post infection for cells included in each of the 8 pseudotime bins in a-l.
Supplementary information
Supplementary Table 1
Stratified MUS expression over time and space. Mouse gene expression of zonally-stratified cells at different time points tagged as uninfected (NI) or infected (INF). Shown are means, SEMs (Units are fraction of cellular UMIs), stratified median ratio per-gene, one-sided Wilcoxon rank-sum test p-value and Benjamini–Hochberg FDR q-value (left: up regulation in INF, right: down regulation).
Supplementary Table 2
DGE. Differential Plasmodium/Mouse gene expression of (1) 36hpi cells tagged as either abortive or productive (2) zonally-stratified infected cells in late pseudotime. Shown are means [Units are fraction of cellular UMIs], ratio between means, two-sided Wilcoxon rank-sum test p-value, and Benjamini–Hochberg FDR q-value.
Supplementary Table 3
Stratified expression over pseudotime. Plasmodium and Mouse gene expression of pseudotemporally-stratified infected cells. Shown are means, SEMs [Units are fraction of cellular UMIs], center of mass (COM) and KEGG pathways that include this gene (for PBANKA).
Supplementary Table 4
Probes used in this study. Sequences of the smFISH probes libraries used in this study.
Supplementary Table 5
Sample statistics. Detailed breakdown of the contribution of different samples to the scRNA-seq dataset.
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Afriat, A., Zuzarte-Luís, V., Bahar Halpern, K. et al. A spatiotemporally resolved single-cell atlas of the Plasmodium liver stage. Nature 611, 563–569 (2022). https://doi.org/10.1038/s41586-022-05406-5
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DOI: https://doi.org/10.1038/s41586-022-05406-5
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