Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution
- Department of Pathobiological Sciences, University of Wisconsin-Madison, United States
- Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, United States
Figures
Figure 1 with 1 supplement
Optimization of single-cell dissociation in B. malayi microfilariae.
(A) Schematic of dissociation protocol using 1 million B. malayi microfilariae as input. (B) Images of 96-well plate wells containing pre- and post- PD-10 filtration of microfilariae-containing …
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Figure 1—source data 1
Comparison of cell counting methods by hemocytometer, flow cytometry, and automated cell counting instrument.
Determining the total cell count in the suspension was difficult due to remnants of debris and small and variable sized cells within the suspension. The recommended tabletop automated cell counting system Countess II (Thermo Fisher, Waltham, MA) was unreliable in confirming total and viable cell counts, likely due to cell sizes below the lower threshold of the instrument and because the cells are in multiple focal planes within the chip. Fluorescent-activated cell sorting (FACS) was also used to estimate cell count and attempt enrichment of viable cells prior to loading the 10x Genomics. The majority of cells did not survive the FACS process and could not be recovered for input into the 10x Genomics Chromium controller. Estimating cell concentration was most reliable using a hemocytometer. Bpa, Brugia pahangi; Bma, Brugia malayi; mf, microfilariae.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig1-data1-v2.docx
Figure 1—figure supplement 1
Optimization of SDS-DTT incubation by time and concentration.
Scale bar = 50 µm.
Figure 2 with 6 supplements
A single-cell transcriptomic atlas of B. malayi microfilariae cell types.
(A) Global Uniform Manifold Approximation and Projection (UMAP) transcriptome clustering of 46,621 cells with cell-type annotations. Inset: comparison of bulk transcript per million counts (tpm) …
Figure 2—figure supplement 1
Histograms of genes (left) and UMIs (right) per cell across all clustered cells.
Red line indicates the mean.
Figure 2—figure supplement 2
Distribution of transcription factors by cell type.
(A) Dot plot of orthologous B. malayi transcription factors based on C. elegans transcription factors confirmed through direct evidence as indicated in the CIS-BP database. Dot size indicates the …
Figure 2—figure supplement 3
Uniform Manifold Approximation and Projection (UMAP) visualization of marker genes used to annotate cell-type clusters.
Figure 2—figure supplement 4
Pseudobulk analysis of scRNA-seq transcriptomic data.
The raw counts for each cluster based on treatment (untreated or ivermectin [IVM]-treated) were aggregated to simulate a bulk transcriptomic library sample. Samples and B. malayi genes are …
Figure 2—figure supplement 5
Distribution of neuronal gene transcripts across all clusters.
Circle size (Total reads [%]) represents the proportion of total reads for each gene located in that cluster. Color indicates neuronal class. MS, muscle; MD, mesoderm; C, coelomocyte; S, secretory; …
Figure 2—figure supplement 6
Integration of single-cell datasets using Scanorama algorithm.
Bma, B. malayi mf. Ben-David, C. elegans L2 stage dataset from Ben-David et al., 2020. CeNGEN, C. elegans L4 stage from Taylor et al., 2021. Integration of the three datasets using the Scanorama …
Figure 3 with 3 supplements
Annotation of the Brugia secretory cell and localization of secretory-related antigens indicates broad distribution of antigen transcriptic origins.
(A) Schematic of fluorescent-activated cell sorting (FACS) enrichment approach for isolating cell populations in B. malayi microfilariae (mf) single-cell dispersions. Viable cells were sorted by …
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Figure 3—source data 1
List of differentially expressed genes identified in the B. malayi microfilariae (mf) ‘largest’ cell population collected via fluorescent-activated cell sorting (FACS) and bulk RNA-seq.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig3-data1-v2.csv
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Figure 3—source data 2
List of differentially expressed genes in secretory cells (cluster 15).
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig3-data2-v2.csv
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Figure 3—source data 3
List of differentially expressed genes identified in C. elegans large and GFP(+) cells collected via fluorescent-activated cell sorting (FACS) and bulk RNA-seq.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig3-data3-v2.csv
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Figure 3—source data 4
Comprehensive table of major antigens, vaccine targets, diagnostic markers, and notable secreted proteins.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig3-data4-v2.csv
Figure 3—figure supplement 1
Representative fluorescent-activated cell sorting (FACS) gating scheme for collecting B. malayi single-cells based on size, granularity, and presence or absence of anti-Wolbachia IgG antibody.
Cells were separated from debris (Gate 1) and live single cells were differentiated from doublets using forward scatter, side scatter, and DAPI/DRAQ5 staining (Gates 2–4). Cells were further …
Figure 3—figure supplement 2
Representative fluorescent-activated cell sorting (FACS) gating scheme for collecting C. elegans strain BK36 cell populations.
Cells were separated from debris (Gate 1) and live single cells differentiated from doublets using forward scatter, side scatter, and DAPI intensity (Gates 2–4). GFP(-) cells were separated for …
Figure 3—figure supplement 3
Distribution of gene transcripts across all clusters.
Circle size (Total reads [%]) represents the proportion of total reads for each gene located in the specified cluster. Transcript abundance distribution across cell types for prominent antigens. MS, …
Figure 4 with 1 supplement
Characterization of the secretory cell indicates an enrichment in signal peptide containing proteins and C2H2 zinc finger transcription factors.
(A) Quantification of signal peptide-containing sequences among all differentially expressed genes (DEGs) (p≤0.05) across major annotated cell types. Differentially expressed transcripts in the …
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Figure 4—source data 1
Summary table of signal peptide sequence predictions in differentially expressed transcripts across major annotated cell types.
S, secretory; MS, muscle; MD, mesoderm; C, coelomocyte; CA, canal-associated; IB, inner body; N, neurons.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig4-data1-v2.csv
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Figure 4—source data 2
Summary of transmembrane domain prediction in differentially expressed transcripts across major annotated cell types.
S, secretory; MS, muscle; MD, mesoderm; C, coelomocyte; CA, canal-associated; IB, inner body; N, neurons.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig4-data2-v2.csv
Figure 4—figure supplement 1
Gene Ontology (GO) term enrichment analysis for differentially expressed genes identified in the secretory cell.
Figure 5 with 1 supplement
Distribution of putative anthelmintic targets and ligand-gated ion channel subunit colocalization.
(A) Transcriptomic and gene expression profiles of major anthelmintic targets in microfilarial cell types. Targets include β-tubulins and cys-loop ligand-gated ion channel (LGIC) subunits …
Figure 5—figure supplement 1
Distribution of gene transcripts associated with anthelmintic targets across all cell clusters.
Circle size (Total reads [%]) represents the proportion of total reads for each gene located in the specified cluster. S, secretory; MS, muscle; MD, mesoderm; C, coelomocyte; CA, canal-associated; …
Figure 6
Cell viability and transcriptional shifts in response to anthelmintics.
(A) Schematic and representative flow cytometry contour plots depicting Calcein Violet-AM (viable) fluorescence in control samples (untreated, 0.1% DMSO, and methanol fixed). Methanol fixed cells …
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Figure 6—source data 1
Differential gene expression analysis between untreated and ivermectin (IVM) (1 µM)-treated single-cell suspensions.
Positive average log2 fold-change (avg_log2FC) values indicate the gene is more highly expressed in cells exposed to IVM. Pct.1 and pct.2 correspond to the percentage of cells expressing the gene in the treated and untreated cell populations, respectively. p-Values were adjusted (p_val_adj) using a Bonferroni p-value correction approach.
- https://cdn.elifesciences.org/articles/83100/elife-83100-fig6-data1-v2.csv
Figure 7 with 2 supplements
Cells of dispersed microfilaria are viable in culture 96 hr after dissociation.
(A) Differential interference contrast (DIC) and fluorescence microscopy of cells from microfilarial stage B. malayi parasites on peanut-lectin-coated slides after 24 hr incubation in culture …
Figure 7—figure supplement 1
Representative images of cell viability over 96 hr.
DRAQ5 (red) indicates nucleated objects and Calcein-AM (green) fluorescence indicates viable cells. Scale bar = 20 µm.
Figure 7—figure supplement 2
EdU cell proliferation assay of B.
malayi mf-derived cells and CHO-K1+Gα16 cells as a proliferating cell positive control. B. malayi cells were incubated with different EdU concentrations and EdU was detected using an anti-EdU Alexa …
Tables
Author response table 1
| Paper | Stage | UMIs/cell | genes/cell |
|---|---|---|---|
| Preston et al., 2019 | embryo | 156 | 52 |
| This study | mf | 267 (median) | 230 (median) |
| Cao et al., 2017 | L2 | 575 (median) 1,121 (mean) | 431 (mean) |
| Taylor et al., 2021 | L4 | 893 (median) | 321 (median) |
| Roux et al., 2022 | Adult | 2,175 | 644 |
