Fig 1.
Analysis of immature and mature S. mansoni eggs isolated from mouse livers and intestines with focus on morphology, miracidia viability and differential gene expression.
(A) Microscopy visualization: Images depict the morphology of eggs following isolation and Percoll separation, highlighting the visual differences between immature and mature stages. Scale bar = 200 μm. (B) Size measurements: An analysis of the length and width of S. mansoni eggs (n = 30) after separation by Percoll. The results were statistically evaluated using a Two-way ANOVA in GraphPad Prism; error bars = SD; ***p<0.001; non-significant = ’ns’. (C) Viability of miracidia originating from liver and intestinal eggs: Percentage of viable miracidia 4 hours after hatching. Bar graph shows a mean of 3 biological replicates represented by miracidia originating from three different mice. Number of miracidia observed in each liver/intestine replicate was 16–27. Data were evaluated by t-test in GraphPad Prism; error bars = SD; *p<0.05. (D) Principal Component Analysis (PCA): The PCA plot showcases the clustering of eggs based on organ origin and maturity level, reflecting the underlying variations in gene expression profiles analyzed through RNA-Seq. INT_im: intestinal immature eggs; INT_ma: intestinal mature eggs; LIV_im: liver immature eggs; LIV_ma: liver mature eggs.
Fig 2.
Comparative Gene Ontology (GO) enrichment analysis between immature and mature S. mansoni eggs.
(A) Upregulated pathways of immature and mature eggs developing in the murine intestine. (B) Upregulated pathways of immature and mature eggs developing mouse liver. The plots are segmented into three sections for Molecular function (orange), Biological process (green), and Cellular component (blue). A maximum of ten pathways from each category are shown.
Fig 3.
Differential expression analysis (DESeq2) and Gene Ontology (GO) enrichment analysis comparing S. mansoni eggs isolated from intestine and liver.
(A) Heatmaps show the overview of all differentially expressed genes (DEGs) between eggs isolated from the intestine and liver, encompassing both developmental stages: immature and mature eggs. Notably, intestinal eggs have more upregulated genes in both developmental stages. INT = intestinal eggs; LIV = liver eggs. (B, C) Comparative GO enrichment analysis between intestinal (B) and liver (C) eggs. The plots are segmented into three sections for Molecular function (orange), Biological process (green), and Cellular component (blue). A maximum of ten pathways from each category are shown.
Fig 4.
Comparative gene expression analysis, investigating mitochondrial genes (A) and genes encoding molecules with potential roles in egg-host interaction (B, C, D) within mature S. mansoni eggs obtained from the intestinal and liver tissues of experimentally infected mice.
Volcano plots show genes that were statistically significantly differentially expressed (DESeq2 analysis; padj < 0.05; log2 fold change > 1) This encompasses (A) genes encoded in mitochondrial genome, (B) micro-exon genes (MEGs) and venom allergen-like proteins (VALs), (C) immunomodulatory molecules IPSE/alpha-1 and omega-1 and (D) proteases and inhibitors in S. mansoni eggs. The corresponding heat maps show a mean of gene expression levels normalized to Reads Per Million (RPM), serving to highlight the proportional contributions of each subgroup to the overall gene expression profile in a given sample.
Fig 5.
Immunolocalization of IPSE/alpha-1 in mouse liver (A, C) and intestine sections (B, D) infected with S. mansoni.
(A, B) Overview axioscan images display entire sections of infected tissue, with three highlighted zoomed areas. (C, D) Confocal close-ups illustrate mature eggs with developed miracidia. Labels are represented by red for anti-IPSE/alpha-1 antibody-specific sites, green for eggshell autofluorescence, and blue for DAPI-labeled cell nuclei. The red anti-IPSE/alpha-1 signal is particularly intense in the subshell area and around liver eggs, while it is weaker or absent in the intestinal eggs. Scale bars: whole-organ section = 1000 μm, zoomed square areas = 50 μm, and confocal close-ups = 20 μm. For negative control images see S2 Fig.
Fig 6.
Soluble egg antigens (SEA) derived from liver and intestinal eggs differentially affect the host immune response.
Mice were infected with 150 cercariae of S. mansoni. Serum, spleen, and mesenteric lymph nodes (mLNs) were collected at 7 weeks post infection. Uninfected (healthy) age-matched mice were used as controls. (A) Reactivity of serum antibodies (IgG and IgG1) with liver and intestinal SEA. (B) Cytokine production by splenocytes or mLN cells treated by liver or intestinal SEA (20 μg/ml) for 72 hours. Values obtained from individual mice are shown (n = 5–7). Data were evaluated by 2-way ANOVA followed by Holm-Šídák’s multiple comparisons test; *p<0.05, **p<0.01, ***p<0.001.